Article

Coniferous-Broadleaf Mixture Increases SoilMicrobial Biomass and Functions Accompanied byImproved Stand Biomass and Litter Production inSubtropical China

Wenxiang Wu 1 Xiaoguo Zhou 1 Yuanguang Wen 12 Hongguang Zhu 1 Yeming You 1Zhiwei Qin 3 Yunchou Li 1 Xueman Huang 1 Li Yan 1 Haiyan Li 4 and Xiaoqiong Li 1

1 Guangxi Key Laboratory of Forest Ecology and Conservation Forestry College Guangxi University100 Daxuedong Road Nanning 530004 China wenxiangwufoxmailcom (WW)xgzhou2014126com (XZ) xu980307163com (HZ) youyeming163com (YY)franklee1235163com (YL) huangxm168168163com (XH) 13647886805163com (LY)lixiaoqiong100163com (XL)

2 Guangxi Youyiguan Forest Ecosystem National Research Station Pingxiang 532600 China3 Guangxi Laibin Forest Inventory and Planning Institute 289 Yaru Road Liuzhou 545001 China

qzw118590578163com4 Guangxi Yulin Forest Inventory and Planning Institute 332 Renmin East Road Yulin 537000 China

lihaiyan6084foxmailcom Correspondence wenyg263net Tel +86-0771-3271-048

Received 16 August 2019 Accepted 2 October 2019 Published 6 October 2019

Abstract Although the advantages of multi-species plantations over single-species plantations havebeen widely recognized the mechanisms driving these advantages remain unclear In this study wecompared stand biomass litter production and quality soil properties soil microbial communityand functions in a Pinus massoniana Lamb and Castanopsis hystrix Miq mixed plantation and theircorresponding mono-specific plantations after 34 years afforestation in subtropical China The resultshave shown that a coniferous-broadleaf mixture created significantly positive effects on stand biomasslitter production soil microbial biomass and activities Firstly the tree shrub and herb biomass andlitter production were significantly higher in the coniferous-broadleaf mixed plantation Secondlyalthough the concentrations of soil organic carbon (SOC) and total nitrogen (TN) were lower inthe mixed stand the concentrations of soil microbial biomass carbon (MBC) and nitrogen (MBN)along with MBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands withmarkedly positive admixing effects We also found higher carbon source utilization ability andβ-14-N-acetylglucosaminidase urease and acid phosphatase activities in mixed stands comparedwith the mono-species stands Our results highlight that establishment of coniferous-broadleaf mixedforests may be a good management practice as coniferous-broadleaf mixture could accumulate higherstand biomass and return more litter resulting in increasing soil microbial biomass and relatedfunctions for the long term in subtropical China

Keywords coniferous-broadleaf mixture soil microbial biomass extracellular enzymes phospholipidfatty acids (PLFA) carbon source utilization ability

1 Introduction

Planted forests have been rapidly expanding around the world to meet the ever-growing demandfor timber and other ecosystem services while avoiding excessive harvesting of natural forests [12]Most planted forests are monospecific plantations In China 85 of the countryrsquos planted forests are

Forests 2019 10 879 doi103390f10100879 wwwmdpicomjournalforests

Forests 2019 10 879 2 of 20

monospecific plantations as reported [3] However there are serious concerns regarding monospecificplantations including stand productivity decrease [4] soil quality degradation [5] and serious exoticplant invasion (eg Eucalyptus plantations) [6] In contrast multi-species plantations often havehigher biodiversity higher productivity and better soil quality than single-species plantations [7ndash9]Theoretically higher productivity in multi-species compared to single-species plantations can onlybe expected when species interactions in the multi-species plantations increase the efficiency ofresource use or one species increases the supply of a resource [10] However interactions betweenspecies growing in mixtures eg competition competitive reduction and facilitation [11] are oftendynamic changing through space and time as resource availability or climatic conditions change [1213]Therefore it is necessary to understand how tree species interact in mixed plantations and to furtherdevelop these systems to minimize competition while maximizing both wood production and otherecosystem services in the long term

Masson pine (Pinus massoniana Lamb) and Castanopsis hystrix Miq are two afforestation treespecies that are most widely planted in subtropical China usually making up monoculture plantationsSuch monospecific plantations have caused serious ecological concerns including declines in standproductivity litter quality and soil fertility resulting from long-term single litter input increased pestand disease attack self-allelopathy and site degradation [14ndash16] To mitigate these disadvantagesmixed P massoniana and C hystrix plantations were established about 40 years ago at the ExperimentalCenter of Tropical Forestry Chinese Academy of Forestry in subtropical China At present thearea of these plantations is more than 5000 ha [1] Previous studies have suggested that mixedplantations of these two genera could lead to more plant species diversity more productivity fasterlitter decomposition rate and higher carbon stocks compared to monospecific stands [7141617]Interactions between aboveground plant communities and belowground microbial communities mayunderlie some of these positive effects while little is known about these interactions in mixed stands ofP massoniana and C hystrix

In general there are enormously important interactions between the composition of abovegroundplant communities and belowground microbial communities in forest ecosystems [18] Managementstrategies such as tree species transformation adding N2-fixing species or broad-leaf species inconiferous monospecific stands may change the amount and chemical composition of litterfall rootmass and exudates [1419] then affect soil organic matter quantity and dynamics [8] along withmicrosite conditions [20] As a result these changes may control the composition and functions of the soilmicroorganisms which acquire carbon from primarily producer-derived organic resources [21] At thesame time soil microbes are important regulators of plant productivity through the mineralizationof and competition for nutrients that sustain plant productivity [22] Moreover soil microbialdiversity and abundancebiomass play key roles in ecosystem sustainability by maintaining essentialfunctions of soil health through carbon and nutrient turnover [23] Meta-analysis has shown thatplant mixtures have increased soil total microbial bacterial and fungal biomass across a diverse rangeof terrestrial ecosystems and the increase in microbial biomass was more pronounced in older andmore diverse mixtures [24] This may be attributable to higher productivity inducing more carbonand nutrient inputs to the soil in mixtures benefitting both fungi and bacteria [2425] Moreoverdifferent tree species in mixtures produce litter of varying quality interacting with the rhizosphere andcausing microenvironmental changes that affect microbial activity and therefore soil nutrients [26ndash28]However the effects of tree species mixtures on soil microorganisms may change with ecosystem typesenvironment and time Whether coniferous-broadleaf mixture increases soil microbial biomass andactivity in subtropical China in long-term afforestation remains unexplored

In this study we explored stand biomass litter soil physicochemical properties and soil microbialcommunity structure and function in a mixed P massoniana and C hystrix plantation and correspondingsingle species plantations at an age of 34 years in subtropical China Our goal was to determine theeffects of coniferous-broadleaf mixture on soil microbial communities and functions in long termWe hypothesized that (1) stand biomass litter production and quality are higher in the mixed stands

Forests 2019 10 879 3 of 20

than those in corresponding monocultures in long term (2) positive admixing effects on soil microbialbiomass and functions are accompanied by improved stand biomass and litter production

2 Materials and Methods

21 Study Sites and Experimental Design

This study was conducted at the Experimental Center of Tropical Forestry (2210prime N 10650prime E)Chinese Academy of Forestry Pingxiang City Guangxi Zhuang Autonomous Region PR ChinaThis region belongs to a subtropical monsoon climate zone and has a humid-to-semi-humid climatewith dry-cool (from October to April) and wet-warm seasons (from May to September) according to thevariation of precipitation and temperature [29] The mean annual precipitation is about 1400 mm mostrainfall occurs from April to September The mean annual temperature is 21 C The loamy-texturedsoil in this area was formed from granitic parent geological material and is classified as red soil in theChinese system of soil classification which is equivalent to an oxisol in the USDA Soil Taxonomy [814]

Our study site consisted of a 33 ha plantation of pure C hystrix (PCH) a 33 ha plantation of pureP massoniana (PPM) and an 18 ha plantation of mixed C hystrix and P massoniana (MIX) Prior to theestablishment of the current plantations the area was covered by a Cunninghamia lanceolata monospecificplantation which was established in 1958 on a historically deforested hill and clear harvested in1983 After clearcutting the prior plantations the PCH PPM and MIX plantations were establishedin 1983 with an initial planting density of 2500 tree haminus1 and a 11 mixed proportion in the MIX forcomparison of tree productivity and the ecological benefits of mixed plantations Buffer zones of150ndash800 m were set up to separate these plantations These stands had similar soil types topographiesand management histories

In June 2017 34 years after the establishment of the plantations we randomly established fivesample plots (20 m times 20 m) in each plantation The five sampling plots within a single experimentalstand were well separated with distances of at least 200 m between adjacent plots Elevations of allplots were 530ndash650 m and the degree of slopes was 30ndash39 The stand density ranged from 355 to400 tree haminus1 and average height from 22 m to 25 m The diameter at breast height (DBH) and basalarea of MIX was significantly higher than that of PCH and PPM (Table 1)

Table 1 Characteristics of pure Castanopsis hystrix Miq (PCH) plantation pure Pinus massoniana Lamb(PPM) plantation and mixed C hystrix and P massoniana (MIX) plantation (mean plusmn standard errorn = 5)

Stand Density (Tree haminus1) Height (m) DBH (cm) Basal Area (m2 haminus1)

PCH 355 plusmn 15 a 2395 plusmn 080 a 2824 plusmn 097 b 2315 plusmn 150 b

MIX 380 plusmn 10 a 2452 plusmn 037 a 3431 plusmn 102 a 3967 plusmn 227 a

P massoniana 95 plusmn 9 2201 plusmn 067 2616 plusmn 075 535 plusmn 085C hystrix 285 plusmn 15 2533 plusmn 039 3695 plusmn 163 3432 plusmn 291

PPM 400 plusmn 24 a 2332 plusmn 071 a 2816 plusmn 104 b 2660 plusmn 257 b

Different superscripts indicate significant differences among stand types (p lt 005) DBH diameter at breast height

22 Stand Biomass Measurements

Three 2 m times 2 m subplots and three 1 m times 1 m subplots were randomly placed in each plotfor shrub and herb biomass measurements respectively in the wet-warm (July 2017) and dry-coolseasons (January 2018) In each season shrubs and herbs were destructively harvested and dividedinto aboveground and belowground parts All understory vegetation samples were transported to thelaboratory and oven-dried at 75 C for dry biomass determination (Mg haminus1) [30]

In addition we measured tree height with a digital clinometer (Vertex IV Hagloumlf Inc LangseleSweden) and DBH with a diameter tape of each tree in each plot and in each season Tree aboveground

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biomass (TAB) and tree belowground biomass (TBB) were calculated by the allometric equationsdescribed by You et al (2018) [7]

23 Litter Production and Quality Measurements

A litter collector with an effective collection area of 4 m2 was randomly set in each plot Each littercollector was made with nylon net with a mesh size of 1 mm and set 05 m above the ground withPVC (polyvinyl chloride) tube Litter was collected once a month from August 2017 to July 2018 Littersamples were transported to the laboratory and oven-dried at 75 C for production determination(Mg haminus1) Litter organic carbon (OClitter) concentration was determined by the K2Cr2O7ndashH2SO4

calefaction procedure [30] Litter total nitrogen (TNlitter) total phosphorus (TPlitter) and total potassium(TKlitter) concentrations were measured after heat digestion with 98 H2SO4 [31ndash33]

24 Soil Sampling and Analysis

Soil samples were also collected in the wet-warm (July 2017) and dry-cool seasons (January 2018)In each season we took eight soil cores systematically arranged at a distance of 5 m from the plotcenter in the directions of 0 45 90 135 180 215 270 and 315 Each core was taken using an87 cm inner diameter steel tube at a depth of 0ndash10 cm below the soil surface The eight cores of eachplot were mixed to form a composite sample Each soil sample was divided into two subsamplesOne subsample was air-dried to a constant weight and sieved using a 2 mm sieve for measuring soilproperties The other subsample was sieved to 2 mm and immediately stored at minus4 C for measuringmoisture content soil ammonium nitrogen (NH4

+-N) nitrate nitrogen (NO3minus-N) microbial biomass

carbon (MBC) microbial biomass nitrogen (MBN) concentration phospholipid fatty acid analysis(PLFA) assays carbon source utilization ability of the soil microbial communities and enzyme activityAt the same time three soil cores (100 cm3) from 0ndash10 cm soil layer were sampled from each plot tomeasure bulk density by the cutting ring method [34]

The soil water content (WC) was measured using a sub-sample dried in oven at 105 C untilthe sample reached a constant weight The soil temperatures were measured with an LI-8100AAutomated Soil Gas Flux System (LindashCor Lincoln NE USA) Soil pH was measured in a 125 soilwatersuspension using a pH meter [835] Soil organic carbon (SOC) concentration was determined bythe K2Cr2O7ndashH2SO4 calefaction procedure [31] Total nitrogen (TN) total phosphorus (TP) andtotal potassium (TK) concentration were measured after heat digestion with 98 H2SO4 TN andTK were measured using the liquid supernatant with a continuous flow analytical system (AA3SEAL Analytical Norderstedt Germany) [3133] and an atomic absorption spectrometer (ZEENIT700P Analytik Jena Germany) [32] respectively TP and AP were determined by MondashSbndashVccolorimetry with the liquid supernatant after heat digestion and HCl (005 M)ndashH2SO4 (0025 M)respectively And available potassium (AK) was determined by atomic absorption spectrometry with1 M NH4OAC Soil ammonium nitrogen (NH4

+-N) and nitrate nitrogen (NO3minus-N) were calorimetrically

determined in a continuous flow analyzer after 10 g fresh mass of soil had been extracted in 50 mLof 2 M KCl [836] The cation exchange capacity (CEC) was determined by [Co(NH3)6]Cl3 extractionspectrophotometry [3738]

Soil microbial biomass carbon (MBC) and nitrogen (MBN) were measured by fumigation-extractionusing 05 M K2SO4 as the extraction agent [3940] with a total organic carbon analyzer (1020A OICollege Station TX USA)

Phospholipid fatty acid analysis (PLFA) assays were employed to analyze soil microbial communitystructure with freeze-dried soil samples using the procedure developed by Bossio et al (1998) [41]The PLFA markers 140 150 170 180 141ω5c 150 3OH 151ω6c 160 2OH 161 2OH 161ω9c161ω7c 181 ω7c 180 2OH 181 ω5c a150 a160 a170 cy170 cy190 i140 i150 i160 i170 i180and i190 were chosen to represent bacteria Gram-positive (GP) bacteria were represented by a150a160 a170 i140 i150 i160 i170 i180 and i190 and gram-negative (GN) bacteria were representedby 141ω5c 150 3OH 151ω6c 160 2OH 161 2OH 161ω7c 161ω9c 180 2OH 181ω5c cy170

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cy190 and 181 ω7c [364243] Actinobacteria were represented by 10-Me170 10-Me180 TBSA11-Me181ω7c and 10-Me160 [124344] Arbuscular mycorrhizal fungi (AMF) were represented by161 ω11c and 161 ω5c [35] and fungi were represented by 181 ω9c 183 ω6c (6912) and 182ω69c [1244] Other PLFAs such as 150 2OH 160 171ω8c 180 iso and i200 were also used for theanalysis of the composition of microbial communities

Carbon source utilization ability of the soil microbial communities was analyzed using BIOLOGreg

Eco plates (Biolog Hayward CA USA) with fresh soil samples Each Biolog Econdashplate comprised96 wells containing triplicates of 31 different carbon sources and a blank [45] We added 10 g dryweight equivalent of fresh soil to 90 mL of 085 sterile saline physiological solution and shook themixture for 1 h on a rotary shaker (100 rpm) at 25 C After 10minus3 dilution we transferred samplescontaining 150 microL of liquid into the 96 wells and read the optical density (OD) at 590 and 750 nm witha Microstationtrade reader (Biolog) after 24 4872 96 120 144 168 and 192 h of incubation [4647]

Enzyme assays were performed with a fully automatic full-wavelength enzyme labeling apparatus(Infinite M200 Pro Tecan Inc Maumlnnedorf Switzerland) using the procedure describe in detailby Saiya-Cork et al (2002) [48] and Steinauer et al (2015) [49] The phenol oxidase andperoxidase assays were colorimetric using L34-dihydroxyphenylalanine (L-DOPA) as the substrateThe colorimetric urease assay used urea as the substrate The α-Glucosidase (AG) β-14-glucosidase (BG)β-Cellobiosidase (CB) acid phosphatase (APH) and β-14-N-acetylglucosaminidase (NAG) activities werefluorometric with substrate 4-methylumbelliferyl-α-D-glucoside 4-methylumbelliferyl-β-D-glucoside4-methylumbelliferyl-β-D-cellobioside 4-methylumbelliferyl-phosphate and 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide respectively

25 Statistical Analysis

One-way analyses of variances (ANOVAs) were used to examine the differences of stand biomasslitter production and quality soil physicochemical properties soil microbial biomass soil microbialPLFAs content and functions among PPM PCH and MIX plantations in each season Two-wayANOVAs were used to determine the interaction effects between stand type and season on the abovevariables Statistical analyses were performed with SPSS software (ver 250 IBM Corp Armonk NYUSA) Significant differences between means were tested by least significant difference (LSD) at the005 level

In order to evaluate admixing effects on soil microbial biomass we calculated the relative effects(RE) of mixing species by comparing the observed value (OV) of one variable in mixed plantationwith the predicted value (PE) based on the respective mono-specific plantations RE was calculated byfollowing Wardle et al (1997) [50]

RE = [(OV minus PE)PE] times 100 (1)

where PE is the mean of the observed value of one variable in respective mono-specific plantationsIf RE differs from zero it would indicate non-additive effects (NAE) of mixing species on soil microbialbiomass Negative and positive deviations from zero are referred to as antagonistic and synergisticeffects respectively One-sample Studentrsquos t-tests with 95 confidence intervals were employed to testwhether RE differed significantly from zero

Redundancy analysis (RDA) was used to determine the relationships among biotic and abioticfactors and soil microbial composition and functions by CANOCO software for Windows 5(Biometris-Plant Research international Wageningen The Netherlands) In RDA the relative abundanceof individual PLFAs (mole present of the total PLFAs) was used as the response variables and soilphysicochemical properties litter and stand biomass were used as the explanatory variables [3551]We also used RDA to determine the linkage between soil microbial community and functions includingcarbon source metabolic function and soil extracellular enzyme activity involved in biogeochemicalcirculation of carbon nitrogen and phosphorus All data were log-transformed before in the analyses

Forests 2019 10 879 6 of 20

The forward selection procedure in RDA based on Monte Carlo permutation with 499 iterations wasperformed to determine the dominant variables that influenced the composition of individual PLFAsand microbial functions A significance level of p lt 005 was used in all analyses

3 Results

31 Stand Biomass

Stand type had significant effects on the -above- and below-ground biomass of trees shrubs andherbs (Table S1 Figure 1) TAB TBB shrub aboveground biomass (SAB) shrub belowground biomass(SBB) and herb aboveground biomass (HAB) in MIX were significantly higher than those in PCHand PPM in both the dry-cool and wet-warm seasons (Figure 1andashe) Except for HAB season had nosignificant effects on the tested biomass variables Moreover significant interaction effects betweenstand type and season only found on HAB and HBB These results supported our first hypothesis thattree species mixture increased stand biomass

Forests 2019 10 x FOR PEER REVIEW 6 of 22

biogeochemical circulation of carbon nitrogen and phosphorus All data were log-transformed before in the analyses The forward selection procedure in RDA based on Monte Carlo permutation with 499 iterations was performed to determine the dominant variables that influenced the composition of individual PLFAs and microbial functions A significance level of p lt 005 was used in all analyses

3 Results

31 Stand Biomass

Stand type had significant effects on the -above- and below-ground biomass of trees shrubs and herbs (Table S1 Figure 1) TAB TBB shrub aboveground biomass (SAB) shrub belowground biomass (SBB) and herb aboveground biomass (HAB) in MIX were significantly higher than those in PCH and PPM in both the dry-cool and wet-warm seasons (Figure 1andashe) Except for HAB season had no significant effects on the tested biomass variables Moreover significant interaction effects between stand type and season only found on HAB and HBB These results supported our first hypothesis that tree species mixture increased stand biomass

Figure 1 Biomass of trees (ab) shrubs (cd) and herbs (ef) in three plantations in different seasons Different letters above the bars denote significant differences at p lt 005 level by LSD (least significant difference) multiple comparisons among stand types (n = 5)

32 Litter Production and Quality

Stand type the interaction between stand type and season significantly influenced litter production at our study sites (Table 2) The MIX had significantly higher litter production than PCH and PPM in both seasons OClitter and CNlitter were significantly affected by both stand type and season

Figure 1 Biomass of trees (ab) shrubs (cd) and herbs (ef) in three plantations in different seasonsDifferent letters above the bars denote significant differences at p lt 005 level by LSD (least significantdifference) multiple comparisons among stand types (n = 5)

Forests 2019 10 879 7 of 20

32 Litter Production and Quality

Stand type the interaction between stand type and season significantly influenced litter productionat our study sites (Table 2) The MIX had significantly higher litter production than PCH and PPMin both seasons OClitter and CNlitter were significantly affected by both stand type and season withhigher value in the wet-warm season than in the dry-cool season in all plantations An opposite trendwas observed in TNlitter TPlitter was significantly affected by stand type season and their interactionwith MIX had significantly lower concentration than PCH and PPM in the dry-cool season TKlitter wassignificantly affected by stand type with MIX had significantly higher concentration than PCH andPPM in the wet-warm season These results partially supported our first hypothesis that tree speciesmixture increased litter production although the increases of litter quality in mixed plantations werenot significant

33 Soil Physical and Chemical Properties

In the dry-cool season the mixed plantations had the highest soil bulk density (BD) and soil watercontent (WC) and the lowest soil temperature and medium pH compared to the pure plantations(Table 3) In the wet-warm season MIX had the medium BD WC and soil temperature and the highestpH compared to PPM and PCH

The PPM plantations had the highest SOC TN TP SOC-to-TN ratio NH4+-N and CEC while

MIX had the highest AP TK and AK and PCH had the highest NO3minus-N and AN in the dry-cool season

(Table 3) On the other hand the PCH had the highest SOC TN and AP while MIX had the highestTP TK AK NO3

minus-N and AN and PPM had the highest SOC-to-TN ratio NO3minus-N and CEC in the

wet-warm seasonTwo-way ANOVAs indicated that season significantly affected all the tested soil variables (Table 3)

In addition the stand type influenced nearly all soil variables except BD TN NH4+-N and AN

Interactions between stand type and season significantly influenced WC temperature pH SOC TNTP AP AK NO3

minus-N and CEC

Forests 2019 10 879 8 of 20

Table 2 The quantity and quality of litter in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM) plantation and mixed C hystrix and P massoniana(MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH MIX PPM PCH MIX PPM F p F p F p

LP 668 plusmn 019 b 836 plusmn 069 a 484 plusmn 019 c 553 plusmn 043 b 921 plusmn 040 a 616 plusmn 023 b 3752 lt0001 093 0343 527 0013OClitter 19064 plusmn 1063 b 24859 plusmn 1848 a 25505 plusmn 1800 a 27757 plusmn 1885 b 2743 plusmn 1142 b 32175 plusmn 544 a 683 0004 2486 lt0001 226 0127TNlitter 1738 plusmn 078 a 1353 plusmn 106 b 1496 plusmn 062 ab 1295 plusmn 059 a 1357 plusmn 090 a 1189 plusmn 050 a 321 0058 1579 0001 445 0023CNlitter 1112 plusmn 095 b 1850 plusmn 081 a 1733 plusmn 181 a 2182 plusmn 223 b 2056 plusmn 156 b 2722 plusmn 096 a 767 0003 3890 lt0001 518 0013TPlitter 134 plusmn 008 a 050 plusmn 007 b 116 plusmn 005 a 119 plusmn 015 a 138 plusmn 015 a 131 plusmn 005 a 667 0005 1281 0002 1417 lt0001TKlitter 116 plusmn 016 a 152 plusmn 010 a 082 plusmn 009 b 107 plusmn 002 b 149 plusmn 013 a 088 plusmn 007 c 2084 lt0001 007 0796 029 0751

Different superscripts indicate significant differences among stand types in each season (p lt 005) LP (Mg haminus1) litter production OClitter (g kgminus1) litter organic carbon TNlitter (g kgminus1)litter total nitrogen CNlitter CN ratio of litter TPlitter (g kgminus1) litter total phosphorus TKlitter (g kgminus1) litter total potassium

Table 3 Soil physical and chemical properties (0ndash10 cm soil depth) in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM) plantation and mixedC hystrix and P massoniana (MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH Mixed PPM PCH Mixed PPM F p F p F p

BD 109 plusmn 003 b 125 plusmn 004 a 119 plusmn 002 a 103 plusmn 001 a 103 plusmn 003 a 104 plusmn 005 a 311 0063 3044 lt0001 297 0070WC 3966 plusmn 136 b 4359 plusmn 061 a 4017 plusmn 158 ab 4053 plusmn 107 c 4440 plusmn 084 b 5048 plusmn 141 a 1035 0001 1681 lt0001 1048 0001Tem 1682 plusmn 021 a 1550 plusmn 009 c 1617 plusmn 005 b 2628 plusmn 043 a 2598 plusmn 022 a 2541 plusmn 008 a 821 0002 283334 lt0001 431 0025pH 432 plusmn 002 a 419 plusmn 003 b 404 plusmn 005 c 402 plusmn 002 b 421 plusmn 003 a 390 plusmn 009 b 1440 lt0001 1370 0001 623 0007

SOC 3742 plusmn 108 b 4082 plusmn 022 b 5698 plusmn 189 a 5331 plusmn 194 a 4839 plusmn 200 a 5083 plusmn 054 a 2460 lt0001 2369 lt0001 2864 lt0001TN 163 plusmn 005 b 160 plusmn 006 b 216 plusmn 022 a 248 plusmn 011 a 213 plusmn 013 a 217 plusmn 014 a 248 0105 4408 lt0001 480 0018

SOCTN 2298 plusmn 059 b 2567 plusmn 103 b 2949 plusmn 159 a 2167 plusmn 133 a 2304 plusmn 162 a 2390 plusmn 202 a 457 0021 722 0013 114 0338TP 031 plusmn 004 c 042 plusmn 002 b 060 plusmn 004 a 052 plusmn 006 b 078 plusmn 004 a 054 plusmn 004 b 2186 lt0001 2889 lt0001 1795 lt0001AP 919 plusmn 058 b 1252 plusmn 069 a 1002 plusmn 030 b 1330 plusmn 040 a 1315 plusmn 057 a 998 plusmn 027 b 1670 lt0001 1525 0001 1029 0001TK 414 plusmn 015 b 545 plusmn 049 a 242 plusmn 016 c 540 plusmn 050 a 694 plusmn 082 a 338 plusmn 013 b 3226 lt0001 1034 0004 039 0680AK 2990 plusmn 149 b 4629 plusmn 096 a 2846 plusmn 108 b 4719 plusmn 228 a 5279 plusmn 365 a 2837 plusmn 144 b 5375 lt0001 2257 lt0001 927 0001

NO3minus-N 355 plusmn 024 a 185 plusmn 004 c 286 plusmn 022 b 406 plusmn 019 c 530 plusmn 009 b 579 plusmn 009 a 1096 lt0001 29106 lt0001 4547 lt0001

NH4+-N 2129 plusmn 101 a 2334 plusmn 113 a 2447 plusmn 137 a 4224 plusmn 162 a 4350 plusmn 166 a 3883 plusmn 247 a 075 0482 19616 lt0001 247 0105

AN 2734 plusmn 157 a 2519 plusmn 114 a 2485 plusmn 090 a 463 plusmn 155 a 488 plusmn 169 a 4462 plusmn 246 a 120 0319 24555 lt0001 117 0328CEC 4553 plusmn 327 c 5758 plusmn 411 b 8233 plusmn 194 a 5543 plusmn 202a 5446 plusmn 119 a 6224 plusmn 291 a 4514 lt0001 1216 0002 2168 lt0001

Different superscripts indicate significant differences among stand types in each season (p lt 005) BD (g cmminus1) bulk density WC () soil water content Tem (C) temperature SOC(g kgminus1) soil organic carbon TN (g kgminus1) total nitrogen TP (g kgminus1) total phosphorus AP (mg kgminus1) available phosphorus TK (g kgminus1) total potassium AK (mg kgminus1) availablepotassium NO3

minus-N (mg kgminus1) nitrate nitrogen NH4+-N (mg kgminus1) ammonium nitrogen AN (mg kgminus1) Available nitrogen CEC (cmol + kgminus1) cation exchange capacity

Forests 2019 10 879 9 of 20

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantlyaffected by stand type and MBN was also significantly affected by season and interaction betweenstand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a)In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantlyhigher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highestMBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOCand MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significantlevel in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOCand MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-coolseason (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season(Figure 3b) were significant

Forests 2019 10 x FOR PEER REVIEW 10 of 22

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantly affected by stand type and MBN was also significantly affected by season and interaction between stand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a) In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantly higher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highest MBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOC and MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significant level in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOC and MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-cool season (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season (Figure 3b) were significant

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBN MBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a) and wet-warm season (b)

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBNMBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005level by LSD multiple comparisons among stand types (n = 5)

Forests 2019 10 x FOR PEER REVIEW 10 of 22

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantly affected by stand type and MBN was also significantly affected by season and interaction between stand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a) In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantly higher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highest MBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOC and MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significant level in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOC and MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-cool season (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season (Figure 3b) were significant

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBN MBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a) and wet-warm season (b)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a)and wet-warm season (b)

Forests 2019 10 879 10 of 20

In the dry-cool season total PLFAs AMF PLFAs actinobacteria PLFAs bacteria PLFAs fungiPLFAs GP bacteria PLFAs and GP-to-GN PLFAs ratio were smaller in MIX than those in PPM andPCH On the other hand in the wet-warm season these PLFAs were smaller in PPM than thosein PCH and MIX (Table 4) Specifically the ratios of bacteria-to-fungi PLFA and GP-to-GN PLFAwere significantly higher in MIX than those in PPM and PCH in the wet-warm season These resultsindicated that the response of the soil microbial community structure to tree species mixture varied indifferent seasons Two-way ANOVAs showed that both stand type and season significantly affectedall the tested microbial variables while the interaction between stand type and season significantlyinfluenced total PLFAs bacteria PLFAs GP bacteria PLFAs Acti PLFAs bacteria-to-fungi PLFA andGP-to-GN PLFA (Table 4)

Across plantations and seasons bacteria PLFAs were the most abundant followed by fungi andactinobacteria PLFAs (Figure 4 and Table S3) It was worth noting that the relative abundance ofGN bacteria PLFAs was significantly higher in MIX than in other plantations in the dry-cool season(Figure 4) Furthermore the relative abundance of GP bacteria PLFAs was significantly higher andthat of fungi PLFAs was significantly lower in MIX than in other plantations in the wet-warm season

Forests 2019 10 x FOR PEER REVIEW 13 of 22

Across plantations and seasons bacteria PLFAs were the most abundant followed by fungi and actinobacteria PLFAs (Figure 4 and Table S3) It was worth noting that the relative abundance of GN bacteria PLFAs was significantly higher in MIX than in other plantations in the dry-cool season (Figure 4) Furthermore the relative abundance of GP bacteria PLFAs was significantly higher and that of fungi PLFAs was significantly lower in MIX than in other plantations in the wet-warm season

Figure 4 The relative abundance of PLFAs (mol ) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleaf plantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increased APH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increased urease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg) Carbon source utilization ability of the soil microbial community indicated by average well color development (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation (Figure 6)

Figure 4 The relative abundance of PLFAs (mol ) in three plantations in the dry-cool season (a) andwet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level byLSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 11 of 20

Table 4 Soil microbial community PLFAs content and structure (0ndash10 cm soil depth) in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM)plantation and mixed C hystrix and P massoniana (MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH MIX PPM PCH MIX PPM F p F p F p

TPLFAs 3767 plusmn 271 a 3255 plusmn 124 a 3796 plusmn 128 a 4373 plusmn 208 a 4001 plusmn 07 a 3478 plusmn 111 b 464 0020 646 0018 607 0007B 1579 plusmn 081 a 1306 plusmn 043 b 1490 plusmn 051 ab 1764 plusmn 072 a 1723 plusmn 042 a 1276 plusmn 041 b 1274 lt0001 768 0011 1556 lt0001

GP 945 plusmn 043 a 794 plusmn 029 b 918 plusmn 031 a 1066 plusmn 051 a 1155 plusmn 031 a 810 plusmn 031 b 817 0002 1709 lt0001 2030 lt0001GN 591 plusmn 076 a 590 plusmn 016 a 553 plusmn 022 a 619 plusmn 025 a 455 plusmn 037 b 411 plusmn 020 b 526 0013 702 0014 313 0062

F 498 plusmn 034 a 375 plusmn 022 b 424 plusmn 028 ab 492 plusmn 035 a 296 plusmn 043 b 345 plusmn 021 b 1356 lt0001 448 0045 091 0416AMF 104 plusmn 003 a 075 plusmn 003 b 075 plusmn 004 b 085 plusmn 007 a 044 plusmn 008 b 048 plusmn 003 b 2822 lt0001 3483 lt0001 056 0577Acti 426 plusmn 037 ab 377 plusmn 021 b 486 plusmn 020 a 531 plusmn 019 a 487 plusmn 012 b 458 plusmn 010 b 273 0085 1236 0002 650 0006BF 319 plusmn 011 a 351 plusmn 013 a 356 plusmn 017 a 362 plusmn 014 b 635 plusmn 094 a 377 plusmn 033 b 750 0003 1131 0003 598 0008

GPGN 174 plusmn 029 b 135 plusmn 003 b 166 plusmn 004 a 173 plusmn 008 b 263 plusmn 028 a 198 plusmn 011 b 107 0359 1376 0001 733 0003

Different superscripts indicate significant differences among stand types in each season (p lt 005) TPLFAs (nmol gminus1 soil) total PLFAs B (nmol gminus1 soil) bacteria PLFAs GP (nmol gminus1

soil) gram-positive bacteria PLFAs GN (nmol gminus1 soil) gram-negative bacteria PLFAs F (nmol gminus1 soil) fungi PLFAs Acti (nmol gminus1 soil) actinobacteria PLFAs AMF (nmol gminus1 soil)arbuscular mycorrhizal fungi PLFAs BF bacteria-to-fungi PLFA ratio GPGN GP-to-GN ratio

Forests 2019 10 879 12 of 20

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleafplantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increasedAPH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increasedurease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg)Carbon source utilization ability of the soil microbial community indicated by average well colordevelopment (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation(Figure 6)Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in threeplantations in different seasons Error bars are standard error Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 13 of 20

Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in threeplantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in thedry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons(F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pHTK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-coolseason in which SOC and TBB were the major correlates of changes in soil microbial composition(Table S5) A total of 11 biotic and abiotic factors including NO3

minus-N TBB pH TKlitter HBB AP AKTP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warmseason in which NO3

minus-N TBB pH and TKlitter were the major correlates of changes in soil microbialcomposition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

37 Relationship between Soil Microbial Structure and Functions

Soil enzyme activity and soil microbial carbon source utilization ability are both important soilmicrobial functions The first two RDA axes explained 4948 of the variation in soil enzyme activityand carbon utilization ability in the dry-cool season (F = 24 p = 0006 Figure 7b) and 6891 of thevariation in the wet-warm season (F = 64 p = 0002 Figure 7d) AMF and GP were significantlycorrelated with soil microbial functions in the dry-cool season (Table S6) Total bacteria GP bacteriaGN bacteria and actinobacteria were significantly correlated with soil microbial functions in thewet-warm season in which total bacteria GP bacteria and GN bacteria were the major correlates ofchanges of soil enzyme activity (Table S6)

Forests 2019 10 879 14 of 20

Forests 2019 10 x FOR PEER REVIEW 15 of 22

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in the dry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons (F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pH TK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-cool season in which SOC and TBB were the major correlates of changes in soil microbial composition (Table S5) A total of 11 biotic and abiotic factors including NO3minus-N TBB pH TKlitter HBB AP AK TP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warm season in which NO3minus-N TBB pH and TKlitter were the major correlates of changes in soil microbial composition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160 were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant biotic and abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot of soil microbial community structure and soil microbial functions (bd) Above the label represents a significant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowground biomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soil total potassium NO3ndash-N soil nitrate nitrogen HBB herb belowground biomass AP soil available phosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil water

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant bioticand abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot ofsoil microbial community structure and soil microbial functions (bd) Above the label represents asignificant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowgroundbiomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soiltotal potassium NO3

minus-N soil nitrate nitrogen HBB herb belowground biomass AP soil availablephosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil watercontent LP litter production TKlitter litter total potassium GP gram-positive bacteria GN gramnegative bacteria AMF arbuscular mycorrhizal fungi AG α-Glucosidase BG β-14-glucosidaseCB β-Cellobiosidase APH acid phosphatase NAG β-14-N-acetylglucosaminidase PHO phenoloxidase PO peroxidase AWCD average well color development of carbon source metabolism at192 h incubation

4 Discussion

41 Coniferous-Broadleaf Mixture Significantly Increase Stand Biomass and Litter Production

The results of this study indicated that admixing of coniferous and broadleaf tree speciessignificantly increased stand biomass and litter production after 34 years of afforestation in bothdry-cool and wet-warm seasons (Figure 1 Table 2) This may be a consequence of a greater growth of

Forests 2019 10 879 15 of 20

C hystrix in the mixed stands These results confirmed previous findings conducted in our experimentsite measured when the plantations were 26-year-old and 28-year-old respectively [716] A bulkof studies revealed that mixed-species management could affect tree growth stand structure andenvironment [145253] Mixed forests can develop stratified canopies due to niche differences amongdifferent species In the present study trees of the faster growing broad-leaf species (C hystrix) weretaller had a larger DBH and thus formed an upper canopy stratum in the mixed stands (Table 1)While trees of the shorter species (P massoniana) formed a lower stratum with lower DBH and treeheight and the reduced amount of light transmitted through the upper canopy contributed to theirslower growth The dominant tree species within the stand can absorb more sunlight and use itmore efficiently for growth [54] and thus contribute to higher tree biomass and litter productionin mixed stands In addition stratified canopies in mixed plantations can help to maintain levelsof understory species richness and biomass compared with pure plantations due to relatively highlight availabilities [5556] This may be why the mixed stand had higher shrub and herb biomass inour study

Specifically in the present study the broad-leaf species attained greater heights and diameters inmixed stands while masson pine had reduced diameter and height growth in mixed-species comparedto mono-species stands (Table 1) The severe suppression of P massoniana by the faster growingC hystrix may be attributed to differences in the light requirements of the two species [7] The growthpattern of C hystrix and P massoniana in mixed stands in our study (34-year-old) were differed fromwhen they were 26-years-old in which the growth of C hystrix and P massoniana in mixed standswere both greater than in the corresponding mono-species stands [7] This suggests that effects of thetree species mixture could change over time and long-term investigation of mixed forests is of greatsignificance to reveal mixed effects

42 Coniferous-Broadleaf Mixture Significantly Increase Soil Microbial Biomass

The changes of tree species and composition may significantly affect the amount of organic matterinput in soil [57] As the most active part in soil organic matter (SOM) soil microbial biomass isimportant in nutrient cycling and release participating in the circulation of critical elements such ascarbon and nitrogen [23] Because of the quantity and quality of the bulk of SOM changes slowly andthe changes of SOM are difficult to measure due to the spatial heterogeneity of soils soil microbialbiomass and in particular MBC-to-SOC and MBN-to-TN ratio have been suggested as indicators ofthe state and modification of SOM [58ndash60] and as an indicator for carbon (nitrogen) availability insoil [6162] The concentrations of SOC TN and MBC and MBN measured in our study were affectedby stand type and seasons (Table 3 and Table S2 Figure 2) In the dry cool season although the SOCcontent in mixed stands did not significantly differ with that in PCH but significantly lower than thatin PPM the MBC concentration in mixed stands was markedly higher than that in the correspondingmono-species stands Likewise although the TN content in mixed stands was smaller than that in PCHand PPM stands the MBN content in mixed stands was markedly higher than that in the correspondingmono-species stands in the wet-warn season Previous studies found that higher soil microbial biomasscorrelated with higher SOC and TN content that sustain microbial metabolism [2163] However inour study the lower SOC and TN content in MIX was able to maintain a higher microbial biomass thansites where SOC was more abundant Presumably the quality of SOC could explain such a trend [64]and merit further investigation

In this study MBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands(Figure 2) suggesting higher carbon and nitrogen availability in soil and higher soil quality for thegrowth of soil microorganisms by coniferous-broadleaf mixture [21] In addition the effects of admixingon soil microbial biomass MBC-to-SOC and MBN-to-TN ratio were non-additive suggesting theinteraction of P massoniana and C hystrix can have a synergistic effect on improving soil carbon andnitrogen availability (Figure 3) These findings are consistent with many previous studies that havedetermined the effects of tree species mixture on soil microbial biomass [2165] These results may be

Forests 2019 10 879 16 of 20

contributed to (1) greater amount and more diverse organic matter input via litter and rhizodepositionproducts in the mixture of two contrasting tree species [21] (2) the higher use efficiency of soil organiccarbon and nitrogen by microorganisms in mixed stands [66]

We also sought to determine the admixing effects on composition and abundance of soil microbialcommunity with community-level PLFA profiles Surprisingly there were not significant admixingeffects on PLFA content of the major microbial functional groups such as bacteria fungi AMF andactinobacteria (Table 4) However tree species mixture significantly increased the abundance of GNbacteria in the dry-cool season while markedly increased the abundance of GP bacteria but decreasedthe abundance of fungi in the wet-warn season (Figure 4) It seemed that the dominated GN bacteria inthe dry-cool season and GP bacteria in the wet-warn season would correlate with the higher availabilityof soil organic carbon and nitrogen in mixed stands [6768] These correlations are complicated andfurther investigation is required to reveal the underlying mechanisms

43 Effects of Coniferous-Broadleaf Mixture on Soil Microbial Functions

Soil microorganisms excrete soil enzymes to drive nutrient mineralization and litterdecomposition [64] Therefore soil extracellular enzyme activities may be related to the carbonnitrogen and phosphorus cycling in soil [6970] In the present study we found positive admixingeffects on the activities of APH and NAG in the dry-cool season (Figure 5de) On the other handpositive admixing effects on the activities of urease and peroxidase while negative admixing effectson APH was detected in the wet-warn season (Figure 5dfg) This result suggests that the mixtureof C hystrix and P massoniana can accelerate the mineralization rates of soil organic phosphorus andnitrogen These findings were consistent with previous study [67]

The community-level physiological profiling (CLPP) is widely used to characterize microbialcommunities [71ndash73] The average well color development (AWCD) in the CLPP approach reflectsthe ability of soil microorganisms to utilize carbon sources and their metabolic activity The higherAWCD represents the stronger metabolic activity of soil microorganisms [71] In this present study wedetected a significantly positive admixing effect on carbon source utilization ability of the soil microbialcommunities in both dry-cool and wet-warn seasons after 144 h of incubation (Figure 6) Moreover theRDA analysis revealed that carbon source utilization ability of the soil microbial communities werepositive related with APH in the dry-cool season while related with urease and peroxidase activitiesin the wet-warn season (Figure 7) As a whole the higher soil microbial functions in mixed stands arelikely the results of the effects of AMF and GP bacteria in the dry-cool season and the effects of bacteriaincluding GP and GN bacteria the wet-warn season (Table S6)

5 Conclusions

In our study we measured significantly higher tree shrub and herb biomass and litter productionin the coniferous-broadleaf mixed plantation after 34 years of afforestation As expected MBC MBNMBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands suggesting highercarbon and nitrogen availability in soil and higher soil quality for the growth of soil microorganisms inour coniferous-broadleaf mixed stand In addition coniferous-broadleaf mixture can increase carbonsource utilization ability of soil microbial community and accelerate the mineralization rates of soilorganic phosphorus and nitrogen by increasing acid phosphatase β-14-N-acetylglucosaminidaseand urease activities The higher soil microbial biomass and activities are likely the result of greateramount and more diverse organic matter input via litter and rhizodeposition products and the higheruse efficiency of soil organic carbon and nitrogen by microorganisms in mixed stands Howeverthe correlations between soil dominated microbial functional groups and the higher availabilityof soil organic carbon and nitrogen along with the quality of SOC in mixed stands merit furtherinvestigation Our results highlight that establishment of coniferous-broadleaf mixed forests maybe a good management practice as coniferous-broadleaf mixture could accumulate higher stand

Forests 2019 10 879 17 of 20

biomass return more litter and increase soil microbial biomass and functions over the long term insubtropical China

Supplementary Materials The following are available online at httpwwwmdpicom1999-49071010879s1Table S1 Effects of stand type season and their interactions on stand biomass Table S2 Effects of stand type seasonand their interactions on soil microbial structure Table S3 Effects of stand type season and their interactionson relative abundance of the soil microbial community PLFAs Table S4 Effects of stand type season and theirinteractions on soil enzyme activity Table S5 Marginal and conditional effects of biotic and abiotic factors on soilmicrobial composition obtained from the summary of forward selection in Redundancy analysis (RDA) Table S6Marginal and conditional effects of soil microbial functional groups on soil enzyme activity and soil microbialcarbon source utilization ability obtained from the summary of forward selection in Redundancy analysis (RDA)

Author Contributions WW collected data made experiment analysis formal data and drafted the manuscriptYW designed the work XZ revised the manuscript and participated in analyzing the experiment data HZ YYXH and XL ZQ YL LY HL participated in collecting the experiment data

Funding This research was funded by National Natural Science Foundation of China (31860171 3146012131560201) the Guangxi Key Research and Development Program (2018AB40007) Innovation Project of GuangxiGraduate Education (YCBZ2017011) and the Guangxi Universities Science and technology (201201ZD001)

Acknowledgments We gratefully acknowledge the supports in field sampling by the Experimental Center ofTropical Forestry Chinese Academy of Forestry A special acknowledgment is given to Patrick M Finnegan for hiswriting suggestions

Conflicts of Interest The authors declare no conflicts of interest

References

1 Zhang X Liu S Huang Y Fu S Wang J Ming A Li X Yao M Li H Tree species mixture inhibits soilorganic carbon mineralization accompanied by decreased r-selected bacteria Plant Soil 2018 431 203ndash216[CrossRef]

2 Pan Y Birdsey RA Fang J Houghton R Kauppi PE Kurz WA Phillips OL Shvidenko ALewis SL Canadell JG et al A large and persistent carbon sink in the worldrsquos forests Science 2011 333988ndash993 [CrossRef] [PubMed]

3 Chen X Ju Q Lin K Development status issues and countermeasures of Chinarsquo s plantation World For Res2014 27 54ndash59

4 Wang H Lencinas MV Ross Friedman C Wang X Qiu J Understory plant diversity assessment ofEucalyptus plantations over three vegetation types in Yunnan China New For 2011 42 101ndash116 [CrossRef]

5 Sang PM Lamb D Bonner M Schmidt S Carbon sequestration and soil fertility of tropical tree plantationsand secondary forest established on degraded land Plant Soil 2013 362 187ndash200 [CrossRef]

6 Li C Zhou X Wen Y Zhu H Qin Z Li X You Y Huang X Effects of high-generation ratations ofEucalyptus on undergrowth soil fertility and enzyme activities Guangxi Sci 2019 26 176ndash186

7 You Y Huang X Zhu H Liu S Liang H Wen Y Wang H Cai D Ye D Positive interactions betweenPinus massoniana and Castanopsis hystrix species in the uneven-aged mixed plantations can produce moreecosystem carbon in subtropical China For Ecol Manag 2018 410 193ndash200 [CrossRef]

8 Huang X Liu S You Y Wen Y Wang H Wang J Microbial community and associated enzymes activityinfluence soil carbon chemical composition in Eucalyptus urophylla plantation with mixing N2-fixing speciesin sub-tropical China Plant Soil 2017 414 199ndash212 [CrossRef]

9 Kelty MJ The role of species mixtures in plantation forestry For Ecol Manag 2006 233 195ndash204 [CrossRef]10 Bauhus J Khanna P Menden N Aboveground and belowground interactions in mixed plantations of

Eucalyptus globulus and Acacia mearnsii Can J For Res 2000 30 1886ndash1894 [CrossRef]11 Vandermeer J The Ecology of Intercropping Cambridge University Press New York NY USA 1989 p 23712 Forrester DI The spatial and temporal dynamics of species interactions in mixed-species forests From

pattern to process For Ecol Manag 2014 312 282ndash292 [CrossRef]13 Forrester DI Bauhus J A review of processes behind diversity-productivity relationships in forests

Curr For Rep 2016 2 45ndash61 [CrossRef]14 Wang H Liu S Wang J You Y Yang Y Shi Z Huang X Zheng L Li Z Ming A et al Mixed-species

plantation with Pinus massoniana and Castanopsis hystrix accelerates C loss in recalcitrant coniferous litter butslows C loss in labile broadleaf litter in southern China For Ecol Manag 2018 422 207ndash213 [CrossRef]

Forests 2019 10 879 3 of 20

than those in corresponding monocultures in long term (2) positive admixing effects on soil microbialbiomass and functions are accompanied by improved stand biomass and litter production

2 Materials and Methods

21 Study Sites and Experimental Design

This study was conducted at the Experimental Center of Tropical Forestry (2210prime N 10650prime E)Chinese Academy of Forestry Pingxiang City Guangxi Zhuang Autonomous Region PR ChinaThis region belongs to a subtropical monsoon climate zone and has a humid-to-semi-humid climatewith dry-cool (from October to April) and wet-warm seasons (from May to September) according to thevariation of precipitation and temperature [29] The mean annual precipitation is about 1400 mm mostrainfall occurs from April to September The mean annual temperature is 21 C The loamy-texturedsoil in this area was formed from granitic parent geological material and is classified as red soil in theChinese system of soil classification which is equivalent to an oxisol in the USDA Soil Taxonomy [814]

Our study site consisted of a 33 ha plantation of pure C hystrix (PCH) a 33 ha plantation of pureP massoniana (PPM) and an 18 ha plantation of mixed C hystrix and P massoniana (MIX) Prior to theestablishment of the current plantations the area was covered by a Cunninghamia lanceolata monospecificplantation which was established in 1958 on a historically deforested hill and clear harvested in1983 After clearcutting the prior plantations the PCH PPM and MIX plantations were establishedin 1983 with an initial planting density of 2500 tree haminus1 and a 11 mixed proportion in the MIX forcomparison of tree productivity and the ecological benefits of mixed plantations Buffer zones of150ndash800 m were set up to separate these plantations These stands had similar soil types topographiesand management histories

In June 2017 34 years after the establishment of the plantations we randomly established fivesample plots (20 m times 20 m) in each plantation The five sampling plots within a single experimentalstand were well separated with distances of at least 200 m between adjacent plots Elevations of allplots were 530ndash650 m and the degree of slopes was 30ndash39 The stand density ranged from 355 to400 tree haminus1 and average height from 22 m to 25 m The diameter at breast height (DBH) and basalarea of MIX was significantly higher than that of PCH and PPM (Table 1)

Table 1 Characteristics of pure Castanopsis hystrix Miq (PCH) plantation pure Pinus massoniana Lamb(PPM) plantation and mixed C hystrix and P massoniana (MIX) plantation (mean plusmn standard errorn = 5)

Stand Density (Tree haminus1) Height (m) DBH (cm) Basal Area (m2 haminus1)

PCH 355 plusmn 15 a 2395 plusmn 080 a 2824 plusmn 097 b 2315 plusmn 150 b

MIX 380 plusmn 10 a 2452 plusmn 037 a 3431 plusmn 102 a 3967 plusmn 227 a

P massoniana 95 plusmn 9 2201 plusmn 067 2616 plusmn 075 535 plusmn 085C hystrix 285 plusmn 15 2533 plusmn 039 3695 plusmn 163 3432 plusmn 291

PPM 400 plusmn 24 a 2332 plusmn 071 a 2816 plusmn 104 b 2660 plusmn 257 b

Different superscripts indicate significant differences among stand types (p lt 005) DBH diameter at breast height

22 Stand Biomass Measurements

Three 2 m times 2 m subplots and three 1 m times 1 m subplots were randomly placed in each plotfor shrub and herb biomass measurements respectively in the wet-warm (July 2017) and dry-coolseasons (January 2018) In each season shrubs and herbs were destructively harvested and dividedinto aboveground and belowground parts All understory vegetation samples were transported to thelaboratory and oven-dried at 75 C for dry biomass determination (Mg haminus1) [30]

In addition we measured tree height with a digital clinometer (Vertex IV Hagloumlf Inc LangseleSweden) and DBH with a diameter tape of each tree in each plot and in each season Tree aboveground

Forests 2019 10 879 4 of 20

biomass (TAB) and tree belowground biomass (TBB) were calculated by the allometric equationsdescribed by You et al (2018) [7]

23 Litter Production and Quality Measurements

A litter collector with an effective collection area of 4 m2 was randomly set in each plot Each littercollector was made with nylon net with a mesh size of 1 mm and set 05 m above the ground withPVC (polyvinyl chloride) tube Litter was collected once a month from August 2017 to July 2018 Littersamples were transported to the laboratory and oven-dried at 75 C for production determination(Mg haminus1) Litter organic carbon (OClitter) concentration was determined by the K2Cr2O7ndashH2SO4

calefaction procedure [30] Litter total nitrogen (TNlitter) total phosphorus (TPlitter) and total potassium(TKlitter) concentrations were measured after heat digestion with 98 H2SO4 [31ndash33]

24 Soil Sampling and Analysis

Soil samples were also collected in the wet-warm (July 2017) and dry-cool seasons (January 2018)In each season we took eight soil cores systematically arranged at a distance of 5 m from the plotcenter in the directions of 0 45 90 135 180 215 270 and 315 Each core was taken using an87 cm inner diameter steel tube at a depth of 0ndash10 cm below the soil surface The eight cores of eachplot were mixed to form a composite sample Each soil sample was divided into two subsamplesOne subsample was air-dried to a constant weight and sieved using a 2 mm sieve for measuring soilproperties The other subsample was sieved to 2 mm and immediately stored at minus4 C for measuringmoisture content soil ammonium nitrogen (NH4

+-N) nitrate nitrogen (NO3minus-N) microbial biomass

carbon (MBC) microbial biomass nitrogen (MBN) concentration phospholipid fatty acid analysis(PLFA) assays carbon source utilization ability of the soil microbial communities and enzyme activityAt the same time three soil cores (100 cm3) from 0ndash10 cm soil layer were sampled from each plot tomeasure bulk density by the cutting ring method [34]

The soil water content (WC) was measured using a sub-sample dried in oven at 105 C untilthe sample reached a constant weight The soil temperatures were measured with an LI-8100AAutomated Soil Gas Flux System (LindashCor Lincoln NE USA) Soil pH was measured in a 125 soilwatersuspension using a pH meter [835] Soil organic carbon (SOC) concentration was determined bythe K2Cr2O7ndashH2SO4 calefaction procedure [31] Total nitrogen (TN) total phosphorus (TP) andtotal potassium (TK) concentration were measured after heat digestion with 98 H2SO4 TN andTK were measured using the liquid supernatant with a continuous flow analytical system (AA3SEAL Analytical Norderstedt Germany) [3133] and an atomic absorption spectrometer (ZEENIT700P Analytik Jena Germany) [32] respectively TP and AP were determined by MondashSbndashVccolorimetry with the liquid supernatant after heat digestion and HCl (005 M)ndashH2SO4 (0025 M)respectively And available potassium (AK) was determined by atomic absorption spectrometry with1 M NH4OAC Soil ammonium nitrogen (NH4

+-N) and nitrate nitrogen (NO3minus-N) were calorimetrically

determined in a continuous flow analyzer after 10 g fresh mass of soil had been extracted in 50 mLof 2 M KCl [836] The cation exchange capacity (CEC) was determined by [Co(NH3)6]Cl3 extractionspectrophotometry [3738]

Soil microbial biomass carbon (MBC) and nitrogen (MBN) were measured by fumigation-extractionusing 05 M K2SO4 as the extraction agent [3940] with a total organic carbon analyzer (1020A OICollege Station TX USA)

Phospholipid fatty acid analysis (PLFA) assays were employed to analyze soil microbial communitystructure with freeze-dried soil samples using the procedure developed by Bossio et al (1998) [41]The PLFA markers 140 150 170 180 141ω5c 150 3OH 151ω6c 160 2OH 161 2OH 161ω9c161ω7c 181 ω7c 180 2OH 181 ω5c a150 a160 a170 cy170 cy190 i140 i150 i160 i170 i180and i190 were chosen to represent bacteria Gram-positive (GP) bacteria were represented by a150a160 a170 i140 i150 i160 i170 i180 and i190 and gram-negative (GN) bacteria were representedby 141ω5c 150 3OH 151ω6c 160 2OH 161 2OH 161ω7c 161ω9c 180 2OH 181ω5c cy170

Forests 2019 10 879 5 of 20

cy190 and 181 ω7c [364243] Actinobacteria were represented by 10-Me170 10-Me180 TBSA11-Me181ω7c and 10-Me160 [124344] Arbuscular mycorrhizal fungi (AMF) were represented by161 ω11c and 161 ω5c [35] and fungi were represented by 181 ω9c 183 ω6c (6912) and 182ω69c [1244] Other PLFAs such as 150 2OH 160 171ω8c 180 iso and i200 were also used for theanalysis of the composition of microbial communities

Carbon source utilization ability of the soil microbial communities was analyzed using BIOLOGreg

Eco plates (Biolog Hayward CA USA) with fresh soil samples Each Biolog Econdashplate comprised96 wells containing triplicates of 31 different carbon sources and a blank [45] We added 10 g dryweight equivalent of fresh soil to 90 mL of 085 sterile saline physiological solution and shook themixture for 1 h on a rotary shaker (100 rpm) at 25 C After 10minus3 dilution we transferred samplescontaining 150 microL of liquid into the 96 wells and read the optical density (OD) at 590 and 750 nm witha Microstationtrade reader (Biolog) after 24 4872 96 120 144 168 and 192 h of incubation [4647]

Enzyme assays were performed with a fully automatic full-wavelength enzyme labeling apparatus(Infinite M200 Pro Tecan Inc Maumlnnedorf Switzerland) using the procedure describe in detailby Saiya-Cork et al (2002) [48] and Steinauer et al (2015) [49] The phenol oxidase andperoxidase assays were colorimetric using L34-dihydroxyphenylalanine (L-DOPA) as the substrateThe colorimetric urease assay used urea as the substrate The α-Glucosidase (AG) β-14-glucosidase (BG)β-Cellobiosidase (CB) acid phosphatase (APH) and β-14-N-acetylglucosaminidase (NAG) activities werefluorometric with substrate 4-methylumbelliferyl-α-D-glucoside 4-methylumbelliferyl-β-D-glucoside4-methylumbelliferyl-β-D-cellobioside 4-methylumbelliferyl-phosphate and 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide respectively

25 Statistical Analysis

One-way analyses of variances (ANOVAs) were used to examine the differences of stand biomasslitter production and quality soil physicochemical properties soil microbial biomass soil microbialPLFAs content and functions among PPM PCH and MIX plantations in each season Two-wayANOVAs were used to determine the interaction effects between stand type and season on the abovevariables Statistical analyses were performed with SPSS software (ver 250 IBM Corp Armonk NYUSA) Significant differences between means were tested by least significant difference (LSD) at the005 level

In order to evaluate admixing effects on soil microbial biomass we calculated the relative effects(RE) of mixing species by comparing the observed value (OV) of one variable in mixed plantationwith the predicted value (PE) based on the respective mono-specific plantations RE was calculated byfollowing Wardle et al (1997) [50]

RE = [(OV minus PE)PE] times 100 (1)

where PE is the mean of the observed value of one variable in respective mono-specific plantationsIf RE differs from zero it would indicate non-additive effects (NAE) of mixing species on soil microbialbiomass Negative and positive deviations from zero are referred to as antagonistic and synergisticeffects respectively One-sample Studentrsquos t-tests with 95 confidence intervals were employed to testwhether RE differed significantly from zero

Redundancy analysis (RDA) was used to determine the relationships among biotic and abioticfactors and soil microbial composition and functions by CANOCO software for Windows 5(Biometris-Plant Research international Wageningen The Netherlands) In RDA the relative abundanceof individual PLFAs (mole present of the total PLFAs) was used as the response variables and soilphysicochemical properties litter and stand biomass were used as the explanatory variables [3551]We also used RDA to determine the linkage between soil microbial community and functions includingcarbon source metabolic function and soil extracellular enzyme activity involved in biogeochemicalcirculation of carbon nitrogen and phosphorus All data were log-transformed before in the analyses

Forests 2019 10 879 6 of 20

The forward selection procedure in RDA based on Monte Carlo permutation with 499 iterations wasperformed to determine the dominant variables that influenced the composition of individual PLFAsand microbial functions A significance level of p lt 005 was used in all analyses

3 Results

31 Stand Biomass

Stand type had significant effects on the -above- and below-ground biomass of trees shrubs andherbs (Table S1 Figure 1) TAB TBB shrub aboveground biomass (SAB) shrub belowground biomass(SBB) and herb aboveground biomass (HAB) in MIX were significantly higher than those in PCHand PPM in both the dry-cool and wet-warm seasons (Figure 1andashe) Except for HAB season had nosignificant effects on the tested biomass variables Moreover significant interaction effects betweenstand type and season only found on HAB and HBB These results supported our first hypothesis thattree species mixture increased stand biomass

Forests 2019 10 x FOR PEER REVIEW 6 of 22

biogeochemical circulation of carbon nitrogen and phosphorus All data were log-transformed before in the analyses The forward selection procedure in RDA based on Monte Carlo permutation with 499 iterations was performed to determine the dominant variables that influenced the composition of individual PLFAs and microbial functions A significance level of p lt 005 was used in all analyses

3 Results

31 Stand Biomass

Stand type had significant effects on the -above- and below-ground biomass of trees shrubs and herbs (Table S1 Figure 1) TAB TBB shrub aboveground biomass (SAB) shrub belowground biomass (SBB) and herb aboveground biomass (HAB) in MIX were significantly higher than those in PCH and PPM in both the dry-cool and wet-warm seasons (Figure 1andashe) Except for HAB season had no significant effects on the tested biomass variables Moreover significant interaction effects between stand type and season only found on HAB and HBB These results supported our first hypothesis that tree species mixture increased stand biomass

Figure 1 Biomass of trees (ab) shrubs (cd) and herbs (ef) in three plantations in different seasons Different letters above the bars denote significant differences at p lt 005 level by LSD (least significant difference) multiple comparisons among stand types (n = 5)

32 Litter Production and Quality

Stand type the interaction between stand type and season significantly influenced litter production at our study sites (Table 2) The MIX had significantly higher litter production than PCH and PPM in both seasons OClitter and CNlitter were significantly affected by both stand type and season

Figure 1 Biomass of trees (ab) shrubs (cd) and herbs (ef) in three plantations in different seasonsDifferent letters above the bars denote significant differences at p lt 005 level by LSD (least significantdifference) multiple comparisons among stand types (n = 5)

Forests 2019 10 879 7 of 20

32 Litter Production and Quality

Stand type the interaction between stand type and season significantly influenced litter productionat our study sites (Table 2) The MIX had significantly higher litter production than PCH and PPMin both seasons OClitter and CNlitter were significantly affected by both stand type and season withhigher value in the wet-warm season than in the dry-cool season in all plantations An opposite trendwas observed in TNlitter TPlitter was significantly affected by stand type season and their interactionwith MIX had significantly lower concentration than PCH and PPM in the dry-cool season TKlitter wassignificantly affected by stand type with MIX had significantly higher concentration than PCH andPPM in the wet-warm season These results partially supported our first hypothesis that tree speciesmixture increased litter production although the increases of litter quality in mixed plantations werenot significant

33 Soil Physical and Chemical Properties

In the dry-cool season the mixed plantations had the highest soil bulk density (BD) and soil watercontent (WC) and the lowest soil temperature and medium pH compared to the pure plantations(Table 3) In the wet-warm season MIX had the medium BD WC and soil temperature and the highestpH compared to PPM and PCH

The PPM plantations had the highest SOC TN TP SOC-to-TN ratio NH4+-N and CEC while

MIX had the highest AP TK and AK and PCH had the highest NO3minus-N and AN in the dry-cool season

(Table 3) On the other hand the PCH had the highest SOC TN and AP while MIX had the highestTP TK AK NO3

minus-N and AN and PPM had the highest SOC-to-TN ratio NO3minus-N and CEC in the

wet-warm seasonTwo-way ANOVAs indicated that season significantly affected all the tested soil variables (Table 3)

In addition the stand type influenced nearly all soil variables except BD TN NH4+-N and AN

Interactions between stand type and season significantly influenced WC temperature pH SOC TNTP AP AK NO3

minus-N and CEC

Forests 2019 10 879 8 of 20

Table 2 The quantity and quality of litter in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM) plantation and mixed C hystrix and P massoniana(MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH MIX PPM PCH MIX PPM F p F p F p

LP 668 plusmn 019 b 836 plusmn 069 a 484 plusmn 019 c 553 plusmn 043 b 921 plusmn 040 a 616 plusmn 023 b 3752 lt0001 093 0343 527 0013OClitter 19064 plusmn 1063 b 24859 plusmn 1848 a 25505 plusmn 1800 a 27757 plusmn 1885 b 2743 plusmn 1142 b 32175 plusmn 544 a 683 0004 2486 lt0001 226 0127TNlitter 1738 plusmn 078 a 1353 plusmn 106 b 1496 plusmn 062 ab 1295 plusmn 059 a 1357 plusmn 090 a 1189 plusmn 050 a 321 0058 1579 0001 445 0023CNlitter 1112 plusmn 095 b 1850 plusmn 081 a 1733 plusmn 181 a 2182 plusmn 223 b 2056 plusmn 156 b 2722 plusmn 096 a 767 0003 3890 lt0001 518 0013TPlitter 134 plusmn 008 a 050 plusmn 007 b 116 plusmn 005 a 119 plusmn 015 a 138 plusmn 015 a 131 plusmn 005 a 667 0005 1281 0002 1417 lt0001TKlitter 116 plusmn 016 a 152 plusmn 010 a 082 plusmn 009 b 107 plusmn 002 b 149 plusmn 013 a 088 plusmn 007 c 2084 lt0001 007 0796 029 0751

Different superscripts indicate significant differences among stand types in each season (p lt 005) LP (Mg haminus1) litter production OClitter (g kgminus1) litter organic carbon TNlitter (g kgminus1)litter total nitrogen CNlitter CN ratio of litter TPlitter (g kgminus1) litter total phosphorus TKlitter (g kgminus1) litter total potassium

Table 3 Soil physical and chemical properties (0ndash10 cm soil depth) in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM) plantation and mixedC hystrix and P massoniana (MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH Mixed PPM PCH Mixed PPM F p F p F p

BD 109 plusmn 003 b 125 plusmn 004 a 119 plusmn 002 a 103 plusmn 001 a 103 plusmn 003 a 104 plusmn 005 a 311 0063 3044 lt0001 297 0070WC 3966 plusmn 136 b 4359 plusmn 061 a 4017 plusmn 158 ab 4053 plusmn 107 c 4440 plusmn 084 b 5048 plusmn 141 a 1035 0001 1681 lt0001 1048 0001Tem 1682 plusmn 021 a 1550 plusmn 009 c 1617 plusmn 005 b 2628 plusmn 043 a 2598 plusmn 022 a 2541 plusmn 008 a 821 0002 283334 lt0001 431 0025pH 432 plusmn 002 a 419 plusmn 003 b 404 plusmn 005 c 402 plusmn 002 b 421 plusmn 003 a 390 plusmn 009 b 1440 lt0001 1370 0001 623 0007

SOC 3742 plusmn 108 b 4082 plusmn 022 b 5698 plusmn 189 a 5331 plusmn 194 a 4839 plusmn 200 a 5083 plusmn 054 a 2460 lt0001 2369 lt0001 2864 lt0001TN 163 plusmn 005 b 160 plusmn 006 b 216 plusmn 022 a 248 plusmn 011 a 213 plusmn 013 a 217 plusmn 014 a 248 0105 4408 lt0001 480 0018

SOCTN 2298 plusmn 059 b 2567 plusmn 103 b 2949 plusmn 159 a 2167 plusmn 133 a 2304 plusmn 162 a 2390 plusmn 202 a 457 0021 722 0013 114 0338TP 031 plusmn 004 c 042 plusmn 002 b 060 plusmn 004 a 052 plusmn 006 b 078 plusmn 004 a 054 plusmn 004 b 2186 lt0001 2889 lt0001 1795 lt0001AP 919 plusmn 058 b 1252 plusmn 069 a 1002 plusmn 030 b 1330 plusmn 040 a 1315 plusmn 057 a 998 plusmn 027 b 1670 lt0001 1525 0001 1029 0001TK 414 plusmn 015 b 545 plusmn 049 a 242 plusmn 016 c 540 plusmn 050 a 694 plusmn 082 a 338 plusmn 013 b 3226 lt0001 1034 0004 039 0680AK 2990 plusmn 149 b 4629 plusmn 096 a 2846 plusmn 108 b 4719 plusmn 228 a 5279 plusmn 365 a 2837 plusmn 144 b 5375 lt0001 2257 lt0001 927 0001

NO3minus-N 355 plusmn 024 a 185 plusmn 004 c 286 plusmn 022 b 406 plusmn 019 c 530 plusmn 009 b 579 plusmn 009 a 1096 lt0001 29106 lt0001 4547 lt0001

NH4+-N 2129 plusmn 101 a 2334 plusmn 113 a 2447 plusmn 137 a 4224 plusmn 162 a 4350 plusmn 166 a 3883 plusmn 247 a 075 0482 19616 lt0001 247 0105

AN 2734 plusmn 157 a 2519 plusmn 114 a 2485 plusmn 090 a 463 plusmn 155 a 488 plusmn 169 a 4462 plusmn 246 a 120 0319 24555 lt0001 117 0328CEC 4553 plusmn 327 c 5758 plusmn 411 b 8233 plusmn 194 a 5543 plusmn 202a 5446 plusmn 119 a 6224 plusmn 291 a 4514 lt0001 1216 0002 2168 lt0001

Different superscripts indicate significant differences among stand types in each season (p lt 005) BD (g cmminus1) bulk density WC () soil water content Tem (C) temperature SOC(g kgminus1) soil organic carbon TN (g kgminus1) total nitrogen TP (g kgminus1) total phosphorus AP (mg kgminus1) available phosphorus TK (g kgminus1) total potassium AK (mg kgminus1) availablepotassium NO3

minus-N (mg kgminus1) nitrate nitrogen NH4+-N (mg kgminus1) ammonium nitrogen AN (mg kgminus1) Available nitrogen CEC (cmol + kgminus1) cation exchange capacity

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34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantlyaffected by stand type and MBN was also significantly affected by season and interaction betweenstand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a)In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantlyhigher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highestMBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOCand MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significantlevel in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOCand MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-coolseason (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season(Figure 3b) were significant

Forests 2019 10 x FOR PEER REVIEW 10 of 22

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantly affected by stand type and MBN was also significantly affected by season and interaction between stand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a) In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantly higher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highest MBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOC and MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significant level in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOC and MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-cool season (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season (Figure 3b) were significant

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBN MBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a) and wet-warm season (b)

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBNMBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005level by LSD multiple comparisons among stand types (n = 5)

Forests 2019 10 x FOR PEER REVIEW 10 of 22

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantly affected by stand type and MBN was also significantly affected by season and interaction between stand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a) In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantly higher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highest MBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOC and MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significant level in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOC and MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-cool season (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season (Figure 3b) were significant

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBN MBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a) and wet-warm season (b)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a)and wet-warm season (b)

Forests 2019 10 879 10 of 20

In the dry-cool season total PLFAs AMF PLFAs actinobacteria PLFAs bacteria PLFAs fungiPLFAs GP bacteria PLFAs and GP-to-GN PLFAs ratio were smaller in MIX than those in PPM andPCH On the other hand in the wet-warm season these PLFAs were smaller in PPM than thosein PCH and MIX (Table 4) Specifically the ratios of bacteria-to-fungi PLFA and GP-to-GN PLFAwere significantly higher in MIX than those in PPM and PCH in the wet-warm season These resultsindicated that the response of the soil microbial community structure to tree species mixture varied indifferent seasons Two-way ANOVAs showed that both stand type and season significantly affectedall the tested microbial variables while the interaction between stand type and season significantlyinfluenced total PLFAs bacteria PLFAs GP bacteria PLFAs Acti PLFAs bacteria-to-fungi PLFA andGP-to-GN PLFA (Table 4)

Across plantations and seasons bacteria PLFAs were the most abundant followed by fungi andactinobacteria PLFAs (Figure 4 and Table S3) It was worth noting that the relative abundance ofGN bacteria PLFAs was significantly higher in MIX than in other plantations in the dry-cool season(Figure 4) Furthermore the relative abundance of GP bacteria PLFAs was significantly higher andthat of fungi PLFAs was significantly lower in MIX than in other plantations in the wet-warm season

Forests 2019 10 x FOR PEER REVIEW 13 of 22

Across plantations and seasons bacteria PLFAs were the most abundant followed by fungi and actinobacteria PLFAs (Figure 4 and Table S3) It was worth noting that the relative abundance of GN bacteria PLFAs was significantly higher in MIX than in other plantations in the dry-cool season (Figure 4) Furthermore the relative abundance of GP bacteria PLFAs was significantly higher and that of fungi PLFAs was significantly lower in MIX than in other plantations in the wet-warm season

Figure 4 The relative abundance of PLFAs (mol ) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleaf plantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increased APH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increased urease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg) Carbon source utilization ability of the soil microbial community indicated by average well color development (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation (Figure 6)

Figure 4 The relative abundance of PLFAs (mol ) in three plantations in the dry-cool season (a) andwet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level byLSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 11 of 20

Table 4 Soil microbial community PLFAs content and structure (0ndash10 cm soil depth) in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM)plantation and mixed C hystrix and P massoniana (MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH MIX PPM PCH MIX PPM F p F p F p

TPLFAs 3767 plusmn 271 a 3255 plusmn 124 a 3796 plusmn 128 a 4373 plusmn 208 a 4001 plusmn 07 a 3478 plusmn 111 b 464 0020 646 0018 607 0007B 1579 plusmn 081 a 1306 plusmn 043 b 1490 plusmn 051 ab 1764 plusmn 072 a 1723 plusmn 042 a 1276 plusmn 041 b 1274 lt0001 768 0011 1556 lt0001

GP 945 plusmn 043 a 794 plusmn 029 b 918 plusmn 031 a 1066 plusmn 051 a 1155 plusmn 031 a 810 plusmn 031 b 817 0002 1709 lt0001 2030 lt0001GN 591 plusmn 076 a 590 plusmn 016 a 553 plusmn 022 a 619 plusmn 025 a 455 plusmn 037 b 411 plusmn 020 b 526 0013 702 0014 313 0062

F 498 plusmn 034 a 375 plusmn 022 b 424 plusmn 028 ab 492 plusmn 035 a 296 plusmn 043 b 345 plusmn 021 b 1356 lt0001 448 0045 091 0416AMF 104 plusmn 003 a 075 plusmn 003 b 075 plusmn 004 b 085 plusmn 007 a 044 plusmn 008 b 048 plusmn 003 b 2822 lt0001 3483 lt0001 056 0577Acti 426 plusmn 037 ab 377 plusmn 021 b 486 plusmn 020 a 531 plusmn 019 a 487 plusmn 012 b 458 plusmn 010 b 273 0085 1236 0002 650 0006BF 319 plusmn 011 a 351 plusmn 013 a 356 plusmn 017 a 362 plusmn 014 b 635 plusmn 094 a 377 plusmn 033 b 750 0003 1131 0003 598 0008

GPGN 174 plusmn 029 b 135 plusmn 003 b 166 plusmn 004 a 173 plusmn 008 b 263 plusmn 028 a 198 plusmn 011 b 107 0359 1376 0001 733 0003

Different superscripts indicate significant differences among stand types in each season (p lt 005) TPLFAs (nmol gminus1 soil) total PLFAs B (nmol gminus1 soil) bacteria PLFAs GP (nmol gminus1

soil) gram-positive bacteria PLFAs GN (nmol gminus1 soil) gram-negative bacteria PLFAs F (nmol gminus1 soil) fungi PLFAs Acti (nmol gminus1 soil) actinobacteria PLFAs AMF (nmol gminus1 soil)arbuscular mycorrhizal fungi PLFAs BF bacteria-to-fungi PLFA ratio GPGN GP-to-GN ratio

Forests 2019 10 879 12 of 20

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleafplantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increasedAPH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increasedurease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg)Carbon source utilization ability of the soil microbial community indicated by average well colordevelopment (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation(Figure 6)Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in threeplantations in different seasons Error bars are standard error Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 13 of 20

Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in threeplantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in thedry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons(F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pHTK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-coolseason in which SOC and TBB were the major correlates of changes in soil microbial composition(Table S5) A total of 11 biotic and abiotic factors including NO3

minus-N TBB pH TKlitter HBB AP AKTP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warmseason in which NO3

minus-N TBB pH and TKlitter were the major correlates of changes in soil microbialcomposition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

37 Relationship between Soil Microbial Structure and Functions

Soil enzyme activity and soil microbial carbon source utilization ability are both important soilmicrobial functions The first two RDA axes explained 4948 of the variation in soil enzyme activityand carbon utilization ability in the dry-cool season (F = 24 p = 0006 Figure 7b) and 6891 of thevariation in the wet-warm season (F = 64 p = 0002 Figure 7d) AMF and GP were significantlycorrelated with soil microbial functions in the dry-cool season (Table S6) Total bacteria GP bacteriaGN bacteria and actinobacteria were significantly correlated with soil microbial functions in thewet-warm season in which total bacteria GP bacteria and GN bacteria were the major correlates ofchanges of soil enzyme activity (Table S6)

Forests 2019 10 879 14 of 20

Forests 2019 10 x FOR PEER REVIEW 15 of 22

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in the dry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons (F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pH TK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-cool season in which SOC and TBB were the major correlates of changes in soil microbial composition (Table S5) A total of 11 biotic and abiotic factors including NO3minus-N TBB pH TKlitter HBB AP AK TP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warm season in which NO3minus-N TBB pH and TKlitter were the major correlates of changes in soil microbial composition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160 were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant biotic and abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot of soil microbial community structure and soil microbial functions (bd) Above the label represents a significant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowground biomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soil total potassium NO3ndash-N soil nitrate nitrogen HBB herb belowground biomass AP soil available phosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil water

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant bioticand abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot ofsoil microbial community structure and soil microbial functions (bd) Above the label represents asignificant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowgroundbiomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soiltotal potassium NO3

minus-N soil nitrate nitrogen HBB herb belowground biomass AP soil availablephosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil watercontent LP litter production TKlitter litter total potassium GP gram-positive bacteria GN gramnegative bacteria AMF arbuscular mycorrhizal fungi AG α-Glucosidase BG β-14-glucosidaseCB β-Cellobiosidase APH acid phosphatase NAG β-14-N-acetylglucosaminidase PHO phenoloxidase PO peroxidase AWCD average well color development of carbon source metabolism at192 h incubation

4 Discussion

41 Coniferous-Broadleaf Mixture Significantly Increase Stand Biomass and Litter Production

The results of this study indicated that admixing of coniferous and broadleaf tree speciessignificantly increased stand biomass and litter production after 34 years of afforestation in bothdry-cool and wet-warm seasons (Figure 1 Table 2) This may be a consequence of a greater growth of

Forests 2019 10 879 15 of 20

C hystrix in the mixed stands These results confirmed previous findings conducted in our experimentsite measured when the plantations were 26-year-old and 28-year-old respectively [716] A bulkof studies revealed that mixed-species management could affect tree growth stand structure andenvironment [145253] Mixed forests can develop stratified canopies due to niche differences amongdifferent species In the present study trees of the faster growing broad-leaf species (C hystrix) weretaller had a larger DBH and thus formed an upper canopy stratum in the mixed stands (Table 1)While trees of the shorter species (P massoniana) formed a lower stratum with lower DBH and treeheight and the reduced amount of light transmitted through the upper canopy contributed to theirslower growth The dominant tree species within the stand can absorb more sunlight and use itmore efficiently for growth [54] and thus contribute to higher tree biomass and litter productionin mixed stands In addition stratified canopies in mixed plantations can help to maintain levelsof understory species richness and biomass compared with pure plantations due to relatively highlight availabilities [5556] This may be why the mixed stand had higher shrub and herb biomass inour study

Specifically in the present study the broad-leaf species attained greater heights and diameters inmixed stands while masson pine had reduced diameter and height growth in mixed-species comparedto mono-species stands (Table 1) The severe suppression of P massoniana by the faster growingC hystrix may be attributed to differences in the light requirements of the two species [7] The growthpattern of C hystrix and P massoniana in mixed stands in our study (34-year-old) were differed fromwhen they were 26-years-old in which the growth of C hystrix and P massoniana in mixed standswere both greater than in the corresponding mono-species stands [7] This suggests that effects of thetree species mixture could change over time and long-term investigation of mixed forests is of greatsignificance to reveal mixed effects

42 Coniferous-Broadleaf Mixture Significantly Increase Soil Microbial Biomass

The changes of tree species and composition may significantly affect the amount of organic matterinput in soil [57] As the most active part in soil organic matter (SOM) soil microbial biomass isimportant in nutrient cycling and release participating in the circulation of critical elements such ascarbon and nitrogen [23] Because of the quantity and quality of the bulk of SOM changes slowly andthe changes of SOM are difficult to measure due to the spatial heterogeneity of soils soil microbialbiomass and in particular MBC-to-SOC and MBN-to-TN ratio have been suggested as indicators ofthe state and modification of SOM [58ndash60] and as an indicator for carbon (nitrogen) availability insoil [6162] The concentrations of SOC TN and MBC and MBN measured in our study were affectedby stand type and seasons (Table 3 and Table S2 Figure 2) In the dry cool season although the SOCcontent in mixed stands did not significantly differ with that in PCH but significantly lower than thatin PPM the MBC concentration in mixed stands was markedly higher than that in the correspondingmono-species stands Likewise although the TN content in mixed stands was smaller than that in PCHand PPM stands the MBN content in mixed stands was markedly higher than that in the correspondingmono-species stands in the wet-warn season Previous studies found that higher soil microbial biomasscorrelated with higher SOC and TN content that sustain microbial metabolism [2163] However inour study the lower SOC and TN content in MIX was able to maintain a higher microbial biomass thansites where SOC was more abundant Presumably the quality of SOC could explain such a trend [64]and merit further investigation

In this study MBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands(Figure 2) suggesting higher carbon and nitrogen availability in soil and higher soil quality for thegrowth of soil microorganisms by coniferous-broadleaf mixture [21] In addition the effects of admixingon soil microbial biomass MBC-to-SOC and MBN-to-TN ratio were non-additive suggesting theinteraction of P massoniana and C hystrix can have a synergistic effect on improving soil carbon andnitrogen availability (Figure 3) These findings are consistent with many previous studies that havedetermined the effects of tree species mixture on soil microbial biomass [2165] These results may be

Forests 2019 10 879 16 of 20

contributed to (1) greater amount and more diverse organic matter input via litter and rhizodepositionproducts in the mixture of two contrasting tree species [21] (2) the higher use efficiency of soil organiccarbon and nitrogen by microorganisms in mixed stands [66]

We also sought to determine the admixing effects on composition and abundance of soil microbialcommunity with community-level PLFA profiles Surprisingly there were not significant admixingeffects on PLFA content of the major microbial functional groups such as bacteria fungi AMF andactinobacteria (Table 4) However tree species mixture significantly increased the abundance of GNbacteria in the dry-cool season while markedly increased the abundance of GP bacteria but decreasedthe abundance of fungi in the wet-warn season (Figure 4) It seemed that the dominated GN bacteria inthe dry-cool season and GP bacteria in the wet-warn season would correlate with the higher availabilityof soil organic carbon and nitrogen in mixed stands [6768] These correlations are complicated andfurther investigation is required to reveal the underlying mechanisms

43 Effects of Coniferous-Broadleaf Mixture on Soil Microbial Functions

Soil microorganisms excrete soil enzymes to drive nutrient mineralization and litterdecomposition [64] Therefore soil extracellular enzyme activities may be related to the carbonnitrogen and phosphorus cycling in soil [6970] In the present study we found positive admixingeffects on the activities of APH and NAG in the dry-cool season (Figure 5de) On the other handpositive admixing effects on the activities of urease and peroxidase while negative admixing effectson APH was detected in the wet-warn season (Figure 5dfg) This result suggests that the mixtureof C hystrix and P massoniana can accelerate the mineralization rates of soil organic phosphorus andnitrogen These findings were consistent with previous study [67]

The community-level physiological profiling (CLPP) is widely used to characterize microbialcommunities [71ndash73] The average well color development (AWCD) in the CLPP approach reflectsthe ability of soil microorganisms to utilize carbon sources and their metabolic activity The higherAWCD represents the stronger metabolic activity of soil microorganisms [71] In this present study wedetected a significantly positive admixing effect on carbon source utilization ability of the soil microbialcommunities in both dry-cool and wet-warn seasons after 144 h of incubation (Figure 6) Moreover theRDA analysis revealed that carbon source utilization ability of the soil microbial communities werepositive related with APH in the dry-cool season while related with urease and peroxidase activitiesin the wet-warn season (Figure 7) As a whole the higher soil microbial functions in mixed stands arelikely the results of the effects of AMF and GP bacteria in the dry-cool season and the effects of bacteriaincluding GP and GN bacteria the wet-warn season (Table S6)

5 Conclusions

In our study we measured significantly higher tree shrub and herb biomass and litter productionin the coniferous-broadleaf mixed plantation after 34 years of afforestation As expected MBC MBNMBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands suggesting highercarbon and nitrogen availability in soil and higher soil quality for the growth of soil microorganisms inour coniferous-broadleaf mixed stand In addition coniferous-broadleaf mixture can increase carbonsource utilization ability of soil microbial community and accelerate the mineralization rates of soilorganic phosphorus and nitrogen by increasing acid phosphatase β-14-N-acetylglucosaminidaseand urease activities The higher soil microbial biomass and activities are likely the result of greateramount and more diverse organic matter input via litter and rhizodeposition products and the higheruse efficiency of soil organic carbon and nitrogen by microorganisms in mixed stands Howeverthe correlations between soil dominated microbial functional groups and the higher availabilityof soil organic carbon and nitrogen along with the quality of SOC in mixed stands merit furtherinvestigation Our results highlight that establishment of coniferous-broadleaf mixed forests maybe a good management practice as coniferous-broadleaf mixture could accumulate higher stand

Forests 2019 10 879 17 of 20

biomass return more litter and increase soil microbial biomass and functions over the long term insubtropical China

Supplementary Materials The following are available online at httpwwwmdpicom1999-49071010879s1Table S1 Effects of stand type season and their interactions on stand biomass Table S2 Effects of stand type seasonand their interactions on soil microbial structure Table S3 Effects of stand type season and their interactionson relative abundance of the soil microbial community PLFAs Table S4 Effects of stand type season and theirinteractions on soil enzyme activity Table S5 Marginal and conditional effects of biotic and abiotic factors on soilmicrobial composition obtained from the summary of forward selection in Redundancy analysis (RDA) Table S6Marginal and conditional effects of soil microbial functional groups on soil enzyme activity and soil microbialcarbon source utilization ability obtained from the summary of forward selection in Redundancy analysis (RDA)

Author Contributions WW collected data made experiment analysis formal data and drafted the manuscriptYW designed the work XZ revised the manuscript and participated in analyzing the experiment data HZ YYXH and XL ZQ YL LY HL participated in collecting the experiment data

Funding This research was funded by National Natural Science Foundation of China (31860171 3146012131560201) the Guangxi Key Research and Development Program (2018AB40007) Innovation Project of GuangxiGraduate Education (YCBZ2017011) and the Guangxi Universities Science and technology (201201ZD001)

Acknowledgments We gratefully acknowledge the supports in field sampling by the Experimental Center ofTropical Forestry Chinese Academy of Forestry A special acknowledgment is given to Patrick M Finnegan for hiswriting suggestions

Conflicts of Interest The authors declare no conflicts of interest

References

1 Zhang X Liu S Huang Y Fu S Wang J Ming A Li X Yao M Li H Tree species mixture inhibits soilorganic carbon mineralization accompanied by decreased r-selected bacteria Plant Soil 2018 431 203ndash216[CrossRef]

2 Pan Y Birdsey RA Fang J Houghton R Kauppi PE Kurz WA Phillips OL Shvidenko ALewis SL Canadell JG et al A large and persistent carbon sink in the worldrsquos forests Science 2011 333988ndash993 [CrossRef] [PubMed]

3 Chen X Ju Q Lin K Development status issues and countermeasures of Chinarsquo s plantation World For Res2014 27 54ndash59

4 Wang H Lencinas MV Ross Friedman C Wang X Qiu J Understory plant diversity assessment ofEucalyptus plantations over three vegetation types in Yunnan China New For 2011 42 101ndash116 [CrossRef]

5 Sang PM Lamb D Bonner M Schmidt S Carbon sequestration and soil fertility of tropical tree plantationsand secondary forest established on degraded land Plant Soil 2013 362 187ndash200 [CrossRef]

6 Li C Zhou X Wen Y Zhu H Qin Z Li X You Y Huang X Effects of high-generation ratations ofEucalyptus on undergrowth soil fertility and enzyme activities Guangxi Sci 2019 26 176ndash186

7 You Y Huang X Zhu H Liu S Liang H Wen Y Wang H Cai D Ye D Positive interactions betweenPinus massoniana and Castanopsis hystrix species in the uneven-aged mixed plantations can produce moreecosystem carbon in subtropical China For Ecol Manag 2018 410 193ndash200 [CrossRef]

8 Huang X Liu S You Y Wen Y Wang H Wang J Microbial community and associated enzymes activityinfluence soil carbon chemical composition in Eucalyptus urophylla plantation with mixing N2-fixing speciesin sub-tropical China Plant Soil 2017 414 199ndash212 [CrossRef]

9 Kelty MJ The role of species mixtures in plantation forestry For Ecol Manag 2006 233 195ndash204 [CrossRef]10 Bauhus J Khanna P Menden N Aboveground and belowground interactions in mixed plantations of

Eucalyptus globulus and Acacia mearnsii Can J For Res 2000 30 1886ndash1894 [CrossRef]11 Vandermeer J The Ecology of Intercropping Cambridge University Press New York NY USA 1989 p 23712 Forrester DI The spatial and temporal dynamics of species interactions in mixed-species forests From

pattern to process For Ecol Manag 2014 312 282ndash292 [CrossRef]13 Forrester DI Bauhus J A review of processes behind diversity-productivity relationships in forests

Curr For Rep 2016 2 45ndash61 [CrossRef]14 Wang H Liu S Wang J You Y Yang Y Shi Z Huang X Zheng L Li Z Ming A et al Mixed-species

plantation with Pinus massoniana and Castanopsis hystrix accelerates C loss in recalcitrant coniferous litter butslows C loss in labile broadleaf litter in southern China For Ecol Manag 2018 422 207ndash213 [CrossRef]

Forests 2019 10 879 4 of 20

biomass (TAB) and tree belowground biomass (TBB) were calculated by the allometric equationsdescribed by You et al (2018) [7]

23 Litter Production and Quality Measurements

A litter collector with an effective collection area of 4 m2 was randomly set in each plot Each littercollector was made with nylon net with a mesh size of 1 mm and set 05 m above the ground withPVC (polyvinyl chloride) tube Litter was collected once a month from August 2017 to July 2018 Littersamples were transported to the laboratory and oven-dried at 75 C for production determination(Mg haminus1) Litter organic carbon (OClitter) concentration was determined by the K2Cr2O7ndashH2SO4

calefaction procedure [30] Litter total nitrogen (TNlitter) total phosphorus (TPlitter) and total potassium(TKlitter) concentrations were measured after heat digestion with 98 H2SO4 [31ndash33]

24 Soil Sampling and Analysis

Soil samples were also collected in the wet-warm (July 2017) and dry-cool seasons (January 2018)In each season we took eight soil cores systematically arranged at a distance of 5 m from the plotcenter in the directions of 0 45 90 135 180 215 270 and 315 Each core was taken using an87 cm inner diameter steel tube at a depth of 0ndash10 cm below the soil surface The eight cores of eachplot were mixed to form a composite sample Each soil sample was divided into two subsamplesOne subsample was air-dried to a constant weight and sieved using a 2 mm sieve for measuring soilproperties The other subsample was sieved to 2 mm and immediately stored at minus4 C for measuringmoisture content soil ammonium nitrogen (NH4

+-N) nitrate nitrogen (NO3minus-N) microbial biomass

carbon (MBC) microbial biomass nitrogen (MBN) concentration phospholipid fatty acid analysis(PLFA) assays carbon source utilization ability of the soil microbial communities and enzyme activityAt the same time three soil cores (100 cm3) from 0ndash10 cm soil layer were sampled from each plot tomeasure bulk density by the cutting ring method [34]

The soil water content (WC) was measured using a sub-sample dried in oven at 105 C untilthe sample reached a constant weight The soil temperatures were measured with an LI-8100AAutomated Soil Gas Flux System (LindashCor Lincoln NE USA) Soil pH was measured in a 125 soilwatersuspension using a pH meter [835] Soil organic carbon (SOC) concentration was determined bythe K2Cr2O7ndashH2SO4 calefaction procedure [31] Total nitrogen (TN) total phosphorus (TP) andtotal potassium (TK) concentration were measured after heat digestion with 98 H2SO4 TN andTK were measured using the liquid supernatant with a continuous flow analytical system (AA3SEAL Analytical Norderstedt Germany) [3133] and an atomic absorption spectrometer (ZEENIT700P Analytik Jena Germany) [32] respectively TP and AP were determined by MondashSbndashVccolorimetry with the liquid supernatant after heat digestion and HCl (005 M)ndashH2SO4 (0025 M)respectively And available potassium (AK) was determined by atomic absorption spectrometry with1 M NH4OAC Soil ammonium nitrogen (NH4

+-N) and nitrate nitrogen (NO3minus-N) were calorimetrically

determined in a continuous flow analyzer after 10 g fresh mass of soil had been extracted in 50 mLof 2 M KCl [836] The cation exchange capacity (CEC) was determined by [Co(NH3)6]Cl3 extractionspectrophotometry [3738]

Soil microbial biomass carbon (MBC) and nitrogen (MBN) were measured by fumigation-extractionusing 05 M K2SO4 as the extraction agent [3940] with a total organic carbon analyzer (1020A OICollege Station TX USA)

Phospholipid fatty acid analysis (PLFA) assays were employed to analyze soil microbial communitystructure with freeze-dried soil samples using the procedure developed by Bossio et al (1998) [41]The PLFA markers 140 150 170 180 141ω5c 150 3OH 151ω6c 160 2OH 161 2OH 161ω9c161ω7c 181 ω7c 180 2OH 181 ω5c a150 a160 a170 cy170 cy190 i140 i150 i160 i170 i180and i190 were chosen to represent bacteria Gram-positive (GP) bacteria were represented by a150a160 a170 i140 i150 i160 i170 i180 and i190 and gram-negative (GN) bacteria were representedby 141ω5c 150 3OH 151ω6c 160 2OH 161 2OH 161ω7c 161ω9c 180 2OH 181ω5c cy170

Forests 2019 10 879 5 of 20

cy190 and 181 ω7c [364243] Actinobacteria were represented by 10-Me170 10-Me180 TBSA11-Me181ω7c and 10-Me160 [124344] Arbuscular mycorrhizal fungi (AMF) were represented by161 ω11c and 161 ω5c [35] and fungi were represented by 181 ω9c 183 ω6c (6912) and 182ω69c [1244] Other PLFAs such as 150 2OH 160 171ω8c 180 iso and i200 were also used for theanalysis of the composition of microbial communities

Carbon source utilization ability of the soil microbial communities was analyzed using BIOLOGreg

Eco plates (Biolog Hayward CA USA) with fresh soil samples Each Biolog Econdashplate comprised96 wells containing triplicates of 31 different carbon sources and a blank [45] We added 10 g dryweight equivalent of fresh soil to 90 mL of 085 sterile saline physiological solution and shook themixture for 1 h on a rotary shaker (100 rpm) at 25 C After 10minus3 dilution we transferred samplescontaining 150 microL of liquid into the 96 wells and read the optical density (OD) at 590 and 750 nm witha Microstationtrade reader (Biolog) after 24 4872 96 120 144 168 and 192 h of incubation [4647]

Enzyme assays were performed with a fully automatic full-wavelength enzyme labeling apparatus(Infinite M200 Pro Tecan Inc Maumlnnedorf Switzerland) using the procedure describe in detailby Saiya-Cork et al (2002) [48] and Steinauer et al (2015) [49] The phenol oxidase andperoxidase assays were colorimetric using L34-dihydroxyphenylalanine (L-DOPA) as the substrateThe colorimetric urease assay used urea as the substrate The α-Glucosidase (AG) β-14-glucosidase (BG)β-Cellobiosidase (CB) acid phosphatase (APH) and β-14-N-acetylglucosaminidase (NAG) activities werefluorometric with substrate 4-methylumbelliferyl-α-D-glucoside 4-methylumbelliferyl-β-D-glucoside4-methylumbelliferyl-β-D-cellobioside 4-methylumbelliferyl-phosphate and 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide respectively

25 Statistical Analysis

One-way analyses of variances (ANOVAs) were used to examine the differences of stand biomasslitter production and quality soil physicochemical properties soil microbial biomass soil microbialPLFAs content and functions among PPM PCH and MIX plantations in each season Two-wayANOVAs were used to determine the interaction effects between stand type and season on the abovevariables Statistical analyses were performed with SPSS software (ver 250 IBM Corp Armonk NYUSA) Significant differences between means were tested by least significant difference (LSD) at the005 level

In order to evaluate admixing effects on soil microbial biomass we calculated the relative effects(RE) of mixing species by comparing the observed value (OV) of one variable in mixed plantationwith the predicted value (PE) based on the respective mono-specific plantations RE was calculated byfollowing Wardle et al (1997) [50]

RE = [(OV minus PE)PE] times 100 (1)

where PE is the mean of the observed value of one variable in respective mono-specific plantationsIf RE differs from zero it would indicate non-additive effects (NAE) of mixing species on soil microbialbiomass Negative and positive deviations from zero are referred to as antagonistic and synergisticeffects respectively One-sample Studentrsquos t-tests with 95 confidence intervals were employed to testwhether RE differed significantly from zero

Redundancy analysis (RDA) was used to determine the relationships among biotic and abioticfactors and soil microbial composition and functions by CANOCO software for Windows 5(Biometris-Plant Research international Wageningen The Netherlands) In RDA the relative abundanceof individual PLFAs (mole present of the total PLFAs) was used as the response variables and soilphysicochemical properties litter and stand biomass were used as the explanatory variables [3551]We also used RDA to determine the linkage between soil microbial community and functions includingcarbon source metabolic function and soil extracellular enzyme activity involved in biogeochemicalcirculation of carbon nitrogen and phosphorus All data were log-transformed before in the analyses

Forests 2019 10 879 6 of 20

The forward selection procedure in RDA based on Monte Carlo permutation with 499 iterations wasperformed to determine the dominant variables that influenced the composition of individual PLFAsand microbial functions A significance level of p lt 005 was used in all analyses

3 Results

31 Stand Biomass

Stand type had significant effects on the -above- and below-ground biomass of trees shrubs andherbs (Table S1 Figure 1) TAB TBB shrub aboveground biomass (SAB) shrub belowground biomass(SBB) and herb aboveground biomass (HAB) in MIX were significantly higher than those in PCHand PPM in both the dry-cool and wet-warm seasons (Figure 1andashe) Except for HAB season had nosignificant effects on the tested biomass variables Moreover significant interaction effects betweenstand type and season only found on HAB and HBB These results supported our first hypothesis thattree species mixture increased stand biomass

Forests 2019 10 x FOR PEER REVIEW 6 of 22

biogeochemical circulation of carbon nitrogen and phosphorus All data were log-transformed before in the analyses The forward selection procedure in RDA based on Monte Carlo permutation with 499 iterations was performed to determine the dominant variables that influenced the composition of individual PLFAs and microbial functions A significance level of p lt 005 was used in all analyses

3 Results

31 Stand Biomass

Stand type had significant effects on the -above- and below-ground biomass of trees shrubs and herbs (Table S1 Figure 1) TAB TBB shrub aboveground biomass (SAB) shrub belowground biomass (SBB) and herb aboveground biomass (HAB) in MIX were significantly higher than those in PCH and PPM in both the dry-cool and wet-warm seasons (Figure 1andashe) Except for HAB season had no significant effects on the tested biomass variables Moreover significant interaction effects between stand type and season only found on HAB and HBB These results supported our first hypothesis that tree species mixture increased stand biomass

Figure 1 Biomass of trees (ab) shrubs (cd) and herbs (ef) in three plantations in different seasons Different letters above the bars denote significant differences at p lt 005 level by LSD (least significant difference) multiple comparisons among stand types (n = 5)

32 Litter Production and Quality

Stand type the interaction between stand type and season significantly influenced litter production at our study sites (Table 2) The MIX had significantly higher litter production than PCH and PPM in both seasons OClitter and CNlitter were significantly affected by both stand type and season

Figure 1 Biomass of trees (ab) shrubs (cd) and herbs (ef) in three plantations in different seasonsDifferent letters above the bars denote significant differences at p lt 005 level by LSD (least significantdifference) multiple comparisons among stand types (n = 5)

Forests 2019 10 879 7 of 20

32 Litter Production and Quality

Stand type the interaction between stand type and season significantly influenced litter productionat our study sites (Table 2) The MIX had significantly higher litter production than PCH and PPMin both seasons OClitter and CNlitter were significantly affected by both stand type and season withhigher value in the wet-warm season than in the dry-cool season in all plantations An opposite trendwas observed in TNlitter TPlitter was significantly affected by stand type season and their interactionwith MIX had significantly lower concentration than PCH and PPM in the dry-cool season TKlitter wassignificantly affected by stand type with MIX had significantly higher concentration than PCH andPPM in the wet-warm season These results partially supported our first hypothesis that tree speciesmixture increased litter production although the increases of litter quality in mixed plantations werenot significant

33 Soil Physical and Chemical Properties

In the dry-cool season the mixed plantations had the highest soil bulk density (BD) and soil watercontent (WC) and the lowest soil temperature and medium pH compared to the pure plantations(Table 3) In the wet-warm season MIX had the medium BD WC and soil temperature and the highestpH compared to PPM and PCH

The PPM plantations had the highest SOC TN TP SOC-to-TN ratio NH4+-N and CEC while

MIX had the highest AP TK and AK and PCH had the highest NO3minus-N and AN in the dry-cool season

(Table 3) On the other hand the PCH had the highest SOC TN and AP while MIX had the highestTP TK AK NO3

minus-N and AN and PPM had the highest SOC-to-TN ratio NO3minus-N and CEC in the

wet-warm seasonTwo-way ANOVAs indicated that season significantly affected all the tested soil variables (Table 3)

In addition the stand type influenced nearly all soil variables except BD TN NH4+-N and AN

Interactions between stand type and season significantly influenced WC temperature pH SOC TNTP AP AK NO3

minus-N and CEC

Forests 2019 10 879 8 of 20

Table 2 The quantity and quality of litter in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM) plantation and mixed C hystrix and P massoniana(MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH MIX PPM PCH MIX PPM F p F p F p

LP 668 plusmn 019 b 836 plusmn 069 a 484 plusmn 019 c 553 plusmn 043 b 921 plusmn 040 a 616 plusmn 023 b 3752 lt0001 093 0343 527 0013OClitter 19064 plusmn 1063 b 24859 plusmn 1848 a 25505 plusmn 1800 a 27757 plusmn 1885 b 2743 plusmn 1142 b 32175 plusmn 544 a 683 0004 2486 lt0001 226 0127TNlitter 1738 plusmn 078 a 1353 plusmn 106 b 1496 plusmn 062 ab 1295 plusmn 059 a 1357 plusmn 090 a 1189 plusmn 050 a 321 0058 1579 0001 445 0023CNlitter 1112 plusmn 095 b 1850 plusmn 081 a 1733 plusmn 181 a 2182 plusmn 223 b 2056 plusmn 156 b 2722 plusmn 096 a 767 0003 3890 lt0001 518 0013TPlitter 134 plusmn 008 a 050 plusmn 007 b 116 plusmn 005 a 119 plusmn 015 a 138 plusmn 015 a 131 plusmn 005 a 667 0005 1281 0002 1417 lt0001TKlitter 116 plusmn 016 a 152 plusmn 010 a 082 plusmn 009 b 107 plusmn 002 b 149 plusmn 013 a 088 plusmn 007 c 2084 lt0001 007 0796 029 0751

Different superscripts indicate significant differences among stand types in each season (p lt 005) LP (Mg haminus1) litter production OClitter (g kgminus1) litter organic carbon TNlitter (g kgminus1)litter total nitrogen CNlitter CN ratio of litter TPlitter (g kgminus1) litter total phosphorus TKlitter (g kgminus1) litter total potassium

Table 3 Soil physical and chemical properties (0ndash10 cm soil depth) in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM) plantation and mixedC hystrix and P massoniana (MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH Mixed PPM PCH Mixed PPM F p F p F p

BD 109 plusmn 003 b 125 plusmn 004 a 119 plusmn 002 a 103 plusmn 001 a 103 plusmn 003 a 104 plusmn 005 a 311 0063 3044 lt0001 297 0070WC 3966 plusmn 136 b 4359 plusmn 061 a 4017 plusmn 158 ab 4053 plusmn 107 c 4440 plusmn 084 b 5048 plusmn 141 a 1035 0001 1681 lt0001 1048 0001Tem 1682 plusmn 021 a 1550 plusmn 009 c 1617 plusmn 005 b 2628 plusmn 043 a 2598 plusmn 022 a 2541 plusmn 008 a 821 0002 283334 lt0001 431 0025pH 432 plusmn 002 a 419 plusmn 003 b 404 plusmn 005 c 402 plusmn 002 b 421 plusmn 003 a 390 plusmn 009 b 1440 lt0001 1370 0001 623 0007

SOC 3742 plusmn 108 b 4082 plusmn 022 b 5698 plusmn 189 a 5331 plusmn 194 a 4839 plusmn 200 a 5083 plusmn 054 a 2460 lt0001 2369 lt0001 2864 lt0001TN 163 plusmn 005 b 160 plusmn 006 b 216 plusmn 022 a 248 plusmn 011 a 213 plusmn 013 a 217 plusmn 014 a 248 0105 4408 lt0001 480 0018

SOCTN 2298 plusmn 059 b 2567 plusmn 103 b 2949 plusmn 159 a 2167 plusmn 133 a 2304 plusmn 162 a 2390 plusmn 202 a 457 0021 722 0013 114 0338TP 031 plusmn 004 c 042 plusmn 002 b 060 plusmn 004 a 052 plusmn 006 b 078 plusmn 004 a 054 plusmn 004 b 2186 lt0001 2889 lt0001 1795 lt0001AP 919 plusmn 058 b 1252 plusmn 069 a 1002 plusmn 030 b 1330 plusmn 040 a 1315 plusmn 057 a 998 plusmn 027 b 1670 lt0001 1525 0001 1029 0001TK 414 plusmn 015 b 545 plusmn 049 a 242 plusmn 016 c 540 plusmn 050 a 694 plusmn 082 a 338 plusmn 013 b 3226 lt0001 1034 0004 039 0680AK 2990 plusmn 149 b 4629 plusmn 096 a 2846 plusmn 108 b 4719 plusmn 228 a 5279 plusmn 365 a 2837 plusmn 144 b 5375 lt0001 2257 lt0001 927 0001

NO3minus-N 355 plusmn 024 a 185 plusmn 004 c 286 plusmn 022 b 406 plusmn 019 c 530 plusmn 009 b 579 plusmn 009 a 1096 lt0001 29106 lt0001 4547 lt0001

NH4+-N 2129 plusmn 101 a 2334 plusmn 113 a 2447 plusmn 137 a 4224 plusmn 162 a 4350 plusmn 166 a 3883 plusmn 247 a 075 0482 19616 lt0001 247 0105

AN 2734 plusmn 157 a 2519 plusmn 114 a 2485 plusmn 090 a 463 plusmn 155 a 488 plusmn 169 a 4462 plusmn 246 a 120 0319 24555 lt0001 117 0328CEC 4553 plusmn 327 c 5758 plusmn 411 b 8233 plusmn 194 a 5543 plusmn 202a 5446 plusmn 119 a 6224 plusmn 291 a 4514 lt0001 1216 0002 2168 lt0001

Different superscripts indicate significant differences among stand types in each season (p lt 005) BD (g cmminus1) bulk density WC () soil water content Tem (C) temperature SOC(g kgminus1) soil organic carbon TN (g kgminus1) total nitrogen TP (g kgminus1) total phosphorus AP (mg kgminus1) available phosphorus TK (g kgminus1) total potassium AK (mg kgminus1) availablepotassium NO3

minus-N (mg kgminus1) nitrate nitrogen NH4+-N (mg kgminus1) ammonium nitrogen AN (mg kgminus1) Available nitrogen CEC (cmol + kgminus1) cation exchange capacity

Forests 2019 10 879 9 of 20

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantlyaffected by stand type and MBN was also significantly affected by season and interaction betweenstand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a)In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantlyhigher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highestMBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOCand MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significantlevel in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOCand MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-coolseason (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season(Figure 3b) were significant

Forests 2019 10 x FOR PEER REVIEW 10 of 22

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantly affected by stand type and MBN was also significantly affected by season and interaction between stand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a) In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantly higher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highest MBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOC and MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significant level in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOC and MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-cool season (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season (Figure 3b) were significant

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBN MBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a) and wet-warm season (b)

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBNMBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005level by LSD multiple comparisons among stand types (n = 5)

Forests 2019 10 x FOR PEER REVIEW 10 of 22

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantly affected by stand type and MBN was also significantly affected by season and interaction between stand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a) In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantly higher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highest MBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOC and MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significant level in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOC and MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-cool season (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season (Figure 3b) were significant

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBN MBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a) and wet-warm season (b)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a)and wet-warm season (b)

Forests 2019 10 879 10 of 20

In the dry-cool season total PLFAs AMF PLFAs actinobacteria PLFAs bacteria PLFAs fungiPLFAs GP bacteria PLFAs and GP-to-GN PLFAs ratio were smaller in MIX than those in PPM andPCH On the other hand in the wet-warm season these PLFAs were smaller in PPM than thosein PCH and MIX (Table 4) Specifically the ratios of bacteria-to-fungi PLFA and GP-to-GN PLFAwere significantly higher in MIX than those in PPM and PCH in the wet-warm season These resultsindicated that the response of the soil microbial community structure to tree species mixture varied indifferent seasons Two-way ANOVAs showed that both stand type and season significantly affectedall the tested microbial variables while the interaction between stand type and season significantlyinfluenced total PLFAs bacteria PLFAs GP bacteria PLFAs Acti PLFAs bacteria-to-fungi PLFA andGP-to-GN PLFA (Table 4)

Across plantations and seasons bacteria PLFAs were the most abundant followed by fungi andactinobacteria PLFAs (Figure 4 and Table S3) It was worth noting that the relative abundance ofGN bacteria PLFAs was significantly higher in MIX than in other plantations in the dry-cool season(Figure 4) Furthermore the relative abundance of GP bacteria PLFAs was significantly higher andthat of fungi PLFAs was significantly lower in MIX than in other plantations in the wet-warm season

Forests 2019 10 x FOR PEER REVIEW 13 of 22

Across plantations and seasons bacteria PLFAs were the most abundant followed by fungi and actinobacteria PLFAs (Figure 4 and Table S3) It was worth noting that the relative abundance of GN bacteria PLFAs was significantly higher in MIX than in other plantations in the dry-cool season (Figure 4) Furthermore the relative abundance of GP bacteria PLFAs was significantly higher and that of fungi PLFAs was significantly lower in MIX than in other plantations in the wet-warm season

Figure 4 The relative abundance of PLFAs (mol ) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleaf plantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increased APH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increased urease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg) Carbon source utilization ability of the soil microbial community indicated by average well color development (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation (Figure 6)

Figure 4 The relative abundance of PLFAs (mol ) in three plantations in the dry-cool season (a) andwet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level byLSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 11 of 20

Table 4 Soil microbial community PLFAs content and structure (0ndash10 cm soil depth) in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM)plantation and mixed C hystrix and P massoniana (MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH MIX PPM PCH MIX PPM F p F p F p

TPLFAs 3767 plusmn 271 a 3255 plusmn 124 a 3796 plusmn 128 a 4373 plusmn 208 a 4001 plusmn 07 a 3478 plusmn 111 b 464 0020 646 0018 607 0007B 1579 plusmn 081 a 1306 plusmn 043 b 1490 plusmn 051 ab 1764 plusmn 072 a 1723 plusmn 042 a 1276 plusmn 041 b 1274 lt0001 768 0011 1556 lt0001

GP 945 plusmn 043 a 794 plusmn 029 b 918 plusmn 031 a 1066 plusmn 051 a 1155 plusmn 031 a 810 plusmn 031 b 817 0002 1709 lt0001 2030 lt0001GN 591 plusmn 076 a 590 plusmn 016 a 553 plusmn 022 a 619 plusmn 025 a 455 plusmn 037 b 411 plusmn 020 b 526 0013 702 0014 313 0062

F 498 plusmn 034 a 375 plusmn 022 b 424 plusmn 028 ab 492 plusmn 035 a 296 plusmn 043 b 345 plusmn 021 b 1356 lt0001 448 0045 091 0416AMF 104 plusmn 003 a 075 plusmn 003 b 075 plusmn 004 b 085 plusmn 007 a 044 plusmn 008 b 048 plusmn 003 b 2822 lt0001 3483 lt0001 056 0577Acti 426 plusmn 037 ab 377 plusmn 021 b 486 plusmn 020 a 531 plusmn 019 a 487 plusmn 012 b 458 plusmn 010 b 273 0085 1236 0002 650 0006BF 319 plusmn 011 a 351 plusmn 013 a 356 plusmn 017 a 362 plusmn 014 b 635 plusmn 094 a 377 plusmn 033 b 750 0003 1131 0003 598 0008

GPGN 174 plusmn 029 b 135 plusmn 003 b 166 plusmn 004 a 173 plusmn 008 b 263 plusmn 028 a 198 plusmn 011 b 107 0359 1376 0001 733 0003

Different superscripts indicate significant differences among stand types in each season (p lt 005) TPLFAs (nmol gminus1 soil) total PLFAs B (nmol gminus1 soil) bacteria PLFAs GP (nmol gminus1

soil) gram-positive bacteria PLFAs GN (nmol gminus1 soil) gram-negative bacteria PLFAs F (nmol gminus1 soil) fungi PLFAs Acti (nmol gminus1 soil) actinobacteria PLFAs AMF (nmol gminus1 soil)arbuscular mycorrhizal fungi PLFAs BF bacteria-to-fungi PLFA ratio GPGN GP-to-GN ratio

Forests 2019 10 879 12 of 20

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleafplantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increasedAPH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increasedurease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg)Carbon source utilization ability of the soil microbial community indicated by average well colordevelopment (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation(Figure 6)Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in threeplantations in different seasons Error bars are standard error Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 13 of 20

Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in threeplantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in thedry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons(F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pHTK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-coolseason in which SOC and TBB were the major correlates of changes in soil microbial composition(Table S5) A total of 11 biotic and abiotic factors including NO3

minus-N TBB pH TKlitter HBB AP AKTP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warmseason in which NO3

minus-N TBB pH and TKlitter were the major correlates of changes in soil microbialcomposition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

37 Relationship between Soil Microbial Structure and Functions

Soil enzyme activity and soil microbial carbon source utilization ability are both important soilmicrobial functions The first two RDA axes explained 4948 of the variation in soil enzyme activityand carbon utilization ability in the dry-cool season (F = 24 p = 0006 Figure 7b) and 6891 of thevariation in the wet-warm season (F = 64 p = 0002 Figure 7d) AMF and GP were significantlycorrelated with soil microbial functions in the dry-cool season (Table S6) Total bacteria GP bacteriaGN bacteria and actinobacteria were significantly correlated with soil microbial functions in thewet-warm season in which total bacteria GP bacteria and GN bacteria were the major correlates ofchanges of soil enzyme activity (Table S6)

Forests 2019 10 879 14 of 20

Forests 2019 10 x FOR PEER REVIEW 15 of 22

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in the dry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons (F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pH TK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-cool season in which SOC and TBB were the major correlates of changes in soil microbial composition (Table S5) A total of 11 biotic and abiotic factors including NO3minus-N TBB pH TKlitter HBB AP AK TP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warm season in which NO3minus-N TBB pH and TKlitter were the major correlates of changes in soil microbial composition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160 were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant biotic and abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot of soil microbial community structure and soil microbial functions (bd) Above the label represents a significant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowground biomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soil total potassium NO3ndash-N soil nitrate nitrogen HBB herb belowground biomass AP soil available phosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil water

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant bioticand abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot ofsoil microbial community structure and soil microbial functions (bd) Above the label represents asignificant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowgroundbiomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soiltotal potassium NO3

minus-N soil nitrate nitrogen HBB herb belowground biomass AP soil availablephosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil watercontent LP litter production TKlitter litter total potassium GP gram-positive bacteria GN gramnegative bacteria AMF arbuscular mycorrhizal fungi AG α-Glucosidase BG β-14-glucosidaseCB β-Cellobiosidase APH acid phosphatase NAG β-14-N-acetylglucosaminidase PHO phenoloxidase PO peroxidase AWCD average well color development of carbon source metabolism at192 h incubation

4 Discussion

41 Coniferous-Broadleaf Mixture Significantly Increase Stand Biomass and Litter Production

The results of this study indicated that admixing of coniferous and broadleaf tree speciessignificantly increased stand biomass and litter production after 34 years of afforestation in bothdry-cool and wet-warm seasons (Figure 1 Table 2) This may be a consequence of a greater growth of

Forests 2019 10 879 15 of 20

C hystrix in the mixed stands These results confirmed previous findings conducted in our experimentsite measured when the plantations were 26-year-old and 28-year-old respectively [716] A bulkof studies revealed that mixed-species management could affect tree growth stand structure andenvironment [145253] Mixed forests can develop stratified canopies due to niche differences amongdifferent species In the present study trees of the faster growing broad-leaf species (C hystrix) weretaller had a larger DBH and thus formed an upper canopy stratum in the mixed stands (Table 1)While trees of the shorter species (P massoniana) formed a lower stratum with lower DBH and treeheight and the reduced amount of light transmitted through the upper canopy contributed to theirslower growth The dominant tree species within the stand can absorb more sunlight and use itmore efficiently for growth [54] and thus contribute to higher tree biomass and litter productionin mixed stands In addition stratified canopies in mixed plantations can help to maintain levelsof understory species richness and biomass compared with pure plantations due to relatively highlight availabilities [5556] This may be why the mixed stand had higher shrub and herb biomass inour study

Specifically in the present study the broad-leaf species attained greater heights and diameters inmixed stands while masson pine had reduced diameter and height growth in mixed-species comparedto mono-species stands (Table 1) The severe suppression of P massoniana by the faster growingC hystrix may be attributed to differences in the light requirements of the two species [7] The growthpattern of C hystrix and P massoniana in mixed stands in our study (34-year-old) were differed fromwhen they were 26-years-old in which the growth of C hystrix and P massoniana in mixed standswere both greater than in the corresponding mono-species stands [7] This suggests that effects of thetree species mixture could change over time and long-term investigation of mixed forests is of greatsignificance to reveal mixed effects

42 Coniferous-Broadleaf Mixture Significantly Increase Soil Microbial Biomass

The changes of tree species and composition may significantly affect the amount of organic matterinput in soil [57] As the most active part in soil organic matter (SOM) soil microbial biomass isimportant in nutrient cycling and release participating in the circulation of critical elements such ascarbon and nitrogen [23] Because of the quantity and quality of the bulk of SOM changes slowly andthe changes of SOM are difficult to measure due to the spatial heterogeneity of soils soil microbialbiomass and in particular MBC-to-SOC and MBN-to-TN ratio have been suggested as indicators ofthe state and modification of SOM [58ndash60] and as an indicator for carbon (nitrogen) availability insoil [6162] The concentrations of SOC TN and MBC and MBN measured in our study were affectedby stand type and seasons (Table 3 and Table S2 Figure 2) In the dry cool season although the SOCcontent in mixed stands did not significantly differ with that in PCH but significantly lower than thatin PPM the MBC concentration in mixed stands was markedly higher than that in the correspondingmono-species stands Likewise although the TN content in mixed stands was smaller than that in PCHand PPM stands the MBN content in mixed stands was markedly higher than that in the correspondingmono-species stands in the wet-warn season Previous studies found that higher soil microbial biomasscorrelated with higher SOC and TN content that sustain microbial metabolism [2163] However inour study the lower SOC and TN content in MIX was able to maintain a higher microbial biomass thansites where SOC was more abundant Presumably the quality of SOC could explain such a trend [64]and merit further investigation

In this study MBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands(Figure 2) suggesting higher carbon and nitrogen availability in soil and higher soil quality for thegrowth of soil microorganisms by coniferous-broadleaf mixture [21] In addition the effects of admixingon soil microbial biomass MBC-to-SOC and MBN-to-TN ratio were non-additive suggesting theinteraction of P massoniana and C hystrix can have a synergistic effect on improving soil carbon andnitrogen availability (Figure 3) These findings are consistent with many previous studies that havedetermined the effects of tree species mixture on soil microbial biomass [2165] These results may be

Forests 2019 10 879 16 of 20

contributed to (1) greater amount and more diverse organic matter input via litter and rhizodepositionproducts in the mixture of two contrasting tree species [21] (2) the higher use efficiency of soil organiccarbon and nitrogen by microorganisms in mixed stands [66]

We also sought to determine the admixing effects on composition and abundance of soil microbialcommunity with community-level PLFA profiles Surprisingly there were not significant admixingeffects on PLFA content of the major microbial functional groups such as bacteria fungi AMF andactinobacteria (Table 4) However tree species mixture significantly increased the abundance of GNbacteria in the dry-cool season while markedly increased the abundance of GP bacteria but decreasedthe abundance of fungi in the wet-warn season (Figure 4) It seemed that the dominated GN bacteria inthe dry-cool season and GP bacteria in the wet-warn season would correlate with the higher availabilityof soil organic carbon and nitrogen in mixed stands [6768] These correlations are complicated andfurther investigation is required to reveal the underlying mechanisms

43 Effects of Coniferous-Broadleaf Mixture on Soil Microbial Functions

Soil microorganisms excrete soil enzymes to drive nutrient mineralization and litterdecomposition [64] Therefore soil extracellular enzyme activities may be related to the carbonnitrogen and phosphorus cycling in soil [6970] In the present study we found positive admixingeffects on the activities of APH and NAG in the dry-cool season (Figure 5de) On the other handpositive admixing effects on the activities of urease and peroxidase while negative admixing effectson APH was detected in the wet-warn season (Figure 5dfg) This result suggests that the mixtureof C hystrix and P massoniana can accelerate the mineralization rates of soil organic phosphorus andnitrogen These findings were consistent with previous study [67]

The community-level physiological profiling (CLPP) is widely used to characterize microbialcommunities [71ndash73] The average well color development (AWCD) in the CLPP approach reflectsthe ability of soil microorganisms to utilize carbon sources and their metabolic activity The higherAWCD represents the stronger metabolic activity of soil microorganisms [71] In this present study wedetected a significantly positive admixing effect on carbon source utilization ability of the soil microbialcommunities in both dry-cool and wet-warn seasons after 144 h of incubation (Figure 6) Moreover theRDA analysis revealed that carbon source utilization ability of the soil microbial communities werepositive related with APH in the dry-cool season while related with urease and peroxidase activitiesin the wet-warn season (Figure 7) As a whole the higher soil microbial functions in mixed stands arelikely the results of the effects of AMF and GP bacteria in the dry-cool season and the effects of bacteriaincluding GP and GN bacteria the wet-warn season (Table S6)

5 Conclusions

In our study we measured significantly higher tree shrub and herb biomass and litter productionin the coniferous-broadleaf mixed plantation after 34 years of afforestation As expected MBC MBNMBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands suggesting highercarbon and nitrogen availability in soil and higher soil quality for the growth of soil microorganisms inour coniferous-broadleaf mixed stand In addition coniferous-broadleaf mixture can increase carbonsource utilization ability of soil microbial community and accelerate the mineralization rates of soilorganic phosphorus and nitrogen by increasing acid phosphatase β-14-N-acetylglucosaminidaseand urease activities The higher soil microbial biomass and activities are likely the result of greateramount and more diverse organic matter input via litter and rhizodeposition products and the higheruse efficiency of soil organic carbon and nitrogen by microorganisms in mixed stands Howeverthe correlations between soil dominated microbial functional groups and the higher availabilityof soil organic carbon and nitrogen along with the quality of SOC in mixed stands merit furtherinvestigation Our results highlight that establishment of coniferous-broadleaf mixed forests maybe a good management practice as coniferous-broadleaf mixture could accumulate higher stand

Forests 2019 10 879 17 of 20

biomass return more litter and increase soil microbial biomass and functions over the long term insubtropical China

Supplementary Materials The following are available online at httpwwwmdpicom1999-49071010879s1Table S1 Effects of stand type season and their interactions on stand biomass Table S2 Effects of stand type seasonand their interactions on soil microbial structure Table S3 Effects of stand type season and their interactionson relative abundance of the soil microbial community PLFAs Table S4 Effects of stand type season and theirinteractions on soil enzyme activity Table S5 Marginal and conditional effects of biotic and abiotic factors on soilmicrobial composition obtained from the summary of forward selection in Redundancy analysis (RDA) Table S6Marginal and conditional effects of soil microbial functional groups on soil enzyme activity and soil microbialcarbon source utilization ability obtained from the summary of forward selection in Redundancy analysis (RDA)

Author Contributions WW collected data made experiment analysis formal data and drafted the manuscriptYW designed the work XZ revised the manuscript and participated in analyzing the experiment data HZ YYXH and XL ZQ YL LY HL participated in collecting the experiment data

Funding This research was funded by National Natural Science Foundation of China (31860171 3146012131560201) the Guangxi Key Research and Development Program (2018AB40007) Innovation Project of GuangxiGraduate Education (YCBZ2017011) and the Guangxi Universities Science and technology (201201ZD001)

Acknowledgments We gratefully acknowledge the supports in field sampling by the Experimental Center ofTropical Forestry Chinese Academy of Forestry A special acknowledgment is given to Patrick M Finnegan for hiswriting suggestions

Conflicts of Interest The authors declare no conflicts of interest

References

1 Zhang X Liu S Huang Y Fu S Wang J Ming A Li X Yao M Li H Tree species mixture inhibits soilorganic carbon mineralization accompanied by decreased r-selected bacteria Plant Soil 2018 431 203ndash216[CrossRef]

2 Pan Y Birdsey RA Fang J Houghton R Kauppi PE Kurz WA Phillips OL Shvidenko ALewis SL Canadell JG et al A large and persistent carbon sink in the worldrsquos forests Science 2011 333988ndash993 [CrossRef] [PubMed]

3 Chen X Ju Q Lin K Development status issues and countermeasures of Chinarsquo s plantation World For Res2014 27 54ndash59

4 Wang H Lencinas MV Ross Friedman C Wang X Qiu J Understory plant diversity assessment ofEucalyptus plantations over three vegetation types in Yunnan China New For 2011 42 101ndash116 [CrossRef]

5 Sang PM Lamb D Bonner M Schmidt S Carbon sequestration and soil fertility of tropical tree plantationsand secondary forest established on degraded land Plant Soil 2013 362 187ndash200 [CrossRef]

6 Li C Zhou X Wen Y Zhu H Qin Z Li X You Y Huang X Effects of high-generation ratations ofEucalyptus on undergrowth soil fertility and enzyme activities Guangxi Sci 2019 26 176ndash186

7 You Y Huang X Zhu H Liu S Liang H Wen Y Wang H Cai D Ye D Positive interactions betweenPinus massoniana and Castanopsis hystrix species in the uneven-aged mixed plantations can produce moreecosystem carbon in subtropical China For Ecol Manag 2018 410 193ndash200 [CrossRef]

8 Huang X Liu S You Y Wen Y Wang H Wang J Microbial community and associated enzymes activityinfluence soil carbon chemical composition in Eucalyptus urophylla plantation with mixing N2-fixing speciesin sub-tropical China Plant Soil 2017 414 199ndash212 [CrossRef]

9 Kelty MJ The role of species mixtures in plantation forestry For Ecol Manag 2006 233 195ndash204 [CrossRef]10 Bauhus J Khanna P Menden N Aboveground and belowground interactions in mixed plantations of

Eucalyptus globulus and Acacia mearnsii Can J For Res 2000 30 1886ndash1894 [CrossRef]11 Vandermeer J The Ecology of Intercropping Cambridge University Press New York NY USA 1989 p 23712 Forrester DI The spatial and temporal dynamics of species interactions in mixed-species forests From

pattern to process For Ecol Manag 2014 312 282ndash292 [CrossRef]13 Forrester DI Bauhus J A review of processes behind diversity-productivity relationships in forests

Curr For Rep 2016 2 45ndash61 [CrossRef]14 Wang H Liu S Wang J You Y Yang Y Shi Z Huang X Zheng L Li Z Ming A et al Mixed-species

plantation with Pinus massoniana and Castanopsis hystrix accelerates C loss in recalcitrant coniferous litter butslows C loss in labile broadleaf litter in southern China For Ecol Manag 2018 422 207ndash213 [CrossRef]

Forests 2019 10 879 5 of 20

cy190 and 181 ω7c [364243] Actinobacteria were represented by 10-Me170 10-Me180 TBSA11-Me181ω7c and 10-Me160 [124344] Arbuscular mycorrhizal fungi (AMF) were represented by161 ω11c and 161 ω5c [35] and fungi were represented by 181 ω9c 183 ω6c (6912) and 182ω69c [1244] Other PLFAs such as 150 2OH 160 171ω8c 180 iso and i200 were also used for theanalysis of the composition of microbial communities

Carbon source utilization ability of the soil microbial communities was analyzed using BIOLOGreg

Eco plates (Biolog Hayward CA USA) with fresh soil samples Each Biolog Econdashplate comprised96 wells containing triplicates of 31 different carbon sources and a blank [45] We added 10 g dryweight equivalent of fresh soil to 90 mL of 085 sterile saline physiological solution and shook themixture for 1 h on a rotary shaker (100 rpm) at 25 C After 10minus3 dilution we transferred samplescontaining 150 microL of liquid into the 96 wells and read the optical density (OD) at 590 and 750 nm witha Microstationtrade reader (Biolog) after 24 4872 96 120 144 168 and 192 h of incubation [4647]

Enzyme assays were performed with a fully automatic full-wavelength enzyme labeling apparatus(Infinite M200 Pro Tecan Inc Maumlnnedorf Switzerland) using the procedure describe in detailby Saiya-Cork et al (2002) [48] and Steinauer et al (2015) [49] The phenol oxidase andperoxidase assays were colorimetric using L34-dihydroxyphenylalanine (L-DOPA) as the substrateThe colorimetric urease assay used urea as the substrate The α-Glucosidase (AG) β-14-glucosidase (BG)β-Cellobiosidase (CB) acid phosphatase (APH) and β-14-N-acetylglucosaminidase (NAG) activities werefluorometric with substrate 4-methylumbelliferyl-α-D-glucoside 4-methylumbelliferyl-β-D-glucoside4-methylumbelliferyl-β-D-cellobioside 4-methylumbelliferyl-phosphate and 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide respectively

25 Statistical Analysis

One-way analyses of variances (ANOVAs) were used to examine the differences of stand biomasslitter production and quality soil physicochemical properties soil microbial biomass soil microbialPLFAs content and functions among PPM PCH and MIX plantations in each season Two-wayANOVAs were used to determine the interaction effects between stand type and season on the abovevariables Statistical analyses were performed with SPSS software (ver 250 IBM Corp Armonk NYUSA) Significant differences between means were tested by least significant difference (LSD) at the005 level

In order to evaluate admixing effects on soil microbial biomass we calculated the relative effects(RE) of mixing species by comparing the observed value (OV) of one variable in mixed plantationwith the predicted value (PE) based on the respective mono-specific plantations RE was calculated byfollowing Wardle et al (1997) [50]

RE = [(OV minus PE)PE] times 100 (1)

where PE is the mean of the observed value of one variable in respective mono-specific plantationsIf RE differs from zero it would indicate non-additive effects (NAE) of mixing species on soil microbialbiomass Negative and positive deviations from zero are referred to as antagonistic and synergisticeffects respectively One-sample Studentrsquos t-tests with 95 confidence intervals were employed to testwhether RE differed significantly from zero

Redundancy analysis (RDA) was used to determine the relationships among biotic and abioticfactors and soil microbial composition and functions by CANOCO software for Windows 5(Biometris-Plant Research international Wageningen The Netherlands) In RDA the relative abundanceof individual PLFAs (mole present of the total PLFAs) was used as the response variables and soilphysicochemical properties litter and stand biomass were used as the explanatory variables [3551]We also used RDA to determine the linkage between soil microbial community and functions includingcarbon source metabolic function and soil extracellular enzyme activity involved in biogeochemicalcirculation of carbon nitrogen and phosphorus All data were log-transformed before in the analyses

Forests 2019 10 879 6 of 20

The forward selection procedure in RDA based on Monte Carlo permutation with 499 iterations wasperformed to determine the dominant variables that influenced the composition of individual PLFAsand microbial functions A significance level of p lt 005 was used in all analyses

3 Results

31 Stand Biomass

Stand type had significant effects on the -above- and below-ground biomass of trees shrubs andherbs (Table S1 Figure 1) TAB TBB shrub aboveground biomass (SAB) shrub belowground biomass(SBB) and herb aboveground biomass (HAB) in MIX were significantly higher than those in PCHand PPM in both the dry-cool and wet-warm seasons (Figure 1andashe) Except for HAB season had nosignificant effects on the tested biomass variables Moreover significant interaction effects betweenstand type and season only found on HAB and HBB These results supported our first hypothesis thattree species mixture increased stand biomass

Forests 2019 10 x FOR PEER REVIEW 6 of 22

biogeochemical circulation of carbon nitrogen and phosphorus All data were log-transformed before in the analyses The forward selection procedure in RDA based on Monte Carlo permutation with 499 iterations was performed to determine the dominant variables that influenced the composition of individual PLFAs and microbial functions A significance level of p lt 005 was used in all analyses

3 Results

31 Stand Biomass

Stand type had significant effects on the -above- and below-ground biomass of trees shrubs and herbs (Table S1 Figure 1) TAB TBB shrub aboveground biomass (SAB) shrub belowground biomass (SBB) and herb aboveground biomass (HAB) in MIX were significantly higher than those in PCH and PPM in both the dry-cool and wet-warm seasons (Figure 1andashe) Except for HAB season had no significant effects on the tested biomass variables Moreover significant interaction effects between stand type and season only found on HAB and HBB These results supported our first hypothesis that tree species mixture increased stand biomass

Figure 1 Biomass of trees (ab) shrubs (cd) and herbs (ef) in three plantations in different seasons Different letters above the bars denote significant differences at p lt 005 level by LSD (least significant difference) multiple comparisons among stand types (n = 5)

32 Litter Production and Quality

Stand type the interaction between stand type and season significantly influenced litter production at our study sites (Table 2) The MIX had significantly higher litter production than PCH and PPM in both seasons OClitter and CNlitter were significantly affected by both stand type and season

Figure 1 Biomass of trees (ab) shrubs (cd) and herbs (ef) in three plantations in different seasonsDifferent letters above the bars denote significant differences at p lt 005 level by LSD (least significantdifference) multiple comparisons among stand types (n = 5)

Forests 2019 10 879 7 of 20

32 Litter Production and Quality

Stand type the interaction between stand type and season significantly influenced litter productionat our study sites (Table 2) The MIX had significantly higher litter production than PCH and PPMin both seasons OClitter and CNlitter were significantly affected by both stand type and season withhigher value in the wet-warm season than in the dry-cool season in all plantations An opposite trendwas observed in TNlitter TPlitter was significantly affected by stand type season and their interactionwith MIX had significantly lower concentration than PCH and PPM in the dry-cool season TKlitter wassignificantly affected by stand type with MIX had significantly higher concentration than PCH andPPM in the wet-warm season These results partially supported our first hypothesis that tree speciesmixture increased litter production although the increases of litter quality in mixed plantations werenot significant

33 Soil Physical and Chemical Properties

In the dry-cool season the mixed plantations had the highest soil bulk density (BD) and soil watercontent (WC) and the lowest soil temperature and medium pH compared to the pure plantations(Table 3) In the wet-warm season MIX had the medium BD WC and soil temperature and the highestpH compared to PPM and PCH

The PPM plantations had the highest SOC TN TP SOC-to-TN ratio NH4+-N and CEC while

MIX had the highest AP TK and AK and PCH had the highest NO3minus-N and AN in the dry-cool season

(Table 3) On the other hand the PCH had the highest SOC TN and AP while MIX had the highestTP TK AK NO3

minus-N and AN and PPM had the highest SOC-to-TN ratio NO3minus-N and CEC in the

wet-warm seasonTwo-way ANOVAs indicated that season significantly affected all the tested soil variables (Table 3)

In addition the stand type influenced nearly all soil variables except BD TN NH4+-N and AN

Interactions between stand type and season significantly influenced WC temperature pH SOC TNTP AP AK NO3

minus-N and CEC

Forests 2019 10 879 8 of 20

Table 2 The quantity and quality of litter in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM) plantation and mixed C hystrix and P massoniana(MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH MIX PPM PCH MIX PPM F p F p F p

LP 668 plusmn 019 b 836 plusmn 069 a 484 plusmn 019 c 553 plusmn 043 b 921 plusmn 040 a 616 plusmn 023 b 3752 lt0001 093 0343 527 0013OClitter 19064 plusmn 1063 b 24859 plusmn 1848 a 25505 plusmn 1800 a 27757 plusmn 1885 b 2743 plusmn 1142 b 32175 plusmn 544 a 683 0004 2486 lt0001 226 0127TNlitter 1738 plusmn 078 a 1353 plusmn 106 b 1496 plusmn 062 ab 1295 plusmn 059 a 1357 plusmn 090 a 1189 plusmn 050 a 321 0058 1579 0001 445 0023CNlitter 1112 plusmn 095 b 1850 plusmn 081 a 1733 plusmn 181 a 2182 plusmn 223 b 2056 plusmn 156 b 2722 plusmn 096 a 767 0003 3890 lt0001 518 0013TPlitter 134 plusmn 008 a 050 plusmn 007 b 116 plusmn 005 a 119 plusmn 015 a 138 plusmn 015 a 131 plusmn 005 a 667 0005 1281 0002 1417 lt0001TKlitter 116 plusmn 016 a 152 plusmn 010 a 082 plusmn 009 b 107 plusmn 002 b 149 plusmn 013 a 088 plusmn 007 c 2084 lt0001 007 0796 029 0751

Different superscripts indicate significant differences among stand types in each season (p lt 005) LP (Mg haminus1) litter production OClitter (g kgminus1) litter organic carbon TNlitter (g kgminus1)litter total nitrogen CNlitter CN ratio of litter TPlitter (g kgminus1) litter total phosphorus TKlitter (g kgminus1) litter total potassium

Table 3 Soil physical and chemical properties (0ndash10 cm soil depth) in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM) plantation and mixedC hystrix and P massoniana (MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH Mixed PPM PCH Mixed PPM F p F p F p

BD 109 plusmn 003 b 125 plusmn 004 a 119 plusmn 002 a 103 plusmn 001 a 103 plusmn 003 a 104 plusmn 005 a 311 0063 3044 lt0001 297 0070WC 3966 plusmn 136 b 4359 plusmn 061 a 4017 plusmn 158 ab 4053 plusmn 107 c 4440 plusmn 084 b 5048 plusmn 141 a 1035 0001 1681 lt0001 1048 0001Tem 1682 plusmn 021 a 1550 plusmn 009 c 1617 plusmn 005 b 2628 plusmn 043 a 2598 plusmn 022 a 2541 plusmn 008 a 821 0002 283334 lt0001 431 0025pH 432 plusmn 002 a 419 plusmn 003 b 404 plusmn 005 c 402 plusmn 002 b 421 plusmn 003 a 390 plusmn 009 b 1440 lt0001 1370 0001 623 0007

SOC 3742 plusmn 108 b 4082 plusmn 022 b 5698 plusmn 189 a 5331 plusmn 194 a 4839 plusmn 200 a 5083 plusmn 054 a 2460 lt0001 2369 lt0001 2864 lt0001TN 163 plusmn 005 b 160 plusmn 006 b 216 plusmn 022 a 248 plusmn 011 a 213 plusmn 013 a 217 plusmn 014 a 248 0105 4408 lt0001 480 0018

SOCTN 2298 plusmn 059 b 2567 plusmn 103 b 2949 plusmn 159 a 2167 plusmn 133 a 2304 plusmn 162 a 2390 plusmn 202 a 457 0021 722 0013 114 0338TP 031 plusmn 004 c 042 plusmn 002 b 060 plusmn 004 a 052 plusmn 006 b 078 plusmn 004 a 054 plusmn 004 b 2186 lt0001 2889 lt0001 1795 lt0001AP 919 plusmn 058 b 1252 plusmn 069 a 1002 plusmn 030 b 1330 plusmn 040 a 1315 plusmn 057 a 998 plusmn 027 b 1670 lt0001 1525 0001 1029 0001TK 414 plusmn 015 b 545 plusmn 049 a 242 plusmn 016 c 540 plusmn 050 a 694 plusmn 082 a 338 plusmn 013 b 3226 lt0001 1034 0004 039 0680AK 2990 plusmn 149 b 4629 plusmn 096 a 2846 plusmn 108 b 4719 plusmn 228 a 5279 plusmn 365 a 2837 plusmn 144 b 5375 lt0001 2257 lt0001 927 0001

NO3minus-N 355 plusmn 024 a 185 plusmn 004 c 286 plusmn 022 b 406 plusmn 019 c 530 plusmn 009 b 579 plusmn 009 a 1096 lt0001 29106 lt0001 4547 lt0001

NH4+-N 2129 plusmn 101 a 2334 plusmn 113 a 2447 plusmn 137 a 4224 plusmn 162 a 4350 plusmn 166 a 3883 plusmn 247 a 075 0482 19616 lt0001 247 0105

AN 2734 plusmn 157 a 2519 plusmn 114 a 2485 plusmn 090 a 463 plusmn 155 a 488 plusmn 169 a 4462 plusmn 246 a 120 0319 24555 lt0001 117 0328CEC 4553 plusmn 327 c 5758 plusmn 411 b 8233 plusmn 194 a 5543 plusmn 202a 5446 plusmn 119 a 6224 plusmn 291 a 4514 lt0001 1216 0002 2168 lt0001

Different superscripts indicate significant differences among stand types in each season (p lt 005) BD (g cmminus1) bulk density WC () soil water content Tem (C) temperature SOC(g kgminus1) soil organic carbon TN (g kgminus1) total nitrogen TP (g kgminus1) total phosphorus AP (mg kgminus1) available phosphorus TK (g kgminus1) total potassium AK (mg kgminus1) availablepotassium NO3

minus-N (mg kgminus1) nitrate nitrogen NH4+-N (mg kgminus1) ammonium nitrogen AN (mg kgminus1) Available nitrogen CEC (cmol + kgminus1) cation exchange capacity

Forests 2019 10 879 9 of 20

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantlyaffected by stand type and MBN was also significantly affected by season and interaction betweenstand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a)In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantlyhigher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highestMBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOCand MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significantlevel in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOCand MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-coolseason (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season(Figure 3b) were significant

Forests 2019 10 x FOR PEER REVIEW 10 of 22

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantly affected by stand type and MBN was also significantly affected by season and interaction between stand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a) In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantly higher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highest MBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOC and MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significant level in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOC and MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-cool season (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season (Figure 3b) were significant

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBN MBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a) and wet-warm season (b)

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBNMBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005level by LSD multiple comparisons among stand types (n = 5)

Forests 2019 10 x FOR PEER REVIEW 10 of 22

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantly affected by stand type and MBN was also significantly affected by season and interaction between stand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a) In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantly higher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highest MBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOC and MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significant level in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOC and MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-cool season (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season (Figure 3b) were significant

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBN MBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a) and wet-warm season (b)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a)and wet-warm season (b)

Forests 2019 10 879 10 of 20

In the dry-cool season total PLFAs AMF PLFAs actinobacteria PLFAs bacteria PLFAs fungiPLFAs GP bacteria PLFAs and GP-to-GN PLFAs ratio were smaller in MIX than those in PPM andPCH On the other hand in the wet-warm season these PLFAs were smaller in PPM than thosein PCH and MIX (Table 4) Specifically the ratios of bacteria-to-fungi PLFA and GP-to-GN PLFAwere significantly higher in MIX than those in PPM and PCH in the wet-warm season These resultsindicated that the response of the soil microbial community structure to tree species mixture varied indifferent seasons Two-way ANOVAs showed that both stand type and season significantly affectedall the tested microbial variables while the interaction between stand type and season significantlyinfluenced total PLFAs bacteria PLFAs GP bacteria PLFAs Acti PLFAs bacteria-to-fungi PLFA andGP-to-GN PLFA (Table 4)

Across plantations and seasons bacteria PLFAs were the most abundant followed by fungi andactinobacteria PLFAs (Figure 4 and Table S3) It was worth noting that the relative abundance ofGN bacteria PLFAs was significantly higher in MIX than in other plantations in the dry-cool season(Figure 4) Furthermore the relative abundance of GP bacteria PLFAs was significantly higher andthat of fungi PLFAs was significantly lower in MIX than in other plantations in the wet-warm season

Forests 2019 10 x FOR PEER REVIEW 13 of 22

Across plantations and seasons bacteria PLFAs were the most abundant followed by fungi and actinobacteria PLFAs (Figure 4 and Table S3) It was worth noting that the relative abundance of GN bacteria PLFAs was significantly higher in MIX than in other plantations in the dry-cool season (Figure 4) Furthermore the relative abundance of GP bacteria PLFAs was significantly higher and that of fungi PLFAs was significantly lower in MIX than in other plantations in the wet-warm season

Figure 4 The relative abundance of PLFAs (mol ) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleaf plantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increased APH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increased urease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg) Carbon source utilization ability of the soil microbial community indicated by average well color development (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation (Figure 6)

Figure 4 The relative abundance of PLFAs (mol ) in three plantations in the dry-cool season (a) andwet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level byLSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 11 of 20

Table 4 Soil microbial community PLFAs content and structure (0ndash10 cm soil depth) in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM)plantation and mixed C hystrix and P massoniana (MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH MIX PPM PCH MIX PPM F p F p F p

TPLFAs 3767 plusmn 271 a 3255 plusmn 124 a 3796 plusmn 128 a 4373 plusmn 208 a 4001 plusmn 07 a 3478 plusmn 111 b 464 0020 646 0018 607 0007B 1579 plusmn 081 a 1306 plusmn 043 b 1490 plusmn 051 ab 1764 plusmn 072 a 1723 plusmn 042 a 1276 plusmn 041 b 1274 lt0001 768 0011 1556 lt0001

GP 945 plusmn 043 a 794 plusmn 029 b 918 plusmn 031 a 1066 plusmn 051 a 1155 plusmn 031 a 810 plusmn 031 b 817 0002 1709 lt0001 2030 lt0001GN 591 plusmn 076 a 590 plusmn 016 a 553 plusmn 022 a 619 plusmn 025 a 455 plusmn 037 b 411 plusmn 020 b 526 0013 702 0014 313 0062

F 498 plusmn 034 a 375 plusmn 022 b 424 plusmn 028 ab 492 plusmn 035 a 296 plusmn 043 b 345 plusmn 021 b 1356 lt0001 448 0045 091 0416AMF 104 plusmn 003 a 075 plusmn 003 b 075 plusmn 004 b 085 plusmn 007 a 044 plusmn 008 b 048 plusmn 003 b 2822 lt0001 3483 lt0001 056 0577Acti 426 plusmn 037 ab 377 plusmn 021 b 486 plusmn 020 a 531 plusmn 019 a 487 plusmn 012 b 458 plusmn 010 b 273 0085 1236 0002 650 0006BF 319 plusmn 011 a 351 plusmn 013 a 356 plusmn 017 a 362 plusmn 014 b 635 plusmn 094 a 377 plusmn 033 b 750 0003 1131 0003 598 0008

GPGN 174 plusmn 029 b 135 plusmn 003 b 166 plusmn 004 a 173 plusmn 008 b 263 plusmn 028 a 198 plusmn 011 b 107 0359 1376 0001 733 0003

Different superscripts indicate significant differences among stand types in each season (p lt 005) TPLFAs (nmol gminus1 soil) total PLFAs B (nmol gminus1 soil) bacteria PLFAs GP (nmol gminus1

soil) gram-positive bacteria PLFAs GN (nmol gminus1 soil) gram-negative bacteria PLFAs F (nmol gminus1 soil) fungi PLFAs Acti (nmol gminus1 soil) actinobacteria PLFAs AMF (nmol gminus1 soil)arbuscular mycorrhizal fungi PLFAs BF bacteria-to-fungi PLFA ratio GPGN GP-to-GN ratio

Forests 2019 10 879 12 of 20

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleafplantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increasedAPH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increasedurease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg)Carbon source utilization ability of the soil microbial community indicated by average well colordevelopment (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation(Figure 6)Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in threeplantations in different seasons Error bars are standard error Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 13 of 20

Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in threeplantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in thedry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons(F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pHTK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-coolseason in which SOC and TBB were the major correlates of changes in soil microbial composition(Table S5) A total of 11 biotic and abiotic factors including NO3

minus-N TBB pH TKlitter HBB AP AKTP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warmseason in which NO3

minus-N TBB pH and TKlitter were the major correlates of changes in soil microbialcomposition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

37 Relationship between Soil Microbial Structure and Functions

Soil enzyme activity and soil microbial carbon source utilization ability are both important soilmicrobial functions The first two RDA axes explained 4948 of the variation in soil enzyme activityand carbon utilization ability in the dry-cool season (F = 24 p = 0006 Figure 7b) and 6891 of thevariation in the wet-warm season (F = 64 p = 0002 Figure 7d) AMF and GP were significantlycorrelated with soil microbial functions in the dry-cool season (Table S6) Total bacteria GP bacteriaGN bacteria and actinobacteria were significantly correlated with soil microbial functions in thewet-warm season in which total bacteria GP bacteria and GN bacteria were the major correlates ofchanges of soil enzyme activity (Table S6)

Forests 2019 10 879 14 of 20

Forests 2019 10 x FOR PEER REVIEW 15 of 22

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in the dry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons (F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pH TK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-cool season in which SOC and TBB were the major correlates of changes in soil microbial composition (Table S5) A total of 11 biotic and abiotic factors including NO3minus-N TBB pH TKlitter HBB AP AK TP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warm season in which NO3minus-N TBB pH and TKlitter were the major correlates of changes in soil microbial composition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160 were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant biotic and abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot of soil microbial community structure and soil microbial functions (bd) Above the label represents a significant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowground biomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soil total potassium NO3ndash-N soil nitrate nitrogen HBB herb belowground biomass AP soil available phosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil water

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant bioticand abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot ofsoil microbial community structure and soil microbial functions (bd) Above the label represents asignificant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowgroundbiomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soiltotal potassium NO3

minus-N soil nitrate nitrogen HBB herb belowground biomass AP soil availablephosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil watercontent LP litter production TKlitter litter total potassium GP gram-positive bacteria GN gramnegative bacteria AMF arbuscular mycorrhizal fungi AG α-Glucosidase BG β-14-glucosidaseCB β-Cellobiosidase APH acid phosphatase NAG β-14-N-acetylglucosaminidase PHO phenoloxidase PO peroxidase AWCD average well color development of carbon source metabolism at192 h incubation

4 Discussion

41 Coniferous-Broadleaf Mixture Significantly Increase Stand Biomass and Litter Production

The results of this study indicated that admixing of coniferous and broadleaf tree speciessignificantly increased stand biomass and litter production after 34 years of afforestation in bothdry-cool and wet-warm seasons (Figure 1 Table 2) This may be a consequence of a greater growth of

Forests 2019 10 879 15 of 20

C hystrix in the mixed stands These results confirmed previous findings conducted in our experimentsite measured when the plantations were 26-year-old and 28-year-old respectively [716] A bulkof studies revealed that mixed-species management could affect tree growth stand structure andenvironment [145253] Mixed forests can develop stratified canopies due to niche differences amongdifferent species In the present study trees of the faster growing broad-leaf species (C hystrix) weretaller had a larger DBH and thus formed an upper canopy stratum in the mixed stands (Table 1)While trees of the shorter species (P massoniana) formed a lower stratum with lower DBH and treeheight and the reduced amount of light transmitted through the upper canopy contributed to theirslower growth The dominant tree species within the stand can absorb more sunlight and use itmore efficiently for growth [54] and thus contribute to higher tree biomass and litter productionin mixed stands In addition stratified canopies in mixed plantations can help to maintain levelsof understory species richness and biomass compared with pure plantations due to relatively highlight availabilities [5556] This may be why the mixed stand had higher shrub and herb biomass inour study

Specifically in the present study the broad-leaf species attained greater heights and diameters inmixed stands while masson pine had reduced diameter and height growth in mixed-species comparedto mono-species stands (Table 1) The severe suppression of P massoniana by the faster growingC hystrix may be attributed to differences in the light requirements of the two species [7] The growthpattern of C hystrix and P massoniana in mixed stands in our study (34-year-old) were differed fromwhen they were 26-years-old in which the growth of C hystrix and P massoniana in mixed standswere both greater than in the corresponding mono-species stands [7] This suggests that effects of thetree species mixture could change over time and long-term investigation of mixed forests is of greatsignificance to reveal mixed effects

42 Coniferous-Broadleaf Mixture Significantly Increase Soil Microbial Biomass

The changes of tree species and composition may significantly affect the amount of organic matterinput in soil [57] As the most active part in soil organic matter (SOM) soil microbial biomass isimportant in nutrient cycling and release participating in the circulation of critical elements such ascarbon and nitrogen [23] Because of the quantity and quality of the bulk of SOM changes slowly andthe changes of SOM are difficult to measure due to the spatial heterogeneity of soils soil microbialbiomass and in particular MBC-to-SOC and MBN-to-TN ratio have been suggested as indicators ofthe state and modification of SOM [58ndash60] and as an indicator for carbon (nitrogen) availability insoil [6162] The concentrations of SOC TN and MBC and MBN measured in our study were affectedby stand type and seasons (Table 3 and Table S2 Figure 2) In the dry cool season although the SOCcontent in mixed stands did not significantly differ with that in PCH but significantly lower than thatin PPM the MBC concentration in mixed stands was markedly higher than that in the correspondingmono-species stands Likewise although the TN content in mixed stands was smaller than that in PCHand PPM stands the MBN content in mixed stands was markedly higher than that in the correspondingmono-species stands in the wet-warn season Previous studies found that higher soil microbial biomasscorrelated with higher SOC and TN content that sustain microbial metabolism [2163] However inour study the lower SOC and TN content in MIX was able to maintain a higher microbial biomass thansites where SOC was more abundant Presumably the quality of SOC could explain such a trend [64]and merit further investigation

In this study MBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands(Figure 2) suggesting higher carbon and nitrogen availability in soil and higher soil quality for thegrowth of soil microorganisms by coniferous-broadleaf mixture [21] In addition the effects of admixingon soil microbial biomass MBC-to-SOC and MBN-to-TN ratio were non-additive suggesting theinteraction of P massoniana and C hystrix can have a synergistic effect on improving soil carbon andnitrogen availability (Figure 3) These findings are consistent with many previous studies that havedetermined the effects of tree species mixture on soil microbial biomass [2165] These results may be

Forests 2019 10 879 16 of 20

contributed to (1) greater amount and more diverse organic matter input via litter and rhizodepositionproducts in the mixture of two contrasting tree species [21] (2) the higher use efficiency of soil organiccarbon and nitrogen by microorganisms in mixed stands [66]

We also sought to determine the admixing effects on composition and abundance of soil microbialcommunity with community-level PLFA profiles Surprisingly there were not significant admixingeffects on PLFA content of the major microbial functional groups such as bacteria fungi AMF andactinobacteria (Table 4) However tree species mixture significantly increased the abundance of GNbacteria in the dry-cool season while markedly increased the abundance of GP bacteria but decreasedthe abundance of fungi in the wet-warn season (Figure 4) It seemed that the dominated GN bacteria inthe dry-cool season and GP bacteria in the wet-warn season would correlate with the higher availabilityof soil organic carbon and nitrogen in mixed stands [6768] These correlations are complicated andfurther investigation is required to reveal the underlying mechanisms

43 Effects of Coniferous-Broadleaf Mixture on Soil Microbial Functions

Soil microorganisms excrete soil enzymes to drive nutrient mineralization and litterdecomposition [64] Therefore soil extracellular enzyme activities may be related to the carbonnitrogen and phosphorus cycling in soil [6970] In the present study we found positive admixingeffects on the activities of APH and NAG in the dry-cool season (Figure 5de) On the other handpositive admixing effects on the activities of urease and peroxidase while negative admixing effectson APH was detected in the wet-warn season (Figure 5dfg) This result suggests that the mixtureof C hystrix and P massoniana can accelerate the mineralization rates of soil organic phosphorus andnitrogen These findings were consistent with previous study [67]

The community-level physiological profiling (CLPP) is widely used to characterize microbialcommunities [71ndash73] The average well color development (AWCD) in the CLPP approach reflectsthe ability of soil microorganisms to utilize carbon sources and their metabolic activity The higherAWCD represents the stronger metabolic activity of soil microorganisms [71] In this present study wedetected a significantly positive admixing effect on carbon source utilization ability of the soil microbialcommunities in both dry-cool and wet-warn seasons after 144 h of incubation (Figure 6) Moreover theRDA analysis revealed that carbon source utilization ability of the soil microbial communities werepositive related with APH in the dry-cool season while related with urease and peroxidase activitiesin the wet-warn season (Figure 7) As a whole the higher soil microbial functions in mixed stands arelikely the results of the effects of AMF and GP bacteria in the dry-cool season and the effects of bacteriaincluding GP and GN bacteria the wet-warn season (Table S6)

5 Conclusions

In our study we measured significantly higher tree shrub and herb biomass and litter productionin the coniferous-broadleaf mixed plantation after 34 years of afforestation As expected MBC MBNMBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands suggesting highercarbon and nitrogen availability in soil and higher soil quality for the growth of soil microorganisms inour coniferous-broadleaf mixed stand In addition coniferous-broadleaf mixture can increase carbonsource utilization ability of soil microbial community and accelerate the mineralization rates of soilorganic phosphorus and nitrogen by increasing acid phosphatase β-14-N-acetylglucosaminidaseand urease activities The higher soil microbial biomass and activities are likely the result of greateramount and more diverse organic matter input via litter and rhizodeposition products and the higheruse efficiency of soil organic carbon and nitrogen by microorganisms in mixed stands Howeverthe correlations between soil dominated microbial functional groups and the higher availabilityof soil organic carbon and nitrogen along with the quality of SOC in mixed stands merit furtherinvestigation Our results highlight that establishment of coniferous-broadleaf mixed forests maybe a good management practice as coniferous-broadleaf mixture could accumulate higher stand

Forests 2019 10 879 17 of 20

biomass return more litter and increase soil microbial biomass and functions over the long term insubtropical China

Supplementary Materials The following are available online at httpwwwmdpicom1999-49071010879s1Table S1 Effects of stand type season and their interactions on stand biomass Table S2 Effects of stand type seasonand their interactions on soil microbial structure Table S3 Effects of stand type season and their interactionson relative abundance of the soil microbial community PLFAs Table S4 Effects of stand type season and theirinteractions on soil enzyme activity Table S5 Marginal and conditional effects of biotic and abiotic factors on soilmicrobial composition obtained from the summary of forward selection in Redundancy analysis (RDA) Table S6Marginal and conditional effects of soil microbial functional groups on soil enzyme activity and soil microbialcarbon source utilization ability obtained from the summary of forward selection in Redundancy analysis (RDA)

Author Contributions WW collected data made experiment analysis formal data and drafted the manuscriptYW designed the work XZ revised the manuscript and participated in analyzing the experiment data HZ YYXH and XL ZQ YL LY HL participated in collecting the experiment data

Funding This research was funded by National Natural Science Foundation of China (31860171 3146012131560201) the Guangxi Key Research and Development Program (2018AB40007) Innovation Project of GuangxiGraduate Education (YCBZ2017011) and the Guangxi Universities Science and technology (201201ZD001)

Acknowledgments We gratefully acknowledge the supports in field sampling by the Experimental Center ofTropical Forestry Chinese Academy of Forestry A special acknowledgment is given to Patrick M Finnegan for hiswriting suggestions

Conflicts of Interest The authors declare no conflicts of interest

References

1 Zhang X Liu S Huang Y Fu S Wang J Ming A Li X Yao M Li H Tree species mixture inhibits soilorganic carbon mineralization accompanied by decreased r-selected bacteria Plant Soil 2018 431 203ndash216[CrossRef]

2 Pan Y Birdsey RA Fang J Houghton R Kauppi PE Kurz WA Phillips OL Shvidenko ALewis SL Canadell JG et al A large and persistent carbon sink in the worldrsquos forests Science 2011 333988ndash993 [CrossRef] [PubMed]

3 Chen X Ju Q Lin K Development status issues and countermeasures of Chinarsquo s plantation World For Res2014 27 54ndash59

4 Wang H Lencinas MV Ross Friedman C Wang X Qiu J Understory plant diversity assessment ofEucalyptus plantations over three vegetation types in Yunnan China New For 2011 42 101ndash116 [CrossRef]

5 Sang PM Lamb D Bonner M Schmidt S Carbon sequestration and soil fertility of tropical tree plantationsand secondary forest established on degraded land Plant Soil 2013 362 187ndash200 [CrossRef]

6 Li C Zhou X Wen Y Zhu H Qin Z Li X You Y Huang X Effects of high-generation ratations ofEucalyptus on undergrowth soil fertility and enzyme activities Guangxi Sci 2019 26 176ndash186

7 You Y Huang X Zhu H Liu S Liang H Wen Y Wang H Cai D Ye D Positive interactions betweenPinus massoniana and Castanopsis hystrix species in the uneven-aged mixed plantations can produce moreecosystem carbon in subtropical China For Ecol Manag 2018 410 193ndash200 [CrossRef]

8 Huang X Liu S You Y Wen Y Wang H Wang J Microbial community and associated enzymes activityinfluence soil carbon chemical composition in Eucalyptus urophylla plantation with mixing N2-fixing speciesin sub-tropical China Plant Soil 2017 414 199ndash212 [CrossRef]

9 Kelty MJ The role of species mixtures in plantation forestry For Ecol Manag 2006 233 195ndash204 [CrossRef]10 Bauhus J Khanna P Menden N Aboveground and belowground interactions in mixed plantations of

Eucalyptus globulus and Acacia mearnsii Can J For Res 2000 30 1886ndash1894 [CrossRef]11 Vandermeer J The Ecology of Intercropping Cambridge University Press New York NY USA 1989 p 23712 Forrester DI The spatial and temporal dynamics of species interactions in mixed-species forests From

pattern to process For Ecol Manag 2014 312 282ndash292 [CrossRef]13 Forrester DI Bauhus J A review of processes behind diversity-productivity relationships in forests

Curr For Rep 2016 2 45ndash61 [CrossRef]14 Wang H Liu S Wang J You Y Yang Y Shi Z Huang X Zheng L Li Z Ming A et al Mixed-species

plantation with Pinus massoniana and Castanopsis hystrix accelerates C loss in recalcitrant coniferous litter butslows C loss in labile broadleaf litter in southern China For Ecol Manag 2018 422 207ndash213 [CrossRef]

Forests 2019 10 879 6 of 20

The forward selection procedure in RDA based on Monte Carlo permutation with 499 iterations wasperformed to determine the dominant variables that influenced the composition of individual PLFAsand microbial functions A significance level of p lt 005 was used in all analyses

3 Results

31 Stand Biomass

Stand type had significant effects on the -above- and below-ground biomass of trees shrubs andherbs (Table S1 Figure 1) TAB TBB shrub aboveground biomass (SAB) shrub belowground biomass(SBB) and herb aboveground biomass (HAB) in MIX were significantly higher than those in PCHand PPM in both the dry-cool and wet-warm seasons (Figure 1andashe) Except for HAB season had nosignificant effects on the tested biomass variables Moreover significant interaction effects betweenstand type and season only found on HAB and HBB These results supported our first hypothesis thattree species mixture increased stand biomass

Forests 2019 10 x FOR PEER REVIEW 6 of 22

biogeochemical circulation of carbon nitrogen and phosphorus All data were log-transformed before in the analyses The forward selection procedure in RDA based on Monte Carlo permutation with 499 iterations was performed to determine the dominant variables that influenced the composition of individual PLFAs and microbial functions A significance level of p lt 005 was used in all analyses

3 Results

31 Stand Biomass

Stand type had significant effects on the -above- and below-ground biomass of trees shrubs and herbs (Table S1 Figure 1) TAB TBB shrub aboveground biomass (SAB) shrub belowground biomass (SBB) and herb aboveground biomass (HAB) in MIX were significantly higher than those in PCH and PPM in both the dry-cool and wet-warm seasons (Figure 1andashe) Except for HAB season had no significant effects on the tested biomass variables Moreover significant interaction effects between stand type and season only found on HAB and HBB These results supported our first hypothesis that tree species mixture increased stand biomass

Figure 1 Biomass of trees (ab) shrubs (cd) and herbs (ef) in three plantations in different seasons Different letters above the bars denote significant differences at p lt 005 level by LSD (least significant difference) multiple comparisons among stand types (n = 5)

32 Litter Production and Quality

Stand type the interaction between stand type and season significantly influenced litter production at our study sites (Table 2) The MIX had significantly higher litter production than PCH and PPM in both seasons OClitter and CNlitter were significantly affected by both stand type and season

Figure 1 Biomass of trees (ab) shrubs (cd) and herbs (ef) in three plantations in different seasonsDifferent letters above the bars denote significant differences at p lt 005 level by LSD (least significantdifference) multiple comparisons among stand types (n = 5)

Forests 2019 10 879 7 of 20

32 Litter Production and Quality

Stand type the interaction between stand type and season significantly influenced litter productionat our study sites (Table 2) The MIX had significantly higher litter production than PCH and PPMin both seasons OClitter and CNlitter were significantly affected by both stand type and season withhigher value in the wet-warm season than in the dry-cool season in all plantations An opposite trendwas observed in TNlitter TPlitter was significantly affected by stand type season and their interactionwith MIX had significantly lower concentration than PCH and PPM in the dry-cool season TKlitter wassignificantly affected by stand type with MIX had significantly higher concentration than PCH andPPM in the wet-warm season These results partially supported our first hypothesis that tree speciesmixture increased litter production although the increases of litter quality in mixed plantations werenot significant

33 Soil Physical and Chemical Properties

In the dry-cool season the mixed plantations had the highest soil bulk density (BD) and soil watercontent (WC) and the lowest soil temperature and medium pH compared to the pure plantations(Table 3) In the wet-warm season MIX had the medium BD WC and soil temperature and the highestpH compared to PPM and PCH

The PPM plantations had the highest SOC TN TP SOC-to-TN ratio NH4+-N and CEC while

MIX had the highest AP TK and AK and PCH had the highest NO3minus-N and AN in the dry-cool season

(Table 3) On the other hand the PCH had the highest SOC TN and AP while MIX had the highestTP TK AK NO3

minus-N and AN and PPM had the highest SOC-to-TN ratio NO3minus-N and CEC in the

wet-warm seasonTwo-way ANOVAs indicated that season significantly affected all the tested soil variables (Table 3)

In addition the stand type influenced nearly all soil variables except BD TN NH4+-N and AN

Interactions between stand type and season significantly influenced WC temperature pH SOC TNTP AP AK NO3

minus-N and CEC

Forests 2019 10 879 8 of 20

Table 2 The quantity and quality of litter in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM) plantation and mixed C hystrix and P massoniana(MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH MIX PPM PCH MIX PPM F p F p F p

LP 668 plusmn 019 b 836 plusmn 069 a 484 plusmn 019 c 553 plusmn 043 b 921 plusmn 040 a 616 plusmn 023 b 3752 lt0001 093 0343 527 0013OClitter 19064 plusmn 1063 b 24859 plusmn 1848 a 25505 plusmn 1800 a 27757 plusmn 1885 b 2743 plusmn 1142 b 32175 plusmn 544 a 683 0004 2486 lt0001 226 0127TNlitter 1738 plusmn 078 a 1353 plusmn 106 b 1496 plusmn 062 ab 1295 plusmn 059 a 1357 plusmn 090 a 1189 plusmn 050 a 321 0058 1579 0001 445 0023CNlitter 1112 plusmn 095 b 1850 plusmn 081 a 1733 plusmn 181 a 2182 plusmn 223 b 2056 plusmn 156 b 2722 plusmn 096 a 767 0003 3890 lt0001 518 0013TPlitter 134 plusmn 008 a 050 plusmn 007 b 116 plusmn 005 a 119 plusmn 015 a 138 plusmn 015 a 131 plusmn 005 a 667 0005 1281 0002 1417 lt0001TKlitter 116 plusmn 016 a 152 plusmn 010 a 082 plusmn 009 b 107 plusmn 002 b 149 plusmn 013 a 088 plusmn 007 c 2084 lt0001 007 0796 029 0751

Different superscripts indicate significant differences among stand types in each season (p lt 005) LP (Mg haminus1) litter production OClitter (g kgminus1) litter organic carbon TNlitter (g kgminus1)litter total nitrogen CNlitter CN ratio of litter TPlitter (g kgminus1) litter total phosphorus TKlitter (g kgminus1) litter total potassium

Table 3 Soil physical and chemical properties (0ndash10 cm soil depth) in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM) plantation and mixedC hystrix and P massoniana (MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH Mixed PPM PCH Mixed PPM F p F p F p

BD 109 plusmn 003 b 125 plusmn 004 a 119 plusmn 002 a 103 plusmn 001 a 103 plusmn 003 a 104 plusmn 005 a 311 0063 3044 lt0001 297 0070WC 3966 plusmn 136 b 4359 plusmn 061 a 4017 plusmn 158 ab 4053 plusmn 107 c 4440 plusmn 084 b 5048 plusmn 141 a 1035 0001 1681 lt0001 1048 0001Tem 1682 plusmn 021 a 1550 plusmn 009 c 1617 plusmn 005 b 2628 plusmn 043 a 2598 plusmn 022 a 2541 plusmn 008 a 821 0002 283334 lt0001 431 0025pH 432 plusmn 002 a 419 plusmn 003 b 404 plusmn 005 c 402 plusmn 002 b 421 plusmn 003 a 390 plusmn 009 b 1440 lt0001 1370 0001 623 0007

SOC 3742 plusmn 108 b 4082 plusmn 022 b 5698 plusmn 189 a 5331 plusmn 194 a 4839 plusmn 200 a 5083 plusmn 054 a 2460 lt0001 2369 lt0001 2864 lt0001TN 163 plusmn 005 b 160 plusmn 006 b 216 plusmn 022 a 248 plusmn 011 a 213 plusmn 013 a 217 plusmn 014 a 248 0105 4408 lt0001 480 0018

SOCTN 2298 plusmn 059 b 2567 plusmn 103 b 2949 plusmn 159 a 2167 plusmn 133 a 2304 plusmn 162 a 2390 plusmn 202 a 457 0021 722 0013 114 0338TP 031 plusmn 004 c 042 plusmn 002 b 060 plusmn 004 a 052 plusmn 006 b 078 plusmn 004 a 054 plusmn 004 b 2186 lt0001 2889 lt0001 1795 lt0001AP 919 plusmn 058 b 1252 plusmn 069 a 1002 plusmn 030 b 1330 plusmn 040 a 1315 plusmn 057 a 998 plusmn 027 b 1670 lt0001 1525 0001 1029 0001TK 414 plusmn 015 b 545 plusmn 049 a 242 plusmn 016 c 540 plusmn 050 a 694 plusmn 082 a 338 plusmn 013 b 3226 lt0001 1034 0004 039 0680AK 2990 plusmn 149 b 4629 plusmn 096 a 2846 plusmn 108 b 4719 plusmn 228 a 5279 plusmn 365 a 2837 plusmn 144 b 5375 lt0001 2257 lt0001 927 0001

NO3minus-N 355 plusmn 024 a 185 plusmn 004 c 286 plusmn 022 b 406 plusmn 019 c 530 plusmn 009 b 579 plusmn 009 a 1096 lt0001 29106 lt0001 4547 lt0001

NH4+-N 2129 plusmn 101 a 2334 plusmn 113 a 2447 plusmn 137 a 4224 plusmn 162 a 4350 plusmn 166 a 3883 plusmn 247 a 075 0482 19616 lt0001 247 0105

AN 2734 plusmn 157 a 2519 plusmn 114 a 2485 plusmn 090 a 463 plusmn 155 a 488 plusmn 169 a 4462 plusmn 246 a 120 0319 24555 lt0001 117 0328CEC 4553 plusmn 327 c 5758 plusmn 411 b 8233 plusmn 194 a 5543 plusmn 202a 5446 plusmn 119 a 6224 plusmn 291 a 4514 lt0001 1216 0002 2168 lt0001

Different superscripts indicate significant differences among stand types in each season (p lt 005) BD (g cmminus1) bulk density WC () soil water content Tem (C) temperature SOC(g kgminus1) soil organic carbon TN (g kgminus1) total nitrogen TP (g kgminus1) total phosphorus AP (mg kgminus1) available phosphorus TK (g kgminus1) total potassium AK (mg kgminus1) availablepotassium NO3

minus-N (mg kgminus1) nitrate nitrogen NH4+-N (mg kgminus1) ammonium nitrogen AN (mg kgminus1) Available nitrogen CEC (cmol + kgminus1) cation exchange capacity

Forests 2019 10 879 9 of 20

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantlyaffected by stand type and MBN was also significantly affected by season and interaction betweenstand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a)In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantlyhigher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highestMBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOCand MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significantlevel in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOCand MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-coolseason (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season(Figure 3b) were significant

Forests 2019 10 x FOR PEER REVIEW 10 of 22

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantly affected by stand type and MBN was also significantly affected by season and interaction between stand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a) In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantly higher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highest MBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOC and MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significant level in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOC and MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-cool season (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season (Figure 3b) were significant

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBN MBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a) and wet-warm season (b)

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBNMBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005level by LSD multiple comparisons among stand types (n = 5)

Forests 2019 10 x FOR PEER REVIEW 10 of 22

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantly affected by stand type and MBN was also significantly affected by season and interaction between stand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a) In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantly higher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highest MBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOC and MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significant level in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOC and MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-cool season (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season (Figure 3b) were significant

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBN MBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a) and wet-warm season (b)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a)and wet-warm season (b)

Forests 2019 10 879 10 of 20

In the dry-cool season total PLFAs AMF PLFAs actinobacteria PLFAs bacteria PLFAs fungiPLFAs GP bacteria PLFAs and GP-to-GN PLFAs ratio were smaller in MIX than those in PPM andPCH On the other hand in the wet-warm season these PLFAs were smaller in PPM than thosein PCH and MIX (Table 4) Specifically the ratios of bacteria-to-fungi PLFA and GP-to-GN PLFAwere significantly higher in MIX than those in PPM and PCH in the wet-warm season These resultsindicated that the response of the soil microbial community structure to tree species mixture varied indifferent seasons Two-way ANOVAs showed that both stand type and season significantly affectedall the tested microbial variables while the interaction between stand type and season significantlyinfluenced total PLFAs bacteria PLFAs GP bacteria PLFAs Acti PLFAs bacteria-to-fungi PLFA andGP-to-GN PLFA (Table 4)

Across plantations and seasons bacteria PLFAs were the most abundant followed by fungi andactinobacteria PLFAs (Figure 4 and Table S3) It was worth noting that the relative abundance ofGN bacteria PLFAs was significantly higher in MIX than in other plantations in the dry-cool season(Figure 4) Furthermore the relative abundance of GP bacteria PLFAs was significantly higher andthat of fungi PLFAs was significantly lower in MIX than in other plantations in the wet-warm season

Forests 2019 10 x FOR PEER REVIEW 13 of 22

Across plantations and seasons bacteria PLFAs were the most abundant followed by fungi and actinobacteria PLFAs (Figure 4 and Table S3) It was worth noting that the relative abundance of GN bacteria PLFAs was significantly higher in MIX than in other plantations in the dry-cool season (Figure 4) Furthermore the relative abundance of GP bacteria PLFAs was significantly higher and that of fungi PLFAs was significantly lower in MIX than in other plantations in the wet-warm season

Figure 4 The relative abundance of PLFAs (mol ) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleaf plantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increased APH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increased urease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg) Carbon source utilization ability of the soil microbial community indicated by average well color development (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation (Figure 6)

Figure 4 The relative abundance of PLFAs (mol ) in three plantations in the dry-cool season (a) andwet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level byLSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 11 of 20

Table 4 Soil microbial community PLFAs content and structure (0ndash10 cm soil depth) in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM)plantation and mixed C hystrix and P massoniana (MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH MIX PPM PCH MIX PPM F p F p F p

TPLFAs 3767 plusmn 271 a 3255 plusmn 124 a 3796 plusmn 128 a 4373 plusmn 208 a 4001 plusmn 07 a 3478 plusmn 111 b 464 0020 646 0018 607 0007B 1579 plusmn 081 a 1306 plusmn 043 b 1490 plusmn 051 ab 1764 plusmn 072 a 1723 plusmn 042 a 1276 plusmn 041 b 1274 lt0001 768 0011 1556 lt0001

GP 945 plusmn 043 a 794 plusmn 029 b 918 plusmn 031 a 1066 plusmn 051 a 1155 plusmn 031 a 810 plusmn 031 b 817 0002 1709 lt0001 2030 lt0001GN 591 plusmn 076 a 590 plusmn 016 a 553 plusmn 022 a 619 plusmn 025 a 455 plusmn 037 b 411 plusmn 020 b 526 0013 702 0014 313 0062

F 498 plusmn 034 a 375 plusmn 022 b 424 plusmn 028 ab 492 plusmn 035 a 296 plusmn 043 b 345 plusmn 021 b 1356 lt0001 448 0045 091 0416AMF 104 plusmn 003 a 075 plusmn 003 b 075 plusmn 004 b 085 plusmn 007 a 044 plusmn 008 b 048 plusmn 003 b 2822 lt0001 3483 lt0001 056 0577Acti 426 plusmn 037 ab 377 plusmn 021 b 486 plusmn 020 a 531 plusmn 019 a 487 plusmn 012 b 458 plusmn 010 b 273 0085 1236 0002 650 0006BF 319 plusmn 011 a 351 plusmn 013 a 356 plusmn 017 a 362 plusmn 014 b 635 plusmn 094 a 377 plusmn 033 b 750 0003 1131 0003 598 0008

GPGN 174 plusmn 029 b 135 plusmn 003 b 166 plusmn 004 a 173 plusmn 008 b 263 plusmn 028 a 198 plusmn 011 b 107 0359 1376 0001 733 0003

Different superscripts indicate significant differences among stand types in each season (p lt 005) TPLFAs (nmol gminus1 soil) total PLFAs B (nmol gminus1 soil) bacteria PLFAs GP (nmol gminus1

soil) gram-positive bacteria PLFAs GN (nmol gminus1 soil) gram-negative bacteria PLFAs F (nmol gminus1 soil) fungi PLFAs Acti (nmol gminus1 soil) actinobacteria PLFAs AMF (nmol gminus1 soil)arbuscular mycorrhizal fungi PLFAs BF bacteria-to-fungi PLFA ratio GPGN GP-to-GN ratio

Forests 2019 10 879 12 of 20

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleafplantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increasedAPH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increasedurease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg)Carbon source utilization ability of the soil microbial community indicated by average well colordevelopment (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation(Figure 6)Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in threeplantations in different seasons Error bars are standard error Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 13 of 20

Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in threeplantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in thedry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons(F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pHTK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-coolseason in which SOC and TBB were the major correlates of changes in soil microbial composition(Table S5) A total of 11 biotic and abiotic factors including NO3

minus-N TBB pH TKlitter HBB AP AKTP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warmseason in which NO3

minus-N TBB pH and TKlitter were the major correlates of changes in soil microbialcomposition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

37 Relationship between Soil Microbial Structure and Functions

Soil enzyme activity and soil microbial carbon source utilization ability are both important soilmicrobial functions The first two RDA axes explained 4948 of the variation in soil enzyme activityand carbon utilization ability in the dry-cool season (F = 24 p = 0006 Figure 7b) and 6891 of thevariation in the wet-warm season (F = 64 p = 0002 Figure 7d) AMF and GP were significantlycorrelated with soil microbial functions in the dry-cool season (Table S6) Total bacteria GP bacteriaGN bacteria and actinobacteria were significantly correlated with soil microbial functions in thewet-warm season in which total bacteria GP bacteria and GN bacteria were the major correlates ofchanges of soil enzyme activity (Table S6)

Forests 2019 10 879 14 of 20

Forests 2019 10 x FOR PEER REVIEW 15 of 22

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in the dry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons (F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pH TK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-cool season in which SOC and TBB were the major correlates of changes in soil microbial composition (Table S5) A total of 11 biotic and abiotic factors including NO3minus-N TBB pH TKlitter HBB AP AK TP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warm season in which NO3minus-N TBB pH and TKlitter were the major correlates of changes in soil microbial composition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160 were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant biotic and abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot of soil microbial community structure and soil microbial functions (bd) Above the label represents a significant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowground biomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soil total potassium NO3ndash-N soil nitrate nitrogen HBB herb belowground biomass AP soil available phosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil water

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant bioticand abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot ofsoil microbial community structure and soil microbial functions (bd) Above the label represents asignificant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowgroundbiomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soiltotal potassium NO3

minus-N soil nitrate nitrogen HBB herb belowground biomass AP soil availablephosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil watercontent LP litter production TKlitter litter total potassium GP gram-positive bacteria GN gramnegative bacteria AMF arbuscular mycorrhizal fungi AG α-Glucosidase BG β-14-glucosidaseCB β-Cellobiosidase APH acid phosphatase NAG β-14-N-acetylglucosaminidase PHO phenoloxidase PO peroxidase AWCD average well color development of carbon source metabolism at192 h incubation

4 Discussion

41 Coniferous-Broadleaf Mixture Significantly Increase Stand Biomass and Litter Production

The results of this study indicated that admixing of coniferous and broadleaf tree speciessignificantly increased stand biomass and litter production after 34 years of afforestation in bothdry-cool and wet-warm seasons (Figure 1 Table 2) This may be a consequence of a greater growth of

Forests 2019 10 879 15 of 20

C hystrix in the mixed stands These results confirmed previous findings conducted in our experimentsite measured when the plantations were 26-year-old and 28-year-old respectively [716] A bulkof studies revealed that mixed-species management could affect tree growth stand structure andenvironment [145253] Mixed forests can develop stratified canopies due to niche differences amongdifferent species In the present study trees of the faster growing broad-leaf species (C hystrix) weretaller had a larger DBH and thus formed an upper canopy stratum in the mixed stands (Table 1)While trees of the shorter species (P massoniana) formed a lower stratum with lower DBH and treeheight and the reduced amount of light transmitted through the upper canopy contributed to theirslower growth The dominant tree species within the stand can absorb more sunlight and use itmore efficiently for growth [54] and thus contribute to higher tree biomass and litter productionin mixed stands In addition stratified canopies in mixed plantations can help to maintain levelsof understory species richness and biomass compared with pure plantations due to relatively highlight availabilities [5556] This may be why the mixed stand had higher shrub and herb biomass inour study

Specifically in the present study the broad-leaf species attained greater heights and diameters inmixed stands while masson pine had reduced diameter and height growth in mixed-species comparedto mono-species stands (Table 1) The severe suppression of P massoniana by the faster growingC hystrix may be attributed to differences in the light requirements of the two species [7] The growthpattern of C hystrix and P massoniana in mixed stands in our study (34-year-old) were differed fromwhen they were 26-years-old in which the growth of C hystrix and P massoniana in mixed standswere both greater than in the corresponding mono-species stands [7] This suggests that effects of thetree species mixture could change over time and long-term investigation of mixed forests is of greatsignificance to reveal mixed effects

42 Coniferous-Broadleaf Mixture Significantly Increase Soil Microbial Biomass

The changes of tree species and composition may significantly affect the amount of organic matterinput in soil [57] As the most active part in soil organic matter (SOM) soil microbial biomass isimportant in nutrient cycling and release participating in the circulation of critical elements such ascarbon and nitrogen [23] Because of the quantity and quality of the bulk of SOM changes slowly andthe changes of SOM are difficult to measure due to the spatial heterogeneity of soils soil microbialbiomass and in particular MBC-to-SOC and MBN-to-TN ratio have been suggested as indicators ofthe state and modification of SOM [58ndash60] and as an indicator for carbon (nitrogen) availability insoil [6162] The concentrations of SOC TN and MBC and MBN measured in our study were affectedby stand type and seasons (Table 3 and Table S2 Figure 2) In the dry cool season although the SOCcontent in mixed stands did not significantly differ with that in PCH but significantly lower than thatin PPM the MBC concentration in mixed stands was markedly higher than that in the correspondingmono-species stands Likewise although the TN content in mixed stands was smaller than that in PCHand PPM stands the MBN content in mixed stands was markedly higher than that in the correspondingmono-species stands in the wet-warn season Previous studies found that higher soil microbial biomasscorrelated with higher SOC and TN content that sustain microbial metabolism [2163] However inour study the lower SOC and TN content in MIX was able to maintain a higher microbial biomass thansites where SOC was more abundant Presumably the quality of SOC could explain such a trend [64]and merit further investigation

In this study MBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands(Figure 2) suggesting higher carbon and nitrogen availability in soil and higher soil quality for thegrowth of soil microorganisms by coniferous-broadleaf mixture [21] In addition the effects of admixingon soil microbial biomass MBC-to-SOC and MBN-to-TN ratio were non-additive suggesting theinteraction of P massoniana and C hystrix can have a synergistic effect on improving soil carbon andnitrogen availability (Figure 3) These findings are consistent with many previous studies that havedetermined the effects of tree species mixture on soil microbial biomass [2165] These results may be

Forests 2019 10 879 16 of 20

contributed to (1) greater amount and more diverse organic matter input via litter and rhizodepositionproducts in the mixture of two contrasting tree species [21] (2) the higher use efficiency of soil organiccarbon and nitrogen by microorganisms in mixed stands [66]

We also sought to determine the admixing effects on composition and abundance of soil microbialcommunity with community-level PLFA profiles Surprisingly there were not significant admixingeffects on PLFA content of the major microbial functional groups such as bacteria fungi AMF andactinobacteria (Table 4) However tree species mixture significantly increased the abundance of GNbacteria in the dry-cool season while markedly increased the abundance of GP bacteria but decreasedthe abundance of fungi in the wet-warn season (Figure 4) It seemed that the dominated GN bacteria inthe dry-cool season and GP bacteria in the wet-warn season would correlate with the higher availabilityof soil organic carbon and nitrogen in mixed stands [6768] These correlations are complicated andfurther investigation is required to reveal the underlying mechanisms

43 Effects of Coniferous-Broadleaf Mixture on Soil Microbial Functions

Soil microorganisms excrete soil enzymes to drive nutrient mineralization and litterdecomposition [64] Therefore soil extracellular enzyme activities may be related to the carbonnitrogen and phosphorus cycling in soil [6970] In the present study we found positive admixingeffects on the activities of APH and NAG in the dry-cool season (Figure 5de) On the other handpositive admixing effects on the activities of urease and peroxidase while negative admixing effectson APH was detected in the wet-warn season (Figure 5dfg) This result suggests that the mixtureof C hystrix and P massoniana can accelerate the mineralization rates of soil organic phosphorus andnitrogen These findings were consistent with previous study [67]

The community-level physiological profiling (CLPP) is widely used to characterize microbialcommunities [71ndash73] The average well color development (AWCD) in the CLPP approach reflectsthe ability of soil microorganisms to utilize carbon sources and their metabolic activity The higherAWCD represents the stronger metabolic activity of soil microorganisms [71] In this present study wedetected a significantly positive admixing effect on carbon source utilization ability of the soil microbialcommunities in both dry-cool and wet-warn seasons after 144 h of incubation (Figure 6) Moreover theRDA analysis revealed that carbon source utilization ability of the soil microbial communities werepositive related with APH in the dry-cool season while related with urease and peroxidase activitiesin the wet-warn season (Figure 7) As a whole the higher soil microbial functions in mixed stands arelikely the results of the effects of AMF and GP bacteria in the dry-cool season and the effects of bacteriaincluding GP and GN bacteria the wet-warn season (Table S6)

5 Conclusions

In our study we measured significantly higher tree shrub and herb biomass and litter productionin the coniferous-broadleaf mixed plantation after 34 years of afforestation As expected MBC MBNMBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands suggesting highercarbon and nitrogen availability in soil and higher soil quality for the growth of soil microorganisms inour coniferous-broadleaf mixed stand In addition coniferous-broadleaf mixture can increase carbonsource utilization ability of soil microbial community and accelerate the mineralization rates of soilorganic phosphorus and nitrogen by increasing acid phosphatase β-14-N-acetylglucosaminidaseand urease activities The higher soil microbial biomass and activities are likely the result of greateramount and more diverse organic matter input via litter and rhizodeposition products and the higheruse efficiency of soil organic carbon and nitrogen by microorganisms in mixed stands Howeverthe correlations between soil dominated microbial functional groups and the higher availabilityof soil organic carbon and nitrogen along with the quality of SOC in mixed stands merit furtherinvestigation Our results highlight that establishment of coniferous-broadleaf mixed forests maybe a good management practice as coniferous-broadleaf mixture could accumulate higher stand

Forests 2019 10 879 17 of 20

biomass return more litter and increase soil microbial biomass and functions over the long term insubtropical China

Supplementary Materials The following are available online at httpwwwmdpicom1999-49071010879s1Table S1 Effects of stand type season and their interactions on stand biomass Table S2 Effects of stand type seasonand their interactions on soil microbial structure Table S3 Effects of stand type season and their interactionson relative abundance of the soil microbial community PLFAs Table S4 Effects of stand type season and theirinteractions on soil enzyme activity Table S5 Marginal and conditional effects of biotic and abiotic factors on soilmicrobial composition obtained from the summary of forward selection in Redundancy analysis (RDA) Table S6Marginal and conditional effects of soil microbial functional groups on soil enzyme activity and soil microbialcarbon source utilization ability obtained from the summary of forward selection in Redundancy analysis (RDA)

Author Contributions WW collected data made experiment analysis formal data and drafted the manuscriptYW designed the work XZ revised the manuscript and participated in analyzing the experiment data HZ YYXH and XL ZQ YL LY HL participated in collecting the experiment data

Funding This research was funded by National Natural Science Foundation of China (31860171 3146012131560201) the Guangxi Key Research and Development Program (2018AB40007) Innovation Project of GuangxiGraduate Education (YCBZ2017011) and the Guangxi Universities Science and technology (201201ZD001)

Acknowledgments We gratefully acknowledge the supports in field sampling by the Experimental Center ofTropical Forestry Chinese Academy of Forestry A special acknowledgment is given to Patrick M Finnegan for hiswriting suggestions

Conflicts of Interest The authors declare no conflicts of interest

References

1 Zhang X Liu S Huang Y Fu S Wang J Ming A Li X Yao M Li H Tree species mixture inhibits soilorganic carbon mineralization accompanied by decreased r-selected bacteria Plant Soil 2018 431 203ndash216[CrossRef]

2 Pan Y Birdsey RA Fang J Houghton R Kauppi PE Kurz WA Phillips OL Shvidenko ALewis SL Canadell JG et al A large and persistent carbon sink in the worldrsquos forests Science 2011 333988ndash993 [CrossRef] [PubMed]

3 Chen X Ju Q Lin K Development status issues and countermeasures of Chinarsquo s plantation World For Res2014 27 54ndash59

4 Wang H Lencinas MV Ross Friedman C Wang X Qiu J Understory plant diversity assessment ofEucalyptus plantations over three vegetation types in Yunnan China New For 2011 42 101ndash116 [CrossRef]

5 Sang PM Lamb D Bonner M Schmidt S Carbon sequestration and soil fertility of tropical tree plantationsand secondary forest established on degraded land Plant Soil 2013 362 187ndash200 [CrossRef]

6 Li C Zhou X Wen Y Zhu H Qin Z Li X You Y Huang X Effects of high-generation ratations ofEucalyptus on undergrowth soil fertility and enzyme activities Guangxi Sci 2019 26 176ndash186

7 You Y Huang X Zhu H Liu S Liang H Wen Y Wang H Cai D Ye D Positive interactions betweenPinus massoniana and Castanopsis hystrix species in the uneven-aged mixed plantations can produce moreecosystem carbon in subtropical China For Ecol Manag 2018 410 193ndash200 [CrossRef]

8 Huang X Liu S You Y Wen Y Wang H Wang J Microbial community and associated enzymes activityinfluence soil carbon chemical composition in Eucalyptus urophylla plantation with mixing N2-fixing speciesin sub-tropical China Plant Soil 2017 414 199ndash212 [CrossRef]

9 Kelty MJ The role of species mixtures in plantation forestry For Ecol Manag 2006 233 195ndash204 [CrossRef]10 Bauhus J Khanna P Menden N Aboveground and belowground interactions in mixed plantations of

Eucalyptus globulus and Acacia mearnsii Can J For Res 2000 30 1886ndash1894 [CrossRef]11 Vandermeer J The Ecology of Intercropping Cambridge University Press New York NY USA 1989 p 23712 Forrester DI The spatial and temporal dynamics of species interactions in mixed-species forests From

pattern to process For Ecol Manag 2014 312 282ndash292 [CrossRef]13 Forrester DI Bauhus J A review of processes behind diversity-productivity relationships in forests

Curr For Rep 2016 2 45ndash61 [CrossRef]14 Wang H Liu S Wang J You Y Yang Y Shi Z Huang X Zheng L Li Z Ming A et al Mixed-species

plantation with Pinus massoniana and Castanopsis hystrix accelerates C loss in recalcitrant coniferous litter butslows C loss in labile broadleaf litter in southern China For Ecol Manag 2018 422 207ndash213 [CrossRef]

Forests 2019 10 879 7 of 20

32 Litter Production and Quality

Stand type the interaction between stand type and season significantly influenced litter productionat our study sites (Table 2) The MIX had significantly higher litter production than PCH and PPMin both seasons OClitter and CNlitter were significantly affected by both stand type and season withhigher value in the wet-warm season than in the dry-cool season in all plantations An opposite trendwas observed in TNlitter TPlitter was significantly affected by stand type season and their interactionwith MIX had significantly lower concentration than PCH and PPM in the dry-cool season TKlitter wassignificantly affected by stand type with MIX had significantly higher concentration than PCH andPPM in the wet-warm season These results partially supported our first hypothesis that tree speciesmixture increased litter production although the increases of litter quality in mixed plantations werenot significant

33 Soil Physical and Chemical Properties

In the dry-cool season the mixed plantations had the highest soil bulk density (BD) and soil watercontent (WC) and the lowest soil temperature and medium pH compared to the pure plantations(Table 3) In the wet-warm season MIX had the medium BD WC and soil temperature and the highestpH compared to PPM and PCH

The PPM plantations had the highest SOC TN TP SOC-to-TN ratio NH4+-N and CEC while

MIX had the highest AP TK and AK and PCH had the highest NO3minus-N and AN in the dry-cool season

(Table 3) On the other hand the PCH had the highest SOC TN and AP while MIX had the highestTP TK AK NO3

minus-N and AN and PPM had the highest SOC-to-TN ratio NO3minus-N and CEC in the

wet-warm seasonTwo-way ANOVAs indicated that season significantly affected all the tested soil variables (Table 3)

In addition the stand type influenced nearly all soil variables except BD TN NH4+-N and AN

Interactions between stand type and season significantly influenced WC temperature pH SOC TNTP AP AK NO3

minus-N and CEC

Forests 2019 10 879 8 of 20

Table 2 The quantity and quality of litter in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM) plantation and mixed C hystrix and P massoniana(MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH MIX PPM PCH MIX PPM F p F p F p

LP 668 plusmn 019 b 836 plusmn 069 a 484 plusmn 019 c 553 plusmn 043 b 921 plusmn 040 a 616 plusmn 023 b 3752 lt0001 093 0343 527 0013OClitter 19064 plusmn 1063 b 24859 plusmn 1848 a 25505 plusmn 1800 a 27757 plusmn 1885 b 2743 plusmn 1142 b 32175 plusmn 544 a 683 0004 2486 lt0001 226 0127TNlitter 1738 plusmn 078 a 1353 plusmn 106 b 1496 plusmn 062 ab 1295 plusmn 059 a 1357 plusmn 090 a 1189 plusmn 050 a 321 0058 1579 0001 445 0023CNlitter 1112 plusmn 095 b 1850 plusmn 081 a 1733 plusmn 181 a 2182 plusmn 223 b 2056 plusmn 156 b 2722 plusmn 096 a 767 0003 3890 lt0001 518 0013TPlitter 134 plusmn 008 a 050 plusmn 007 b 116 plusmn 005 a 119 plusmn 015 a 138 plusmn 015 a 131 plusmn 005 a 667 0005 1281 0002 1417 lt0001TKlitter 116 plusmn 016 a 152 plusmn 010 a 082 plusmn 009 b 107 plusmn 002 b 149 plusmn 013 a 088 plusmn 007 c 2084 lt0001 007 0796 029 0751

Different superscripts indicate significant differences among stand types in each season (p lt 005) LP (Mg haminus1) litter production OClitter (g kgminus1) litter organic carbon TNlitter (g kgminus1)litter total nitrogen CNlitter CN ratio of litter TPlitter (g kgminus1) litter total phosphorus TKlitter (g kgminus1) litter total potassium

Table 3 Soil physical and chemical properties (0ndash10 cm soil depth) in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM) plantation and mixedC hystrix and P massoniana (MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH Mixed PPM PCH Mixed PPM F p F p F p

BD 109 plusmn 003 b 125 plusmn 004 a 119 plusmn 002 a 103 plusmn 001 a 103 plusmn 003 a 104 plusmn 005 a 311 0063 3044 lt0001 297 0070WC 3966 plusmn 136 b 4359 plusmn 061 a 4017 plusmn 158 ab 4053 plusmn 107 c 4440 plusmn 084 b 5048 plusmn 141 a 1035 0001 1681 lt0001 1048 0001Tem 1682 plusmn 021 a 1550 plusmn 009 c 1617 plusmn 005 b 2628 plusmn 043 a 2598 plusmn 022 a 2541 plusmn 008 a 821 0002 283334 lt0001 431 0025pH 432 plusmn 002 a 419 plusmn 003 b 404 plusmn 005 c 402 plusmn 002 b 421 plusmn 003 a 390 plusmn 009 b 1440 lt0001 1370 0001 623 0007

SOC 3742 plusmn 108 b 4082 plusmn 022 b 5698 plusmn 189 a 5331 plusmn 194 a 4839 plusmn 200 a 5083 plusmn 054 a 2460 lt0001 2369 lt0001 2864 lt0001TN 163 plusmn 005 b 160 plusmn 006 b 216 plusmn 022 a 248 plusmn 011 a 213 plusmn 013 a 217 plusmn 014 a 248 0105 4408 lt0001 480 0018

SOCTN 2298 plusmn 059 b 2567 plusmn 103 b 2949 plusmn 159 a 2167 plusmn 133 a 2304 plusmn 162 a 2390 plusmn 202 a 457 0021 722 0013 114 0338TP 031 plusmn 004 c 042 plusmn 002 b 060 plusmn 004 a 052 plusmn 006 b 078 plusmn 004 a 054 plusmn 004 b 2186 lt0001 2889 lt0001 1795 lt0001AP 919 plusmn 058 b 1252 plusmn 069 a 1002 plusmn 030 b 1330 plusmn 040 a 1315 plusmn 057 a 998 plusmn 027 b 1670 lt0001 1525 0001 1029 0001TK 414 plusmn 015 b 545 plusmn 049 a 242 plusmn 016 c 540 plusmn 050 a 694 plusmn 082 a 338 plusmn 013 b 3226 lt0001 1034 0004 039 0680AK 2990 plusmn 149 b 4629 plusmn 096 a 2846 plusmn 108 b 4719 plusmn 228 a 5279 plusmn 365 a 2837 plusmn 144 b 5375 lt0001 2257 lt0001 927 0001

NO3minus-N 355 plusmn 024 a 185 plusmn 004 c 286 plusmn 022 b 406 plusmn 019 c 530 plusmn 009 b 579 plusmn 009 a 1096 lt0001 29106 lt0001 4547 lt0001

NH4+-N 2129 plusmn 101 a 2334 plusmn 113 a 2447 plusmn 137 a 4224 plusmn 162 a 4350 plusmn 166 a 3883 plusmn 247 a 075 0482 19616 lt0001 247 0105

AN 2734 plusmn 157 a 2519 plusmn 114 a 2485 plusmn 090 a 463 plusmn 155 a 488 plusmn 169 a 4462 plusmn 246 a 120 0319 24555 lt0001 117 0328CEC 4553 plusmn 327 c 5758 plusmn 411 b 8233 plusmn 194 a 5543 plusmn 202a 5446 plusmn 119 a 6224 plusmn 291 a 4514 lt0001 1216 0002 2168 lt0001

Different superscripts indicate significant differences among stand types in each season (p lt 005) BD (g cmminus1) bulk density WC () soil water content Tem (C) temperature SOC(g kgminus1) soil organic carbon TN (g kgminus1) total nitrogen TP (g kgminus1) total phosphorus AP (mg kgminus1) available phosphorus TK (g kgminus1) total potassium AK (mg kgminus1) availablepotassium NO3

minus-N (mg kgminus1) nitrate nitrogen NH4+-N (mg kgminus1) ammonium nitrogen AN (mg kgminus1) Available nitrogen CEC (cmol + kgminus1) cation exchange capacity

Forests 2019 10 879 9 of 20

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantlyaffected by stand type and MBN was also significantly affected by season and interaction betweenstand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a)In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantlyhigher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highestMBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOCand MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significantlevel in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOCand MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-coolseason (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season(Figure 3b) were significant

Forests 2019 10 x FOR PEER REVIEW 10 of 22

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantly affected by stand type and MBN was also significantly affected by season and interaction between stand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a) In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantly higher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highest MBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOC and MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significant level in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOC and MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-cool season (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season (Figure 3b) were significant

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBN MBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a) and wet-warm season (b)

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBNMBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005level by LSD multiple comparisons among stand types (n = 5)

Forests 2019 10 x FOR PEER REVIEW 10 of 22

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantly affected by stand type and MBN was also significantly affected by season and interaction between stand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a) In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantly higher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highest MBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOC and MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significant level in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOC and MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-cool season (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season (Figure 3b) were significant

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBN MBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a) and wet-warm season (b)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a)and wet-warm season (b)

Forests 2019 10 879 10 of 20

In the dry-cool season total PLFAs AMF PLFAs actinobacteria PLFAs bacteria PLFAs fungiPLFAs GP bacteria PLFAs and GP-to-GN PLFAs ratio were smaller in MIX than those in PPM andPCH On the other hand in the wet-warm season these PLFAs were smaller in PPM than thosein PCH and MIX (Table 4) Specifically the ratios of bacteria-to-fungi PLFA and GP-to-GN PLFAwere significantly higher in MIX than those in PPM and PCH in the wet-warm season These resultsindicated that the response of the soil microbial community structure to tree species mixture varied indifferent seasons Two-way ANOVAs showed that both stand type and season significantly affectedall the tested microbial variables while the interaction between stand type and season significantlyinfluenced total PLFAs bacteria PLFAs GP bacteria PLFAs Acti PLFAs bacteria-to-fungi PLFA andGP-to-GN PLFA (Table 4)

Across plantations and seasons bacteria PLFAs were the most abundant followed by fungi andactinobacteria PLFAs (Figure 4 and Table S3) It was worth noting that the relative abundance ofGN bacteria PLFAs was significantly higher in MIX than in other plantations in the dry-cool season(Figure 4) Furthermore the relative abundance of GP bacteria PLFAs was significantly higher andthat of fungi PLFAs was significantly lower in MIX than in other plantations in the wet-warm season

Forests 2019 10 x FOR PEER REVIEW 13 of 22

Across plantations and seasons bacteria PLFAs were the most abundant followed by fungi and actinobacteria PLFAs (Figure 4 and Table S3) It was worth noting that the relative abundance of GN bacteria PLFAs was significantly higher in MIX than in other plantations in the dry-cool season (Figure 4) Furthermore the relative abundance of GP bacteria PLFAs was significantly higher and that of fungi PLFAs was significantly lower in MIX than in other plantations in the wet-warm season

Figure 4 The relative abundance of PLFAs (mol ) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleaf plantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increased APH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increased urease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg) Carbon source utilization ability of the soil microbial community indicated by average well color development (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation (Figure 6)

Figure 4 The relative abundance of PLFAs (mol ) in three plantations in the dry-cool season (a) andwet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level byLSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 11 of 20

Table 4 Soil microbial community PLFAs content and structure (0ndash10 cm soil depth) in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM)plantation and mixed C hystrix and P massoniana (MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH MIX PPM PCH MIX PPM F p F p F p

TPLFAs 3767 plusmn 271 a 3255 plusmn 124 a 3796 plusmn 128 a 4373 plusmn 208 a 4001 plusmn 07 a 3478 plusmn 111 b 464 0020 646 0018 607 0007B 1579 plusmn 081 a 1306 plusmn 043 b 1490 plusmn 051 ab 1764 plusmn 072 a 1723 plusmn 042 a 1276 plusmn 041 b 1274 lt0001 768 0011 1556 lt0001

GP 945 plusmn 043 a 794 plusmn 029 b 918 plusmn 031 a 1066 plusmn 051 a 1155 plusmn 031 a 810 plusmn 031 b 817 0002 1709 lt0001 2030 lt0001GN 591 plusmn 076 a 590 plusmn 016 a 553 plusmn 022 a 619 plusmn 025 a 455 plusmn 037 b 411 plusmn 020 b 526 0013 702 0014 313 0062

F 498 plusmn 034 a 375 plusmn 022 b 424 plusmn 028 ab 492 plusmn 035 a 296 plusmn 043 b 345 plusmn 021 b 1356 lt0001 448 0045 091 0416AMF 104 plusmn 003 a 075 plusmn 003 b 075 plusmn 004 b 085 plusmn 007 a 044 plusmn 008 b 048 plusmn 003 b 2822 lt0001 3483 lt0001 056 0577Acti 426 plusmn 037 ab 377 plusmn 021 b 486 plusmn 020 a 531 plusmn 019 a 487 plusmn 012 b 458 plusmn 010 b 273 0085 1236 0002 650 0006BF 319 plusmn 011 a 351 plusmn 013 a 356 plusmn 017 a 362 plusmn 014 b 635 plusmn 094 a 377 plusmn 033 b 750 0003 1131 0003 598 0008

GPGN 174 plusmn 029 b 135 plusmn 003 b 166 plusmn 004 a 173 plusmn 008 b 263 plusmn 028 a 198 plusmn 011 b 107 0359 1376 0001 733 0003

Different superscripts indicate significant differences among stand types in each season (p lt 005) TPLFAs (nmol gminus1 soil) total PLFAs B (nmol gminus1 soil) bacteria PLFAs GP (nmol gminus1

soil) gram-positive bacteria PLFAs GN (nmol gminus1 soil) gram-negative bacteria PLFAs F (nmol gminus1 soil) fungi PLFAs Acti (nmol gminus1 soil) actinobacteria PLFAs AMF (nmol gminus1 soil)arbuscular mycorrhizal fungi PLFAs BF bacteria-to-fungi PLFA ratio GPGN GP-to-GN ratio

Forests 2019 10 879 12 of 20

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleafplantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increasedAPH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increasedurease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg)Carbon source utilization ability of the soil microbial community indicated by average well colordevelopment (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation(Figure 6)Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in threeplantations in different seasons Error bars are standard error Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 13 of 20

Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in threeplantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in thedry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons(F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pHTK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-coolseason in which SOC and TBB were the major correlates of changes in soil microbial composition(Table S5) A total of 11 biotic and abiotic factors including NO3

minus-N TBB pH TKlitter HBB AP AKTP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warmseason in which NO3

minus-N TBB pH and TKlitter were the major correlates of changes in soil microbialcomposition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

37 Relationship between Soil Microbial Structure and Functions

Soil enzyme activity and soil microbial carbon source utilization ability are both important soilmicrobial functions The first two RDA axes explained 4948 of the variation in soil enzyme activityand carbon utilization ability in the dry-cool season (F = 24 p = 0006 Figure 7b) and 6891 of thevariation in the wet-warm season (F = 64 p = 0002 Figure 7d) AMF and GP were significantlycorrelated with soil microbial functions in the dry-cool season (Table S6) Total bacteria GP bacteriaGN bacteria and actinobacteria were significantly correlated with soil microbial functions in thewet-warm season in which total bacteria GP bacteria and GN bacteria were the major correlates ofchanges of soil enzyme activity (Table S6)

Forests 2019 10 879 14 of 20

Forests 2019 10 x FOR PEER REVIEW 15 of 22

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in the dry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons (F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pH TK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-cool season in which SOC and TBB were the major correlates of changes in soil microbial composition (Table S5) A total of 11 biotic and abiotic factors including NO3minus-N TBB pH TKlitter HBB AP AK TP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warm season in which NO3minus-N TBB pH and TKlitter were the major correlates of changes in soil microbial composition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160 were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant biotic and abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot of soil microbial community structure and soil microbial functions (bd) Above the label represents a significant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowground biomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soil total potassium NO3ndash-N soil nitrate nitrogen HBB herb belowground biomass AP soil available phosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil water

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant bioticand abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot ofsoil microbial community structure and soil microbial functions (bd) Above the label represents asignificant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowgroundbiomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soiltotal potassium NO3

minus-N soil nitrate nitrogen HBB herb belowground biomass AP soil availablephosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil watercontent LP litter production TKlitter litter total potassium GP gram-positive bacteria GN gramnegative bacteria AMF arbuscular mycorrhizal fungi AG α-Glucosidase BG β-14-glucosidaseCB β-Cellobiosidase APH acid phosphatase NAG β-14-N-acetylglucosaminidase PHO phenoloxidase PO peroxidase AWCD average well color development of carbon source metabolism at192 h incubation

4 Discussion

41 Coniferous-Broadleaf Mixture Significantly Increase Stand Biomass and Litter Production

The results of this study indicated that admixing of coniferous and broadleaf tree speciessignificantly increased stand biomass and litter production after 34 years of afforestation in bothdry-cool and wet-warm seasons (Figure 1 Table 2) This may be a consequence of a greater growth of

Forests 2019 10 879 15 of 20

C hystrix in the mixed stands These results confirmed previous findings conducted in our experimentsite measured when the plantations were 26-year-old and 28-year-old respectively [716] A bulkof studies revealed that mixed-species management could affect tree growth stand structure andenvironment [145253] Mixed forests can develop stratified canopies due to niche differences amongdifferent species In the present study trees of the faster growing broad-leaf species (C hystrix) weretaller had a larger DBH and thus formed an upper canopy stratum in the mixed stands (Table 1)While trees of the shorter species (P massoniana) formed a lower stratum with lower DBH and treeheight and the reduced amount of light transmitted through the upper canopy contributed to theirslower growth The dominant tree species within the stand can absorb more sunlight and use itmore efficiently for growth [54] and thus contribute to higher tree biomass and litter productionin mixed stands In addition stratified canopies in mixed plantations can help to maintain levelsof understory species richness and biomass compared with pure plantations due to relatively highlight availabilities [5556] This may be why the mixed stand had higher shrub and herb biomass inour study

Specifically in the present study the broad-leaf species attained greater heights and diameters inmixed stands while masson pine had reduced diameter and height growth in mixed-species comparedto mono-species stands (Table 1) The severe suppression of P massoniana by the faster growingC hystrix may be attributed to differences in the light requirements of the two species [7] The growthpattern of C hystrix and P massoniana in mixed stands in our study (34-year-old) were differed fromwhen they were 26-years-old in which the growth of C hystrix and P massoniana in mixed standswere both greater than in the corresponding mono-species stands [7] This suggests that effects of thetree species mixture could change over time and long-term investigation of mixed forests is of greatsignificance to reveal mixed effects

42 Coniferous-Broadleaf Mixture Significantly Increase Soil Microbial Biomass

The changes of tree species and composition may significantly affect the amount of organic matterinput in soil [57] As the most active part in soil organic matter (SOM) soil microbial biomass isimportant in nutrient cycling and release participating in the circulation of critical elements such ascarbon and nitrogen [23] Because of the quantity and quality of the bulk of SOM changes slowly andthe changes of SOM are difficult to measure due to the spatial heterogeneity of soils soil microbialbiomass and in particular MBC-to-SOC and MBN-to-TN ratio have been suggested as indicators ofthe state and modification of SOM [58ndash60] and as an indicator for carbon (nitrogen) availability insoil [6162] The concentrations of SOC TN and MBC and MBN measured in our study were affectedby stand type and seasons (Table 3 and Table S2 Figure 2) In the dry cool season although the SOCcontent in mixed stands did not significantly differ with that in PCH but significantly lower than thatin PPM the MBC concentration in mixed stands was markedly higher than that in the correspondingmono-species stands Likewise although the TN content in mixed stands was smaller than that in PCHand PPM stands the MBN content in mixed stands was markedly higher than that in the correspondingmono-species stands in the wet-warn season Previous studies found that higher soil microbial biomasscorrelated with higher SOC and TN content that sustain microbial metabolism [2163] However inour study the lower SOC and TN content in MIX was able to maintain a higher microbial biomass thansites where SOC was more abundant Presumably the quality of SOC could explain such a trend [64]and merit further investigation

In this study MBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands(Figure 2) suggesting higher carbon and nitrogen availability in soil and higher soil quality for thegrowth of soil microorganisms by coniferous-broadleaf mixture [21] In addition the effects of admixingon soil microbial biomass MBC-to-SOC and MBN-to-TN ratio were non-additive suggesting theinteraction of P massoniana and C hystrix can have a synergistic effect on improving soil carbon andnitrogen availability (Figure 3) These findings are consistent with many previous studies that havedetermined the effects of tree species mixture on soil microbial biomass [2165] These results may be

Forests 2019 10 879 16 of 20

contributed to (1) greater amount and more diverse organic matter input via litter and rhizodepositionproducts in the mixture of two contrasting tree species [21] (2) the higher use efficiency of soil organiccarbon and nitrogen by microorganisms in mixed stands [66]

We also sought to determine the admixing effects on composition and abundance of soil microbialcommunity with community-level PLFA profiles Surprisingly there were not significant admixingeffects on PLFA content of the major microbial functional groups such as bacteria fungi AMF andactinobacteria (Table 4) However tree species mixture significantly increased the abundance of GNbacteria in the dry-cool season while markedly increased the abundance of GP bacteria but decreasedthe abundance of fungi in the wet-warn season (Figure 4) It seemed that the dominated GN bacteria inthe dry-cool season and GP bacteria in the wet-warn season would correlate with the higher availabilityof soil organic carbon and nitrogen in mixed stands [6768] These correlations are complicated andfurther investigation is required to reveal the underlying mechanisms

43 Effects of Coniferous-Broadleaf Mixture on Soil Microbial Functions

Soil microorganisms excrete soil enzymes to drive nutrient mineralization and litterdecomposition [64] Therefore soil extracellular enzyme activities may be related to the carbonnitrogen and phosphorus cycling in soil [6970] In the present study we found positive admixingeffects on the activities of APH and NAG in the dry-cool season (Figure 5de) On the other handpositive admixing effects on the activities of urease and peroxidase while negative admixing effectson APH was detected in the wet-warn season (Figure 5dfg) This result suggests that the mixtureof C hystrix and P massoniana can accelerate the mineralization rates of soil organic phosphorus andnitrogen These findings were consistent with previous study [67]

The community-level physiological profiling (CLPP) is widely used to characterize microbialcommunities [71ndash73] The average well color development (AWCD) in the CLPP approach reflectsthe ability of soil microorganisms to utilize carbon sources and their metabolic activity The higherAWCD represents the stronger metabolic activity of soil microorganisms [71] In this present study wedetected a significantly positive admixing effect on carbon source utilization ability of the soil microbialcommunities in both dry-cool and wet-warn seasons after 144 h of incubation (Figure 6) Moreover theRDA analysis revealed that carbon source utilization ability of the soil microbial communities werepositive related with APH in the dry-cool season while related with urease and peroxidase activitiesin the wet-warn season (Figure 7) As a whole the higher soil microbial functions in mixed stands arelikely the results of the effects of AMF and GP bacteria in the dry-cool season and the effects of bacteriaincluding GP and GN bacteria the wet-warn season (Table S6)

5 Conclusions

In our study we measured significantly higher tree shrub and herb biomass and litter productionin the coniferous-broadleaf mixed plantation after 34 years of afforestation As expected MBC MBNMBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands suggesting highercarbon and nitrogen availability in soil and higher soil quality for the growth of soil microorganisms inour coniferous-broadleaf mixed stand In addition coniferous-broadleaf mixture can increase carbonsource utilization ability of soil microbial community and accelerate the mineralization rates of soilorganic phosphorus and nitrogen by increasing acid phosphatase β-14-N-acetylglucosaminidaseand urease activities The higher soil microbial biomass and activities are likely the result of greateramount and more diverse organic matter input via litter and rhizodeposition products and the higheruse efficiency of soil organic carbon and nitrogen by microorganisms in mixed stands Howeverthe correlations between soil dominated microbial functional groups and the higher availabilityof soil organic carbon and nitrogen along with the quality of SOC in mixed stands merit furtherinvestigation Our results highlight that establishment of coniferous-broadleaf mixed forests maybe a good management practice as coniferous-broadleaf mixture could accumulate higher stand

Forests 2019 10 879 17 of 20

biomass return more litter and increase soil microbial biomass and functions over the long term insubtropical China

Supplementary Materials The following are available online at httpwwwmdpicom1999-49071010879s1Table S1 Effects of stand type season and their interactions on stand biomass Table S2 Effects of stand type seasonand their interactions on soil microbial structure Table S3 Effects of stand type season and their interactionson relative abundance of the soil microbial community PLFAs Table S4 Effects of stand type season and theirinteractions on soil enzyme activity Table S5 Marginal and conditional effects of biotic and abiotic factors on soilmicrobial composition obtained from the summary of forward selection in Redundancy analysis (RDA) Table S6Marginal and conditional effects of soil microbial functional groups on soil enzyme activity and soil microbialcarbon source utilization ability obtained from the summary of forward selection in Redundancy analysis (RDA)

Author Contributions WW collected data made experiment analysis formal data and drafted the manuscriptYW designed the work XZ revised the manuscript and participated in analyzing the experiment data HZ YYXH and XL ZQ YL LY HL participated in collecting the experiment data

Funding This research was funded by National Natural Science Foundation of China (31860171 3146012131560201) the Guangxi Key Research and Development Program (2018AB40007) Innovation Project of GuangxiGraduate Education (YCBZ2017011) and the Guangxi Universities Science and technology (201201ZD001)

Acknowledgments We gratefully acknowledge the supports in field sampling by the Experimental Center ofTropical Forestry Chinese Academy of Forestry A special acknowledgment is given to Patrick M Finnegan for hiswriting suggestions

Conflicts of Interest The authors declare no conflicts of interest

References

1 Zhang X Liu S Huang Y Fu S Wang J Ming A Li X Yao M Li H Tree species mixture inhibits soilorganic carbon mineralization accompanied by decreased r-selected bacteria Plant Soil 2018 431 203ndash216[CrossRef]

2 Pan Y Birdsey RA Fang J Houghton R Kauppi PE Kurz WA Phillips OL Shvidenko ALewis SL Canadell JG et al A large and persistent carbon sink in the worldrsquos forests Science 2011 333988ndash993 [CrossRef] [PubMed]

3 Chen X Ju Q Lin K Development status issues and countermeasures of Chinarsquo s plantation World For Res2014 27 54ndash59

4 Wang H Lencinas MV Ross Friedman C Wang X Qiu J Understory plant diversity assessment ofEucalyptus plantations over three vegetation types in Yunnan China New For 2011 42 101ndash116 [CrossRef]

5 Sang PM Lamb D Bonner M Schmidt S Carbon sequestration and soil fertility of tropical tree plantationsand secondary forest established on degraded land Plant Soil 2013 362 187ndash200 [CrossRef]

6 Li C Zhou X Wen Y Zhu H Qin Z Li X You Y Huang X Effects of high-generation ratations ofEucalyptus on undergrowth soil fertility and enzyme activities Guangxi Sci 2019 26 176ndash186

7 You Y Huang X Zhu H Liu S Liang H Wen Y Wang H Cai D Ye D Positive interactions betweenPinus massoniana and Castanopsis hystrix species in the uneven-aged mixed plantations can produce moreecosystem carbon in subtropical China For Ecol Manag 2018 410 193ndash200 [CrossRef]

8 Huang X Liu S You Y Wen Y Wang H Wang J Microbial community and associated enzymes activityinfluence soil carbon chemical composition in Eucalyptus urophylla plantation with mixing N2-fixing speciesin sub-tropical China Plant Soil 2017 414 199ndash212 [CrossRef]

9 Kelty MJ The role of species mixtures in plantation forestry For Ecol Manag 2006 233 195ndash204 [CrossRef]10 Bauhus J Khanna P Menden N Aboveground and belowground interactions in mixed plantations of

Eucalyptus globulus and Acacia mearnsii Can J For Res 2000 30 1886ndash1894 [CrossRef]11 Vandermeer J The Ecology of Intercropping Cambridge University Press New York NY USA 1989 p 23712 Forrester DI The spatial and temporal dynamics of species interactions in mixed-species forests From

pattern to process For Ecol Manag 2014 312 282ndash292 [CrossRef]13 Forrester DI Bauhus J A review of processes behind diversity-productivity relationships in forests

Curr For Rep 2016 2 45ndash61 [CrossRef]14 Wang H Liu S Wang J You Y Yang Y Shi Z Huang X Zheng L Li Z Ming A et al Mixed-species

plantation with Pinus massoniana and Castanopsis hystrix accelerates C loss in recalcitrant coniferous litter butslows C loss in labile broadleaf litter in southern China For Ecol Manag 2018 422 207ndash213 [CrossRef]

Forests 2019 10 879 8 of 20

Table 2 The quantity and quality of litter in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM) plantation and mixed C hystrix and P massoniana(MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH MIX PPM PCH MIX PPM F p F p F p

LP 668 plusmn 019 b 836 plusmn 069 a 484 plusmn 019 c 553 plusmn 043 b 921 plusmn 040 a 616 plusmn 023 b 3752 lt0001 093 0343 527 0013OClitter 19064 plusmn 1063 b 24859 plusmn 1848 a 25505 plusmn 1800 a 27757 plusmn 1885 b 2743 plusmn 1142 b 32175 plusmn 544 a 683 0004 2486 lt0001 226 0127TNlitter 1738 plusmn 078 a 1353 plusmn 106 b 1496 plusmn 062 ab 1295 plusmn 059 a 1357 plusmn 090 a 1189 plusmn 050 a 321 0058 1579 0001 445 0023CNlitter 1112 plusmn 095 b 1850 plusmn 081 a 1733 plusmn 181 a 2182 plusmn 223 b 2056 plusmn 156 b 2722 plusmn 096 a 767 0003 3890 lt0001 518 0013TPlitter 134 plusmn 008 a 050 plusmn 007 b 116 plusmn 005 a 119 plusmn 015 a 138 plusmn 015 a 131 plusmn 005 a 667 0005 1281 0002 1417 lt0001TKlitter 116 plusmn 016 a 152 plusmn 010 a 082 plusmn 009 b 107 plusmn 002 b 149 plusmn 013 a 088 plusmn 007 c 2084 lt0001 007 0796 029 0751

Different superscripts indicate significant differences among stand types in each season (p lt 005) LP (Mg haminus1) litter production OClitter (g kgminus1) litter organic carbon TNlitter (g kgminus1)litter total nitrogen CNlitter CN ratio of litter TPlitter (g kgminus1) litter total phosphorus TKlitter (g kgminus1) litter total potassium

Table 3 Soil physical and chemical properties (0ndash10 cm soil depth) in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM) plantation and mixedC hystrix and P massoniana (MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH Mixed PPM PCH Mixed PPM F p F p F p

BD 109 plusmn 003 b 125 plusmn 004 a 119 plusmn 002 a 103 plusmn 001 a 103 plusmn 003 a 104 plusmn 005 a 311 0063 3044 lt0001 297 0070WC 3966 plusmn 136 b 4359 plusmn 061 a 4017 plusmn 158 ab 4053 plusmn 107 c 4440 plusmn 084 b 5048 plusmn 141 a 1035 0001 1681 lt0001 1048 0001Tem 1682 plusmn 021 a 1550 plusmn 009 c 1617 plusmn 005 b 2628 plusmn 043 a 2598 plusmn 022 a 2541 plusmn 008 a 821 0002 283334 lt0001 431 0025pH 432 plusmn 002 a 419 plusmn 003 b 404 plusmn 005 c 402 plusmn 002 b 421 plusmn 003 a 390 plusmn 009 b 1440 lt0001 1370 0001 623 0007

SOC 3742 plusmn 108 b 4082 plusmn 022 b 5698 plusmn 189 a 5331 plusmn 194 a 4839 plusmn 200 a 5083 plusmn 054 a 2460 lt0001 2369 lt0001 2864 lt0001TN 163 plusmn 005 b 160 plusmn 006 b 216 plusmn 022 a 248 plusmn 011 a 213 plusmn 013 a 217 plusmn 014 a 248 0105 4408 lt0001 480 0018

SOCTN 2298 plusmn 059 b 2567 plusmn 103 b 2949 plusmn 159 a 2167 plusmn 133 a 2304 plusmn 162 a 2390 plusmn 202 a 457 0021 722 0013 114 0338TP 031 plusmn 004 c 042 plusmn 002 b 060 plusmn 004 a 052 plusmn 006 b 078 plusmn 004 a 054 plusmn 004 b 2186 lt0001 2889 lt0001 1795 lt0001AP 919 plusmn 058 b 1252 plusmn 069 a 1002 plusmn 030 b 1330 plusmn 040 a 1315 plusmn 057 a 998 plusmn 027 b 1670 lt0001 1525 0001 1029 0001TK 414 plusmn 015 b 545 plusmn 049 a 242 plusmn 016 c 540 plusmn 050 a 694 plusmn 082 a 338 plusmn 013 b 3226 lt0001 1034 0004 039 0680AK 2990 plusmn 149 b 4629 plusmn 096 a 2846 plusmn 108 b 4719 plusmn 228 a 5279 plusmn 365 a 2837 plusmn 144 b 5375 lt0001 2257 lt0001 927 0001

NO3minus-N 355 plusmn 024 a 185 plusmn 004 c 286 plusmn 022 b 406 plusmn 019 c 530 plusmn 009 b 579 plusmn 009 a 1096 lt0001 29106 lt0001 4547 lt0001

NH4+-N 2129 plusmn 101 a 2334 plusmn 113 a 2447 plusmn 137 a 4224 plusmn 162 a 4350 plusmn 166 a 3883 plusmn 247 a 075 0482 19616 lt0001 247 0105

AN 2734 plusmn 157 a 2519 plusmn 114 a 2485 plusmn 090 a 463 plusmn 155 a 488 plusmn 169 a 4462 plusmn 246 a 120 0319 24555 lt0001 117 0328CEC 4553 plusmn 327 c 5758 plusmn 411 b 8233 plusmn 194 a 5543 plusmn 202a 5446 plusmn 119 a 6224 plusmn 291 a 4514 lt0001 1216 0002 2168 lt0001

Different superscripts indicate significant differences among stand types in each season (p lt 005) BD (g cmminus1) bulk density WC () soil water content Tem (C) temperature SOC(g kgminus1) soil organic carbon TN (g kgminus1) total nitrogen TP (g kgminus1) total phosphorus AP (mg kgminus1) available phosphorus TK (g kgminus1) total potassium AK (mg kgminus1) availablepotassium NO3

minus-N (mg kgminus1) nitrate nitrogen NH4+-N (mg kgminus1) ammonium nitrogen AN (mg kgminus1) Available nitrogen CEC (cmol + kgminus1) cation exchange capacity

Forests 2019 10 879 9 of 20

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantlyaffected by stand type and MBN was also significantly affected by season and interaction betweenstand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a)In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantlyhigher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highestMBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOCand MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significantlevel in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOCand MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-coolseason (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season(Figure 3b) were significant

Forests 2019 10 x FOR PEER REVIEW 10 of 22

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantly affected by stand type and MBN was also significantly affected by season and interaction between stand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a) In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantly higher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highest MBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOC and MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significant level in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOC and MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-cool season (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season (Figure 3b) were significant

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBN MBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a) and wet-warm season (b)

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBNMBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005level by LSD multiple comparisons among stand types (n = 5)

Forests 2019 10 x FOR PEER REVIEW 10 of 22

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantly affected by stand type and MBN was also significantly affected by season and interaction between stand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a) In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantly higher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highest MBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOC and MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significant level in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOC and MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-cool season (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season (Figure 3b) were significant

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBN MBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a) and wet-warm season (b)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a)and wet-warm season (b)

Forests 2019 10 879 10 of 20

In the dry-cool season total PLFAs AMF PLFAs actinobacteria PLFAs bacteria PLFAs fungiPLFAs GP bacteria PLFAs and GP-to-GN PLFAs ratio were smaller in MIX than those in PPM andPCH On the other hand in the wet-warm season these PLFAs were smaller in PPM than thosein PCH and MIX (Table 4) Specifically the ratios of bacteria-to-fungi PLFA and GP-to-GN PLFAwere significantly higher in MIX than those in PPM and PCH in the wet-warm season These resultsindicated that the response of the soil microbial community structure to tree species mixture varied indifferent seasons Two-way ANOVAs showed that both stand type and season significantly affectedall the tested microbial variables while the interaction between stand type and season significantlyinfluenced total PLFAs bacteria PLFAs GP bacteria PLFAs Acti PLFAs bacteria-to-fungi PLFA andGP-to-GN PLFA (Table 4)

Across plantations and seasons bacteria PLFAs were the most abundant followed by fungi andactinobacteria PLFAs (Figure 4 and Table S3) It was worth noting that the relative abundance ofGN bacteria PLFAs was significantly higher in MIX than in other plantations in the dry-cool season(Figure 4) Furthermore the relative abundance of GP bacteria PLFAs was significantly higher andthat of fungi PLFAs was significantly lower in MIX than in other plantations in the wet-warm season

Forests 2019 10 x FOR PEER REVIEW 13 of 22

Across plantations and seasons bacteria PLFAs were the most abundant followed by fungi and actinobacteria PLFAs (Figure 4 and Table S3) It was worth noting that the relative abundance of GN bacteria PLFAs was significantly higher in MIX than in other plantations in the dry-cool season (Figure 4) Furthermore the relative abundance of GP bacteria PLFAs was significantly higher and that of fungi PLFAs was significantly lower in MIX than in other plantations in the wet-warm season

Figure 4 The relative abundance of PLFAs (mol ) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleaf plantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increased APH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increased urease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg) Carbon source utilization ability of the soil microbial community indicated by average well color development (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation (Figure 6)

Figure 4 The relative abundance of PLFAs (mol ) in three plantations in the dry-cool season (a) andwet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level byLSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 11 of 20

Table 4 Soil microbial community PLFAs content and structure (0ndash10 cm soil depth) in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM)plantation and mixed C hystrix and P massoniana (MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH MIX PPM PCH MIX PPM F p F p F p

TPLFAs 3767 plusmn 271 a 3255 plusmn 124 a 3796 plusmn 128 a 4373 plusmn 208 a 4001 plusmn 07 a 3478 plusmn 111 b 464 0020 646 0018 607 0007B 1579 plusmn 081 a 1306 plusmn 043 b 1490 plusmn 051 ab 1764 plusmn 072 a 1723 plusmn 042 a 1276 plusmn 041 b 1274 lt0001 768 0011 1556 lt0001

GP 945 plusmn 043 a 794 plusmn 029 b 918 plusmn 031 a 1066 plusmn 051 a 1155 plusmn 031 a 810 plusmn 031 b 817 0002 1709 lt0001 2030 lt0001GN 591 plusmn 076 a 590 plusmn 016 a 553 plusmn 022 a 619 plusmn 025 a 455 plusmn 037 b 411 plusmn 020 b 526 0013 702 0014 313 0062

F 498 plusmn 034 a 375 plusmn 022 b 424 plusmn 028 ab 492 plusmn 035 a 296 plusmn 043 b 345 plusmn 021 b 1356 lt0001 448 0045 091 0416AMF 104 plusmn 003 a 075 plusmn 003 b 075 plusmn 004 b 085 plusmn 007 a 044 plusmn 008 b 048 plusmn 003 b 2822 lt0001 3483 lt0001 056 0577Acti 426 plusmn 037 ab 377 plusmn 021 b 486 plusmn 020 a 531 plusmn 019 a 487 plusmn 012 b 458 plusmn 010 b 273 0085 1236 0002 650 0006BF 319 plusmn 011 a 351 plusmn 013 a 356 plusmn 017 a 362 plusmn 014 b 635 plusmn 094 a 377 plusmn 033 b 750 0003 1131 0003 598 0008

GPGN 174 plusmn 029 b 135 plusmn 003 b 166 plusmn 004 a 173 plusmn 008 b 263 plusmn 028 a 198 plusmn 011 b 107 0359 1376 0001 733 0003

Different superscripts indicate significant differences among stand types in each season (p lt 005) TPLFAs (nmol gminus1 soil) total PLFAs B (nmol gminus1 soil) bacteria PLFAs GP (nmol gminus1

soil) gram-positive bacteria PLFAs GN (nmol gminus1 soil) gram-negative bacteria PLFAs F (nmol gminus1 soil) fungi PLFAs Acti (nmol gminus1 soil) actinobacteria PLFAs AMF (nmol gminus1 soil)arbuscular mycorrhizal fungi PLFAs BF bacteria-to-fungi PLFA ratio GPGN GP-to-GN ratio

Forests 2019 10 879 12 of 20

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleafplantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increasedAPH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increasedurease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg)Carbon source utilization ability of the soil microbial community indicated by average well colordevelopment (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation(Figure 6)Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in threeplantations in different seasons Error bars are standard error Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 13 of 20

Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in threeplantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in thedry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons(F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pHTK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-coolseason in which SOC and TBB were the major correlates of changes in soil microbial composition(Table S5) A total of 11 biotic and abiotic factors including NO3

minus-N TBB pH TKlitter HBB AP AKTP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warmseason in which NO3

minus-N TBB pH and TKlitter were the major correlates of changes in soil microbialcomposition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

37 Relationship between Soil Microbial Structure and Functions

Soil enzyme activity and soil microbial carbon source utilization ability are both important soilmicrobial functions The first two RDA axes explained 4948 of the variation in soil enzyme activityand carbon utilization ability in the dry-cool season (F = 24 p = 0006 Figure 7b) and 6891 of thevariation in the wet-warm season (F = 64 p = 0002 Figure 7d) AMF and GP were significantlycorrelated with soil microbial functions in the dry-cool season (Table S6) Total bacteria GP bacteriaGN bacteria and actinobacteria were significantly correlated with soil microbial functions in thewet-warm season in which total bacteria GP bacteria and GN bacteria were the major correlates ofchanges of soil enzyme activity (Table S6)

Forests 2019 10 879 14 of 20

Forests 2019 10 x FOR PEER REVIEW 15 of 22

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in the dry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons (F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pH TK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-cool season in which SOC and TBB were the major correlates of changes in soil microbial composition (Table S5) A total of 11 biotic and abiotic factors including NO3minus-N TBB pH TKlitter HBB AP AK TP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warm season in which NO3minus-N TBB pH and TKlitter were the major correlates of changes in soil microbial composition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160 were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant biotic and abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot of soil microbial community structure and soil microbial functions (bd) Above the label represents a significant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowground biomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soil total potassium NO3ndash-N soil nitrate nitrogen HBB herb belowground biomass AP soil available phosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil water

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant bioticand abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot ofsoil microbial community structure and soil microbial functions (bd) Above the label represents asignificant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowgroundbiomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soiltotal potassium NO3

minus-N soil nitrate nitrogen HBB herb belowground biomass AP soil availablephosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil watercontent LP litter production TKlitter litter total potassium GP gram-positive bacteria GN gramnegative bacteria AMF arbuscular mycorrhizal fungi AG α-Glucosidase BG β-14-glucosidaseCB β-Cellobiosidase APH acid phosphatase NAG β-14-N-acetylglucosaminidase PHO phenoloxidase PO peroxidase AWCD average well color development of carbon source metabolism at192 h incubation

4 Discussion

41 Coniferous-Broadleaf Mixture Significantly Increase Stand Biomass and Litter Production

The results of this study indicated that admixing of coniferous and broadleaf tree speciessignificantly increased stand biomass and litter production after 34 years of afforestation in bothdry-cool and wet-warm seasons (Figure 1 Table 2) This may be a consequence of a greater growth of

Forests 2019 10 879 15 of 20

C hystrix in the mixed stands These results confirmed previous findings conducted in our experimentsite measured when the plantations were 26-year-old and 28-year-old respectively [716] A bulkof studies revealed that mixed-species management could affect tree growth stand structure andenvironment [145253] Mixed forests can develop stratified canopies due to niche differences amongdifferent species In the present study trees of the faster growing broad-leaf species (C hystrix) weretaller had a larger DBH and thus formed an upper canopy stratum in the mixed stands (Table 1)While trees of the shorter species (P massoniana) formed a lower stratum with lower DBH and treeheight and the reduced amount of light transmitted through the upper canopy contributed to theirslower growth The dominant tree species within the stand can absorb more sunlight and use itmore efficiently for growth [54] and thus contribute to higher tree biomass and litter productionin mixed stands In addition stratified canopies in mixed plantations can help to maintain levelsof understory species richness and biomass compared with pure plantations due to relatively highlight availabilities [5556] This may be why the mixed stand had higher shrub and herb biomass inour study

Specifically in the present study the broad-leaf species attained greater heights and diameters inmixed stands while masson pine had reduced diameter and height growth in mixed-species comparedto mono-species stands (Table 1) The severe suppression of P massoniana by the faster growingC hystrix may be attributed to differences in the light requirements of the two species [7] The growthpattern of C hystrix and P massoniana in mixed stands in our study (34-year-old) were differed fromwhen they were 26-years-old in which the growth of C hystrix and P massoniana in mixed standswere both greater than in the corresponding mono-species stands [7] This suggests that effects of thetree species mixture could change over time and long-term investigation of mixed forests is of greatsignificance to reveal mixed effects

42 Coniferous-Broadleaf Mixture Significantly Increase Soil Microbial Biomass

The changes of tree species and composition may significantly affect the amount of organic matterinput in soil [57] As the most active part in soil organic matter (SOM) soil microbial biomass isimportant in nutrient cycling and release participating in the circulation of critical elements such ascarbon and nitrogen [23] Because of the quantity and quality of the bulk of SOM changes slowly andthe changes of SOM are difficult to measure due to the spatial heterogeneity of soils soil microbialbiomass and in particular MBC-to-SOC and MBN-to-TN ratio have been suggested as indicators ofthe state and modification of SOM [58ndash60] and as an indicator for carbon (nitrogen) availability insoil [6162] The concentrations of SOC TN and MBC and MBN measured in our study were affectedby stand type and seasons (Table 3 and Table S2 Figure 2) In the dry cool season although the SOCcontent in mixed stands did not significantly differ with that in PCH but significantly lower than thatin PPM the MBC concentration in mixed stands was markedly higher than that in the correspondingmono-species stands Likewise although the TN content in mixed stands was smaller than that in PCHand PPM stands the MBN content in mixed stands was markedly higher than that in the correspondingmono-species stands in the wet-warn season Previous studies found that higher soil microbial biomasscorrelated with higher SOC and TN content that sustain microbial metabolism [2163] However inour study the lower SOC and TN content in MIX was able to maintain a higher microbial biomass thansites where SOC was more abundant Presumably the quality of SOC could explain such a trend [64]and merit further investigation

In this study MBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands(Figure 2) suggesting higher carbon and nitrogen availability in soil and higher soil quality for thegrowth of soil microorganisms by coniferous-broadleaf mixture [21] In addition the effects of admixingon soil microbial biomass MBC-to-SOC and MBN-to-TN ratio were non-additive suggesting theinteraction of P massoniana and C hystrix can have a synergistic effect on improving soil carbon andnitrogen availability (Figure 3) These findings are consistent with many previous studies that havedetermined the effects of tree species mixture on soil microbial biomass [2165] These results may be

Forests 2019 10 879 16 of 20

contributed to (1) greater amount and more diverse organic matter input via litter and rhizodepositionproducts in the mixture of two contrasting tree species [21] (2) the higher use efficiency of soil organiccarbon and nitrogen by microorganisms in mixed stands [66]

We also sought to determine the admixing effects on composition and abundance of soil microbialcommunity with community-level PLFA profiles Surprisingly there were not significant admixingeffects on PLFA content of the major microbial functional groups such as bacteria fungi AMF andactinobacteria (Table 4) However tree species mixture significantly increased the abundance of GNbacteria in the dry-cool season while markedly increased the abundance of GP bacteria but decreasedthe abundance of fungi in the wet-warn season (Figure 4) It seemed that the dominated GN bacteria inthe dry-cool season and GP bacteria in the wet-warn season would correlate with the higher availabilityof soil organic carbon and nitrogen in mixed stands [6768] These correlations are complicated andfurther investigation is required to reveal the underlying mechanisms

43 Effects of Coniferous-Broadleaf Mixture on Soil Microbial Functions

Soil microorganisms excrete soil enzymes to drive nutrient mineralization and litterdecomposition [64] Therefore soil extracellular enzyme activities may be related to the carbonnitrogen and phosphorus cycling in soil [6970] In the present study we found positive admixingeffects on the activities of APH and NAG in the dry-cool season (Figure 5de) On the other handpositive admixing effects on the activities of urease and peroxidase while negative admixing effectson APH was detected in the wet-warn season (Figure 5dfg) This result suggests that the mixtureof C hystrix and P massoniana can accelerate the mineralization rates of soil organic phosphorus andnitrogen These findings were consistent with previous study [67]

The community-level physiological profiling (CLPP) is widely used to characterize microbialcommunities [71ndash73] The average well color development (AWCD) in the CLPP approach reflectsthe ability of soil microorganisms to utilize carbon sources and their metabolic activity The higherAWCD represents the stronger metabolic activity of soil microorganisms [71] In this present study wedetected a significantly positive admixing effect on carbon source utilization ability of the soil microbialcommunities in both dry-cool and wet-warn seasons after 144 h of incubation (Figure 6) Moreover theRDA analysis revealed that carbon source utilization ability of the soil microbial communities werepositive related with APH in the dry-cool season while related with urease and peroxidase activitiesin the wet-warn season (Figure 7) As a whole the higher soil microbial functions in mixed stands arelikely the results of the effects of AMF and GP bacteria in the dry-cool season and the effects of bacteriaincluding GP and GN bacteria the wet-warn season (Table S6)

5 Conclusions

In our study we measured significantly higher tree shrub and herb biomass and litter productionin the coniferous-broadleaf mixed plantation after 34 years of afforestation As expected MBC MBNMBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands suggesting highercarbon and nitrogen availability in soil and higher soil quality for the growth of soil microorganisms inour coniferous-broadleaf mixed stand In addition coniferous-broadleaf mixture can increase carbonsource utilization ability of soil microbial community and accelerate the mineralization rates of soilorganic phosphorus and nitrogen by increasing acid phosphatase β-14-N-acetylglucosaminidaseand urease activities The higher soil microbial biomass and activities are likely the result of greateramount and more diverse organic matter input via litter and rhizodeposition products and the higheruse efficiency of soil organic carbon and nitrogen by microorganisms in mixed stands Howeverthe correlations between soil dominated microbial functional groups and the higher availabilityof soil organic carbon and nitrogen along with the quality of SOC in mixed stands merit furtherinvestigation Our results highlight that establishment of coniferous-broadleaf mixed forests maybe a good management practice as coniferous-broadleaf mixture could accumulate higher stand

Forests 2019 10 879 17 of 20

biomass return more litter and increase soil microbial biomass and functions over the long term insubtropical China

Supplementary Materials The following are available online at httpwwwmdpicom1999-49071010879s1Table S1 Effects of stand type season and their interactions on stand biomass Table S2 Effects of stand type seasonand their interactions on soil microbial structure Table S3 Effects of stand type season and their interactionson relative abundance of the soil microbial community PLFAs Table S4 Effects of stand type season and theirinteractions on soil enzyme activity Table S5 Marginal and conditional effects of biotic and abiotic factors on soilmicrobial composition obtained from the summary of forward selection in Redundancy analysis (RDA) Table S6Marginal and conditional effects of soil microbial functional groups on soil enzyme activity and soil microbialcarbon source utilization ability obtained from the summary of forward selection in Redundancy analysis (RDA)

Author Contributions WW collected data made experiment analysis formal data and drafted the manuscriptYW designed the work XZ revised the manuscript and participated in analyzing the experiment data HZ YYXH and XL ZQ YL LY HL participated in collecting the experiment data

Funding This research was funded by National Natural Science Foundation of China (31860171 3146012131560201) the Guangxi Key Research and Development Program (2018AB40007) Innovation Project of GuangxiGraduate Education (YCBZ2017011) and the Guangxi Universities Science and technology (201201ZD001)

Acknowledgments We gratefully acknowledge the supports in field sampling by the Experimental Center ofTropical Forestry Chinese Academy of Forestry A special acknowledgment is given to Patrick M Finnegan for hiswriting suggestions

Conflicts of Interest The authors declare no conflicts of interest

References

1 Zhang X Liu S Huang Y Fu S Wang J Ming A Li X Yao M Li H Tree species mixture inhibits soilorganic carbon mineralization accompanied by decreased r-selected bacteria Plant Soil 2018 431 203ndash216[CrossRef]

2 Pan Y Birdsey RA Fang J Houghton R Kauppi PE Kurz WA Phillips OL Shvidenko ALewis SL Canadell JG et al A large and persistent carbon sink in the worldrsquos forests Science 2011 333988ndash993 [CrossRef] [PubMed]

3 Chen X Ju Q Lin K Development status issues and countermeasures of Chinarsquo s plantation World For Res2014 27 54ndash59

4 Wang H Lencinas MV Ross Friedman C Wang X Qiu J Understory plant diversity assessment ofEucalyptus plantations over three vegetation types in Yunnan China New For 2011 42 101ndash116 [CrossRef]

5 Sang PM Lamb D Bonner M Schmidt S Carbon sequestration and soil fertility of tropical tree plantationsand secondary forest established on degraded land Plant Soil 2013 362 187ndash200 [CrossRef]

6 Li C Zhou X Wen Y Zhu H Qin Z Li X You Y Huang X Effects of high-generation ratations ofEucalyptus on undergrowth soil fertility and enzyme activities Guangxi Sci 2019 26 176ndash186

7 You Y Huang X Zhu H Liu S Liang H Wen Y Wang H Cai D Ye D Positive interactions betweenPinus massoniana and Castanopsis hystrix species in the uneven-aged mixed plantations can produce moreecosystem carbon in subtropical China For Ecol Manag 2018 410 193ndash200 [CrossRef]

8 Huang X Liu S You Y Wen Y Wang H Wang J Microbial community and associated enzymes activityinfluence soil carbon chemical composition in Eucalyptus urophylla plantation with mixing N2-fixing speciesin sub-tropical China Plant Soil 2017 414 199ndash212 [CrossRef]

9 Kelty MJ The role of species mixtures in plantation forestry For Ecol Manag 2006 233 195ndash204 [CrossRef]10 Bauhus J Khanna P Menden N Aboveground and belowground interactions in mixed plantations of

Eucalyptus globulus and Acacia mearnsii Can J For Res 2000 30 1886ndash1894 [CrossRef]11 Vandermeer J The Ecology of Intercropping Cambridge University Press New York NY USA 1989 p 23712 Forrester DI The spatial and temporal dynamics of species interactions in mixed-species forests From

pattern to process For Ecol Manag 2014 312 282ndash292 [CrossRef]13 Forrester DI Bauhus J A review of processes behind diversity-productivity relationships in forests

Curr For Rep 2016 2 45ndash61 [CrossRef]14 Wang H Liu S Wang J You Y Yang Y Shi Z Huang X Zheng L Li Z Ming A et al Mixed-species

plantation with Pinus massoniana and Castanopsis hystrix accelerates C loss in recalcitrant coniferous litter butslows C loss in labile broadleaf litter in southern China For Ecol Manag 2018 422 207ndash213 [CrossRef]

Forests 2019 10 879 9 of 20

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantlyaffected by stand type and MBN was also significantly affected by season and interaction betweenstand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a)In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantlyhigher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highestMBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOCand MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significantlevel in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOCand MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-coolseason (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season(Figure 3b) were significant

Forests 2019 10 x FOR PEER REVIEW 10 of 22

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantly affected by stand type and MBN was also significantly affected by season and interaction between stand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a) In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantly higher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highest MBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOC and MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significant level in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOC and MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-cool season (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season (Figure 3b) were significant

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBN MBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a) and wet-warm season (b)

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBNMBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005level by LSD multiple comparisons among stand types (n = 5)

Forests 2019 10 x FOR PEER REVIEW 10 of 22

34 Soil Microbial Biomass and Community Structure

Two-way ANOVAs indicated that MBC MBN MBC-to-SOC and MBN-to-TN were significantly affected by stand type and MBN was also significantly affected by season and interaction between stand type and season (Table S2)

In the dry-cool season MBC was significantly higher in MIX than in PCH and PPM (Figure 2a) In the wet-warm season MBC was significantly higher in MIX than in PPM and MBN was significantly higher in MIX than in PCH and PPM (Figure 2b) The coniferous-broadleaf mixed stand had the highest MBC-to-SOC and MBN-to-TN ratio in both seasons (Figure 2cd) The differences of the MBC-to-SOC and MBN-to-TN ratio between the mixed stand and the corresponding pure stands reached a significant level in the wet season Non-additive effects (NAE) of species mixing on MBC MBN MBC-to-SOC and MBN-to-TN ratio were recorded Synergistic effects on MBC and MBC-to-SOC ratio in the dry-cool season (Figure 3a) and on MBC MBN MBC-to-SOC and MBN-to-TN ratio in the wet-warm season (Figure 3b) were significant

Figure 2 Soil microbial biomass in three plantations in different seasons ((andashd) refer to MBC MBN MBC-to-SOC and MBN-to-TN) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a) and wet-warm season (b)

Figure 3 Not-additive effects of mixed plantation on soil microbial biomass in the dry-cool season (a)and wet-warm season (b)

Forests 2019 10 879 10 of 20

In the dry-cool season total PLFAs AMF PLFAs actinobacteria PLFAs bacteria PLFAs fungiPLFAs GP bacteria PLFAs and GP-to-GN PLFAs ratio were smaller in MIX than those in PPM andPCH On the other hand in the wet-warm season these PLFAs were smaller in PPM than thosein PCH and MIX (Table 4) Specifically the ratios of bacteria-to-fungi PLFA and GP-to-GN PLFAwere significantly higher in MIX than those in PPM and PCH in the wet-warm season These resultsindicated that the response of the soil microbial community structure to tree species mixture varied indifferent seasons Two-way ANOVAs showed that both stand type and season significantly affectedall the tested microbial variables while the interaction between stand type and season significantlyinfluenced total PLFAs bacteria PLFAs GP bacteria PLFAs Acti PLFAs bacteria-to-fungi PLFA andGP-to-GN PLFA (Table 4)

Across plantations and seasons bacteria PLFAs were the most abundant followed by fungi andactinobacteria PLFAs (Figure 4 and Table S3) It was worth noting that the relative abundance ofGN bacteria PLFAs was significantly higher in MIX than in other plantations in the dry-cool season(Figure 4) Furthermore the relative abundance of GP bacteria PLFAs was significantly higher andthat of fungi PLFAs was significantly lower in MIX than in other plantations in the wet-warm season

Forests 2019 10 x FOR PEER REVIEW 13 of 22

Across plantations and seasons bacteria PLFAs were the most abundant followed by fungi and actinobacteria PLFAs (Figure 4 and Table S3) It was worth noting that the relative abundance of GN bacteria PLFAs was significantly higher in MIX than in other plantations in the dry-cool season (Figure 4) Furthermore the relative abundance of GP bacteria PLFAs was significantly higher and that of fungi PLFAs was significantly lower in MIX than in other plantations in the wet-warm season

Figure 4 The relative abundance of PLFAs (mol ) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleaf plantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increased APH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increased urease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg) Carbon source utilization ability of the soil microbial community indicated by average well color development (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation (Figure 6)

Figure 4 The relative abundance of PLFAs (mol ) in three plantations in the dry-cool season (a) andwet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level byLSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 11 of 20

Table 4 Soil microbial community PLFAs content and structure (0ndash10 cm soil depth) in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM)plantation and mixed C hystrix and P massoniana (MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH MIX PPM PCH MIX PPM F p F p F p

TPLFAs 3767 plusmn 271 a 3255 plusmn 124 a 3796 plusmn 128 a 4373 plusmn 208 a 4001 plusmn 07 a 3478 plusmn 111 b 464 0020 646 0018 607 0007B 1579 plusmn 081 a 1306 plusmn 043 b 1490 plusmn 051 ab 1764 plusmn 072 a 1723 plusmn 042 a 1276 plusmn 041 b 1274 lt0001 768 0011 1556 lt0001

GP 945 plusmn 043 a 794 plusmn 029 b 918 plusmn 031 a 1066 plusmn 051 a 1155 plusmn 031 a 810 plusmn 031 b 817 0002 1709 lt0001 2030 lt0001GN 591 plusmn 076 a 590 plusmn 016 a 553 plusmn 022 a 619 plusmn 025 a 455 plusmn 037 b 411 plusmn 020 b 526 0013 702 0014 313 0062

F 498 plusmn 034 a 375 plusmn 022 b 424 plusmn 028 ab 492 plusmn 035 a 296 plusmn 043 b 345 plusmn 021 b 1356 lt0001 448 0045 091 0416AMF 104 plusmn 003 a 075 plusmn 003 b 075 plusmn 004 b 085 plusmn 007 a 044 plusmn 008 b 048 plusmn 003 b 2822 lt0001 3483 lt0001 056 0577Acti 426 plusmn 037 ab 377 plusmn 021 b 486 plusmn 020 a 531 plusmn 019 a 487 plusmn 012 b 458 plusmn 010 b 273 0085 1236 0002 650 0006BF 319 plusmn 011 a 351 plusmn 013 a 356 plusmn 017 a 362 plusmn 014 b 635 plusmn 094 a 377 plusmn 033 b 750 0003 1131 0003 598 0008

GPGN 174 plusmn 029 b 135 plusmn 003 b 166 plusmn 004 a 173 plusmn 008 b 263 plusmn 028 a 198 plusmn 011 b 107 0359 1376 0001 733 0003

Different superscripts indicate significant differences among stand types in each season (p lt 005) TPLFAs (nmol gminus1 soil) total PLFAs B (nmol gminus1 soil) bacteria PLFAs GP (nmol gminus1

soil) gram-positive bacteria PLFAs GN (nmol gminus1 soil) gram-negative bacteria PLFAs F (nmol gminus1 soil) fungi PLFAs Acti (nmol gminus1 soil) actinobacteria PLFAs AMF (nmol gminus1 soil)arbuscular mycorrhizal fungi PLFAs BF bacteria-to-fungi PLFA ratio GPGN GP-to-GN ratio

Forests 2019 10 879 12 of 20

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleafplantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increasedAPH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increasedurease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg)Carbon source utilization ability of the soil microbial community indicated by average well colordevelopment (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation(Figure 6)Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in threeplantations in different seasons Error bars are standard error Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 13 of 20

Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in threeplantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in thedry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons(F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pHTK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-coolseason in which SOC and TBB were the major correlates of changes in soil microbial composition(Table S5) A total of 11 biotic and abiotic factors including NO3

minus-N TBB pH TKlitter HBB AP AKTP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warmseason in which NO3

minus-N TBB pH and TKlitter were the major correlates of changes in soil microbialcomposition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

37 Relationship between Soil Microbial Structure and Functions

Soil enzyme activity and soil microbial carbon source utilization ability are both important soilmicrobial functions The first two RDA axes explained 4948 of the variation in soil enzyme activityand carbon utilization ability in the dry-cool season (F = 24 p = 0006 Figure 7b) and 6891 of thevariation in the wet-warm season (F = 64 p = 0002 Figure 7d) AMF and GP were significantlycorrelated with soil microbial functions in the dry-cool season (Table S6) Total bacteria GP bacteriaGN bacteria and actinobacteria were significantly correlated with soil microbial functions in thewet-warm season in which total bacteria GP bacteria and GN bacteria were the major correlates ofchanges of soil enzyme activity (Table S6)

Forests 2019 10 879 14 of 20

Forests 2019 10 x FOR PEER REVIEW 15 of 22

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in the dry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons (F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pH TK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-cool season in which SOC and TBB were the major correlates of changes in soil microbial composition (Table S5) A total of 11 biotic and abiotic factors including NO3minus-N TBB pH TKlitter HBB AP AK TP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warm season in which NO3minus-N TBB pH and TKlitter were the major correlates of changes in soil microbial composition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160 were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant biotic and abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot of soil microbial community structure and soil microbial functions (bd) Above the label represents a significant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowground biomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soil total potassium NO3ndash-N soil nitrate nitrogen HBB herb belowground biomass AP soil available phosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil water

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant bioticand abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot ofsoil microbial community structure and soil microbial functions (bd) Above the label represents asignificant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowgroundbiomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soiltotal potassium NO3

minus-N soil nitrate nitrogen HBB herb belowground biomass AP soil availablephosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil watercontent LP litter production TKlitter litter total potassium GP gram-positive bacteria GN gramnegative bacteria AMF arbuscular mycorrhizal fungi AG α-Glucosidase BG β-14-glucosidaseCB β-Cellobiosidase APH acid phosphatase NAG β-14-N-acetylglucosaminidase PHO phenoloxidase PO peroxidase AWCD average well color development of carbon source metabolism at192 h incubation

4 Discussion

41 Coniferous-Broadleaf Mixture Significantly Increase Stand Biomass and Litter Production

The results of this study indicated that admixing of coniferous and broadleaf tree speciessignificantly increased stand biomass and litter production after 34 years of afforestation in bothdry-cool and wet-warm seasons (Figure 1 Table 2) This may be a consequence of a greater growth of

Forests 2019 10 879 15 of 20

C hystrix in the mixed stands These results confirmed previous findings conducted in our experimentsite measured when the plantations were 26-year-old and 28-year-old respectively [716] A bulkof studies revealed that mixed-species management could affect tree growth stand structure andenvironment [145253] Mixed forests can develop stratified canopies due to niche differences amongdifferent species In the present study trees of the faster growing broad-leaf species (C hystrix) weretaller had a larger DBH and thus formed an upper canopy stratum in the mixed stands (Table 1)While trees of the shorter species (P massoniana) formed a lower stratum with lower DBH and treeheight and the reduced amount of light transmitted through the upper canopy contributed to theirslower growth The dominant tree species within the stand can absorb more sunlight and use itmore efficiently for growth [54] and thus contribute to higher tree biomass and litter productionin mixed stands In addition stratified canopies in mixed plantations can help to maintain levelsof understory species richness and biomass compared with pure plantations due to relatively highlight availabilities [5556] This may be why the mixed stand had higher shrub and herb biomass inour study

Specifically in the present study the broad-leaf species attained greater heights and diameters inmixed stands while masson pine had reduced diameter and height growth in mixed-species comparedto mono-species stands (Table 1) The severe suppression of P massoniana by the faster growingC hystrix may be attributed to differences in the light requirements of the two species [7] The growthpattern of C hystrix and P massoniana in mixed stands in our study (34-year-old) were differed fromwhen they were 26-years-old in which the growth of C hystrix and P massoniana in mixed standswere both greater than in the corresponding mono-species stands [7] This suggests that effects of thetree species mixture could change over time and long-term investigation of mixed forests is of greatsignificance to reveal mixed effects

42 Coniferous-Broadleaf Mixture Significantly Increase Soil Microbial Biomass

The changes of tree species and composition may significantly affect the amount of organic matterinput in soil [57] As the most active part in soil organic matter (SOM) soil microbial biomass isimportant in nutrient cycling and release participating in the circulation of critical elements such ascarbon and nitrogen [23] Because of the quantity and quality of the bulk of SOM changes slowly andthe changes of SOM are difficult to measure due to the spatial heterogeneity of soils soil microbialbiomass and in particular MBC-to-SOC and MBN-to-TN ratio have been suggested as indicators ofthe state and modification of SOM [58ndash60] and as an indicator for carbon (nitrogen) availability insoil [6162] The concentrations of SOC TN and MBC and MBN measured in our study were affectedby stand type and seasons (Table 3 and Table S2 Figure 2) In the dry cool season although the SOCcontent in mixed stands did not significantly differ with that in PCH but significantly lower than thatin PPM the MBC concentration in mixed stands was markedly higher than that in the correspondingmono-species stands Likewise although the TN content in mixed stands was smaller than that in PCHand PPM stands the MBN content in mixed stands was markedly higher than that in the correspondingmono-species stands in the wet-warn season Previous studies found that higher soil microbial biomasscorrelated with higher SOC and TN content that sustain microbial metabolism [2163] However inour study the lower SOC and TN content in MIX was able to maintain a higher microbial biomass thansites where SOC was more abundant Presumably the quality of SOC could explain such a trend [64]and merit further investigation

In this study MBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands(Figure 2) suggesting higher carbon and nitrogen availability in soil and higher soil quality for thegrowth of soil microorganisms by coniferous-broadleaf mixture [21] In addition the effects of admixingon soil microbial biomass MBC-to-SOC and MBN-to-TN ratio were non-additive suggesting theinteraction of P massoniana and C hystrix can have a synergistic effect on improving soil carbon andnitrogen availability (Figure 3) These findings are consistent with many previous studies that havedetermined the effects of tree species mixture on soil microbial biomass [2165] These results may be

Forests 2019 10 879 16 of 20

contributed to (1) greater amount and more diverse organic matter input via litter and rhizodepositionproducts in the mixture of two contrasting tree species [21] (2) the higher use efficiency of soil organiccarbon and nitrogen by microorganisms in mixed stands [66]

We also sought to determine the admixing effects on composition and abundance of soil microbialcommunity with community-level PLFA profiles Surprisingly there were not significant admixingeffects on PLFA content of the major microbial functional groups such as bacteria fungi AMF andactinobacteria (Table 4) However tree species mixture significantly increased the abundance of GNbacteria in the dry-cool season while markedly increased the abundance of GP bacteria but decreasedthe abundance of fungi in the wet-warn season (Figure 4) It seemed that the dominated GN bacteria inthe dry-cool season and GP bacteria in the wet-warn season would correlate with the higher availabilityof soil organic carbon and nitrogen in mixed stands [6768] These correlations are complicated andfurther investigation is required to reveal the underlying mechanisms

43 Effects of Coniferous-Broadleaf Mixture on Soil Microbial Functions

Soil microorganisms excrete soil enzymes to drive nutrient mineralization and litterdecomposition [64] Therefore soil extracellular enzyme activities may be related to the carbonnitrogen and phosphorus cycling in soil [6970] In the present study we found positive admixingeffects on the activities of APH and NAG in the dry-cool season (Figure 5de) On the other handpositive admixing effects on the activities of urease and peroxidase while negative admixing effectson APH was detected in the wet-warn season (Figure 5dfg) This result suggests that the mixtureof C hystrix and P massoniana can accelerate the mineralization rates of soil organic phosphorus andnitrogen These findings were consistent with previous study [67]

The community-level physiological profiling (CLPP) is widely used to characterize microbialcommunities [71ndash73] The average well color development (AWCD) in the CLPP approach reflectsthe ability of soil microorganisms to utilize carbon sources and their metabolic activity The higherAWCD represents the stronger metabolic activity of soil microorganisms [71] In this present study wedetected a significantly positive admixing effect on carbon source utilization ability of the soil microbialcommunities in both dry-cool and wet-warn seasons after 144 h of incubation (Figure 6) Moreover theRDA analysis revealed that carbon source utilization ability of the soil microbial communities werepositive related with APH in the dry-cool season while related with urease and peroxidase activitiesin the wet-warn season (Figure 7) As a whole the higher soil microbial functions in mixed stands arelikely the results of the effects of AMF and GP bacteria in the dry-cool season and the effects of bacteriaincluding GP and GN bacteria the wet-warn season (Table S6)

5 Conclusions

In our study we measured significantly higher tree shrub and herb biomass and litter productionin the coniferous-broadleaf mixed plantation after 34 years of afforestation As expected MBC MBNMBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands suggesting highercarbon and nitrogen availability in soil and higher soil quality for the growth of soil microorganisms inour coniferous-broadleaf mixed stand In addition coniferous-broadleaf mixture can increase carbonsource utilization ability of soil microbial community and accelerate the mineralization rates of soilorganic phosphorus and nitrogen by increasing acid phosphatase β-14-N-acetylglucosaminidaseand urease activities The higher soil microbial biomass and activities are likely the result of greateramount and more diverse organic matter input via litter and rhizodeposition products and the higheruse efficiency of soil organic carbon and nitrogen by microorganisms in mixed stands Howeverthe correlations between soil dominated microbial functional groups and the higher availabilityof soil organic carbon and nitrogen along with the quality of SOC in mixed stands merit furtherinvestigation Our results highlight that establishment of coniferous-broadleaf mixed forests maybe a good management practice as coniferous-broadleaf mixture could accumulate higher stand

Forests 2019 10 879 17 of 20

biomass return more litter and increase soil microbial biomass and functions over the long term insubtropical China

Supplementary Materials The following are available online at httpwwwmdpicom1999-49071010879s1Table S1 Effects of stand type season and their interactions on stand biomass Table S2 Effects of stand type seasonand their interactions on soil microbial structure Table S3 Effects of stand type season and their interactionson relative abundance of the soil microbial community PLFAs Table S4 Effects of stand type season and theirinteractions on soil enzyme activity Table S5 Marginal and conditional effects of biotic and abiotic factors on soilmicrobial composition obtained from the summary of forward selection in Redundancy analysis (RDA) Table S6Marginal and conditional effects of soil microbial functional groups on soil enzyme activity and soil microbialcarbon source utilization ability obtained from the summary of forward selection in Redundancy analysis (RDA)

Author Contributions WW collected data made experiment analysis formal data and drafted the manuscriptYW designed the work XZ revised the manuscript and participated in analyzing the experiment data HZ YYXH and XL ZQ YL LY HL participated in collecting the experiment data

Funding This research was funded by National Natural Science Foundation of China (31860171 3146012131560201) the Guangxi Key Research and Development Program (2018AB40007) Innovation Project of GuangxiGraduate Education (YCBZ2017011) and the Guangxi Universities Science and technology (201201ZD001)

Acknowledgments We gratefully acknowledge the supports in field sampling by the Experimental Center ofTropical Forestry Chinese Academy of Forestry A special acknowledgment is given to Patrick M Finnegan for hiswriting suggestions

Conflicts of Interest The authors declare no conflicts of interest

References

1 Zhang X Liu S Huang Y Fu S Wang J Ming A Li X Yao M Li H Tree species mixture inhibits soilorganic carbon mineralization accompanied by decreased r-selected bacteria Plant Soil 2018 431 203ndash216[CrossRef]

2 Pan Y Birdsey RA Fang J Houghton R Kauppi PE Kurz WA Phillips OL Shvidenko ALewis SL Canadell JG et al A large and persistent carbon sink in the worldrsquos forests Science 2011 333988ndash993 [CrossRef] [PubMed]

3 Chen X Ju Q Lin K Development status issues and countermeasures of Chinarsquo s plantation World For Res2014 27 54ndash59

4 Wang H Lencinas MV Ross Friedman C Wang X Qiu J Understory plant diversity assessment ofEucalyptus plantations over three vegetation types in Yunnan China New For 2011 42 101ndash116 [CrossRef]

5 Sang PM Lamb D Bonner M Schmidt S Carbon sequestration and soil fertility of tropical tree plantationsand secondary forest established on degraded land Plant Soil 2013 362 187ndash200 [CrossRef]

6 Li C Zhou X Wen Y Zhu H Qin Z Li X You Y Huang X Effects of high-generation ratations ofEucalyptus on undergrowth soil fertility and enzyme activities Guangxi Sci 2019 26 176ndash186

7 You Y Huang X Zhu H Liu S Liang H Wen Y Wang H Cai D Ye D Positive interactions betweenPinus massoniana and Castanopsis hystrix species in the uneven-aged mixed plantations can produce moreecosystem carbon in subtropical China For Ecol Manag 2018 410 193ndash200 [CrossRef]

8 Huang X Liu S You Y Wen Y Wang H Wang J Microbial community and associated enzymes activityinfluence soil carbon chemical composition in Eucalyptus urophylla plantation with mixing N2-fixing speciesin sub-tropical China Plant Soil 2017 414 199ndash212 [CrossRef]

9 Kelty MJ The role of species mixtures in plantation forestry For Ecol Manag 2006 233 195ndash204 [CrossRef]10 Bauhus J Khanna P Menden N Aboveground and belowground interactions in mixed plantations of

Eucalyptus globulus and Acacia mearnsii Can J For Res 2000 30 1886ndash1894 [CrossRef]11 Vandermeer J The Ecology of Intercropping Cambridge University Press New York NY USA 1989 p 23712 Forrester DI The spatial and temporal dynamics of species interactions in mixed-species forests From

pattern to process For Ecol Manag 2014 312 282ndash292 [CrossRef]13 Forrester DI Bauhus J A review of processes behind diversity-productivity relationships in forests

Curr For Rep 2016 2 45ndash61 [CrossRef]14 Wang H Liu S Wang J You Y Yang Y Shi Z Huang X Zheng L Li Z Ming A et al Mixed-species

plantation with Pinus massoniana and Castanopsis hystrix accelerates C loss in recalcitrant coniferous litter butslows C loss in labile broadleaf litter in southern China For Ecol Manag 2018 422 207ndash213 [CrossRef]

Forests 2019 10 879 10 of 20

In the dry-cool season total PLFAs AMF PLFAs actinobacteria PLFAs bacteria PLFAs fungiPLFAs GP bacteria PLFAs and GP-to-GN PLFAs ratio were smaller in MIX than those in PPM andPCH On the other hand in the wet-warm season these PLFAs were smaller in PPM than thosein PCH and MIX (Table 4) Specifically the ratios of bacteria-to-fungi PLFA and GP-to-GN PLFAwere significantly higher in MIX than those in PPM and PCH in the wet-warm season These resultsindicated that the response of the soil microbial community structure to tree species mixture varied indifferent seasons Two-way ANOVAs showed that both stand type and season significantly affectedall the tested microbial variables while the interaction between stand type and season significantlyinfluenced total PLFAs bacteria PLFAs GP bacteria PLFAs Acti PLFAs bacteria-to-fungi PLFA andGP-to-GN PLFA (Table 4)

Across plantations and seasons bacteria PLFAs were the most abundant followed by fungi andactinobacteria PLFAs (Figure 4 and Table S3) It was worth noting that the relative abundance ofGN bacteria PLFAs was significantly higher in MIX than in other plantations in the dry-cool season(Figure 4) Furthermore the relative abundance of GP bacteria PLFAs was significantly higher andthat of fungi PLFAs was significantly lower in MIX than in other plantations in the wet-warm season

Forests 2019 10 x FOR PEER REVIEW 13 of 22

Across plantations and seasons bacteria PLFAs were the most abundant followed by fungi and actinobacteria PLFAs (Figure 4 and Table S3) It was worth noting that the relative abundance of GN bacteria PLFAs was significantly higher in MIX than in other plantations in the dry-cool season (Figure 4) Furthermore the relative abundance of GP bacteria PLFAs was significantly higher and that of fungi PLFAs was significantly lower in MIX than in other plantations in the wet-warm season

Figure 4 The relative abundance of PLFAs (mol ) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleaf plantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increased APH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increased urease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg) Carbon source utilization ability of the soil microbial community indicated by average well color development (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation (Figure 6)

Figure 4 The relative abundance of PLFAs (mol ) in three plantations in the dry-cool season (a) andwet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level byLSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 11 of 20

Table 4 Soil microbial community PLFAs content and structure (0ndash10 cm soil depth) in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM)plantation and mixed C hystrix and P massoniana (MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH MIX PPM PCH MIX PPM F p F p F p

TPLFAs 3767 plusmn 271 a 3255 plusmn 124 a 3796 plusmn 128 a 4373 plusmn 208 a 4001 plusmn 07 a 3478 plusmn 111 b 464 0020 646 0018 607 0007B 1579 plusmn 081 a 1306 plusmn 043 b 1490 plusmn 051 ab 1764 plusmn 072 a 1723 plusmn 042 a 1276 plusmn 041 b 1274 lt0001 768 0011 1556 lt0001

GP 945 plusmn 043 a 794 plusmn 029 b 918 plusmn 031 a 1066 plusmn 051 a 1155 plusmn 031 a 810 plusmn 031 b 817 0002 1709 lt0001 2030 lt0001GN 591 plusmn 076 a 590 plusmn 016 a 553 plusmn 022 a 619 plusmn 025 a 455 plusmn 037 b 411 plusmn 020 b 526 0013 702 0014 313 0062

F 498 plusmn 034 a 375 plusmn 022 b 424 plusmn 028 ab 492 plusmn 035 a 296 plusmn 043 b 345 plusmn 021 b 1356 lt0001 448 0045 091 0416AMF 104 plusmn 003 a 075 plusmn 003 b 075 plusmn 004 b 085 plusmn 007 a 044 plusmn 008 b 048 plusmn 003 b 2822 lt0001 3483 lt0001 056 0577Acti 426 plusmn 037 ab 377 plusmn 021 b 486 plusmn 020 a 531 plusmn 019 a 487 plusmn 012 b 458 plusmn 010 b 273 0085 1236 0002 650 0006BF 319 plusmn 011 a 351 plusmn 013 a 356 plusmn 017 a 362 plusmn 014 b 635 plusmn 094 a 377 plusmn 033 b 750 0003 1131 0003 598 0008

GPGN 174 plusmn 029 b 135 plusmn 003 b 166 plusmn 004 a 173 plusmn 008 b 263 plusmn 028 a 198 plusmn 011 b 107 0359 1376 0001 733 0003

Different superscripts indicate significant differences among stand types in each season (p lt 005) TPLFAs (nmol gminus1 soil) total PLFAs B (nmol gminus1 soil) bacteria PLFAs GP (nmol gminus1

soil) gram-positive bacteria PLFAs GN (nmol gminus1 soil) gram-negative bacteria PLFAs F (nmol gminus1 soil) fungi PLFAs Acti (nmol gminus1 soil) actinobacteria PLFAs AMF (nmol gminus1 soil)arbuscular mycorrhizal fungi PLFAs BF bacteria-to-fungi PLFA ratio GPGN GP-to-GN ratio

Forests 2019 10 879 12 of 20

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleafplantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increasedAPH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increasedurease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg)Carbon source utilization ability of the soil microbial community indicated by average well colordevelopment (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation(Figure 6)Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in threeplantations in different seasons Error bars are standard error Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 13 of 20

Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in threeplantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in thedry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons(F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pHTK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-coolseason in which SOC and TBB were the major correlates of changes in soil microbial composition(Table S5) A total of 11 biotic and abiotic factors including NO3

minus-N TBB pH TKlitter HBB AP AKTP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warmseason in which NO3

minus-N TBB pH and TKlitter were the major correlates of changes in soil microbialcomposition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

37 Relationship between Soil Microbial Structure and Functions

Soil enzyme activity and soil microbial carbon source utilization ability are both important soilmicrobial functions The first two RDA axes explained 4948 of the variation in soil enzyme activityand carbon utilization ability in the dry-cool season (F = 24 p = 0006 Figure 7b) and 6891 of thevariation in the wet-warm season (F = 64 p = 0002 Figure 7d) AMF and GP were significantlycorrelated with soil microbial functions in the dry-cool season (Table S6) Total bacteria GP bacteriaGN bacteria and actinobacteria were significantly correlated with soil microbial functions in thewet-warm season in which total bacteria GP bacteria and GN bacteria were the major correlates ofchanges of soil enzyme activity (Table S6)

Forests 2019 10 879 14 of 20

Forests 2019 10 x FOR PEER REVIEW 15 of 22

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in the dry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons (F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pH TK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-cool season in which SOC and TBB were the major correlates of changes in soil microbial composition (Table S5) A total of 11 biotic and abiotic factors including NO3minus-N TBB pH TKlitter HBB AP AK TP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warm season in which NO3minus-N TBB pH and TKlitter were the major correlates of changes in soil microbial composition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160 were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant biotic and abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot of soil microbial community structure and soil microbial functions (bd) Above the label represents a significant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowground biomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soil total potassium NO3ndash-N soil nitrate nitrogen HBB herb belowground biomass AP soil available phosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil water

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant bioticand abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot ofsoil microbial community structure and soil microbial functions (bd) Above the label represents asignificant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowgroundbiomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soiltotal potassium NO3

minus-N soil nitrate nitrogen HBB herb belowground biomass AP soil availablephosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil watercontent LP litter production TKlitter litter total potassium GP gram-positive bacteria GN gramnegative bacteria AMF arbuscular mycorrhizal fungi AG α-Glucosidase BG β-14-glucosidaseCB β-Cellobiosidase APH acid phosphatase NAG β-14-N-acetylglucosaminidase PHO phenoloxidase PO peroxidase AWCD average well color development of carbon source metabolism at192 h incubation

4 Discussion

41 Coniferous-Broadleaf Mixture Significantly Increase Stand Biomass and Litter Production

The results of this study indicated that admixing of coniferous and broadleaf tree speciessignificantly increased stand biomass and litter production after 34 years of afforestation in bothdry-cool and wet-warm seasons (Figure 1 Table 2) This may be a consequence of a greater growth of

Forests 2019 10 879 15 of 20

C hystrix in the mixed stands These results confirmed previous findings conducted in our experimentsite measured when the plantations were 26-year-old and 28-year-old respectively [716] A bulkof studies revealed that mixed-species management could affect tree growth stand structure andenvironment [145253] Mixed forests can develop stratified canopies due to niche differences amongdifferent species In the present study trees of the faster growing broad-leaf species (C hystrix) weretaller had a larger DBH and thus formed an upper canopy stratum in the mixed stands (Table 1)While trees of the shorter species (P massoniana) formed a lower stratum with lower DBH and treeheight and the reduced amount of light transmitted through the upper canopy contributed to theirslower growth The dominant tree species within the stand can absorb more sunlight and use itmore efficiently for growth [54] and thus contribute to higher tree biomass and litter productionin mixed stands In addition stratified canopies in mixed plantations can help to maintain levelsof understory species richness and biomass compared with pure plantations due to relatively highlight availabilities [5556] This may be why the mixed stand had higher shrub and herb biomass inour study

Specifically in the present study the broad-leaf species attained greater heights and diameters inmixed stands while masson pine had reduced diameter and height growth in mixed-species comparedto mono-species stands (Table 1) The severe suppression of P massoniana by the faster growingC hystrix may be attributed to differences in the light requirements of the two species [7] The growthpattern of C hystrix and P massoniana in mixed stands in our study (34-year-old) were differed fromwhen they were 26-years-old in which the growth of C hystrix and P massoniana in mixed standswere both greater than in the corresponding mono-species stands [7] This suggests that effects of thetree species mixture could change over time and long-term investigation of mixed forests is of greatsignificance to reveal mixed effects

42 Coniferous-Broadleaf Mixture Significantly Increase Soil Microbial Biomass

The changes of tree species and composition may significantly affect the amount of organic matterinput in soil [57] As the most active part in soil organic matter (SOM) soil microbial biomass isimportant in nutrient cycling and release participating in the circulation of critical elements such ascarbon and nitrogen [23] Because of the quantity and quality of the bulk of SOM changes slowly andthe changes of SOM are difficult to measure due to the spatial heterogeneity of soils soil microbialbiomass and in particular MBC-to-SOC and MBN-to-TN ratio have been suggested as indicators ofthe state and modification of SOM [58ndash60] and as an indicator for carbon (nitrogen) availability insoil [6162] The concentrations of SOC TN and MBC and MBN measured in our study were affectedby stand type and seasons (Table 3 and Table S2 Figure 2) In the dry cool season although the SOCcontent in mixed stands did not significantly differ with that in PCH but significantly lower than thatin PPM the MBC concentration in mixed stands was markedly higher than that in the correspondingmono-species stands Likewise although the TN content in mixed stands was smaller than that in PCHand PPM stands the MBN content in mixed stands was markedly higher than that in the correspondingmono-species stands in the wet-warn season Previous studies found that higher soil microbial biomasscorrelated with higher SOC and TN content that sustain microbial metabolism [2163] However inour study the lower SOC and TN content in MIX was able to maintain a higher microbial biomass thansites where SOC was more abundant Presumably the quality of SOC could explain such a trend [64]and merit further investigation

In this study MBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands(Figure 2) suggesting higher carbon and nitrogen availability in soil and higher soil quality for thegrowth of soil microorganisms by coniferous-broadleaf mixture [21] In addition the effects of admixingon soil microbial biomass MBC-to-SOC and MBN-to-TN ratio were non-additive suggesting theinteraction of P massoniana and C hystrix can have a synergistic effect on improving soil carbon andnitrogen availability (Figure 3) These findings are consistent with many previous studies that havedetermined the effects of tree species mixture on soil microbial biomass [2165] These results may be

Forests 2019 10 879 16 of 20

contributed to (1) greater amount and more diverse organic matter input via litter and rhizodepositionproducts in the mixture of two contrasting tree species [21] (2) the higher use efficiency of soil organiccarbon and nitrogen by microorganisms in mixed stands [66]

We also sought to determine the admixing effects on composition and abundance of soil microbialcommunity with community-level PLFA profiles Surprisingly there were not significant admixingeffects on PLFA content of the major microbial functional groups such as bacteria fungi AMF andactinobacteria (Table 4) However tree species mixture significantly increased the abundance of GNbacteria in the dry-cool season while markedly increased the abundance of GP bacteria but decreasedthe abundance of fungi in the wet-warn season (Figure 4) It seemed that the dominated GN bacteria inthe dry-cool season and GP bacteria in the wet-warn season would correlate with the higher availabilityof soil organic carbon and nitrogen in mixed stands [6768] These correlations are complicated andfurther investigation is required to reveal the underlying mechanisms

43 Effects of Coniferous-Broadleaf Mixture on Soil Microbial Functions

Soil microorganisms excrete soil enzymes to drive nutrient mineralization and litterdecomposition [64] Therefore soil extracellular enzyme activities may be related to the carbonnitrogen and phosphorus cycling in soil [6970] In the present study we found positive admixingeffects on the activities of APH and NAG in the dry-cool season (Figure 5de) On the other handpositive admixing effects on the activities of urease and peroxidase while negative admixing effectson APH was detected in the wet-warn season (Figure 5dfg) This result suggests that the mixtureof C hystrix and P massoniana can accelerate the mineralization rates of soil organic phosphorus andnitrogen These findings were consistent with previous study [67]

The community-level physiological profiling (CLPP) is widely used to characterize microbialcommunities [71ndash73] The average well color development (AWCD) in the CLPP approach reflectsthe ability of soil microorganisms to utilize carbon sources and their metabolic activity The higherAWCD represents the stronger metabolic activity of soil microorganisms [71] In this present study wedetected a significantly positive admixing effect on carbon source utilization ability of the soil microbialcommunities in both dry-cool and wet-warn seasons after 144 h of incubation (Figure 6) Moreover theRDA analysis revealed that carbon source utilization ability of the soil microbial communities werepositive related with APH in the dry-cool season while related with urease and peroxidase activitiesin the wet-warn season (Figure 7) As a whole the higher soil microbial functions in mixed stands arelikely the results of the effects of AMF and GP bacteria in the dry-cool season and the effects of bacteriaincluding GP and GN bacteria the wet-warn season (Table S6)

5 Conclusions

In our study we measured significantly higher tree shrub and herb biomass and litter productionin the coniferous-broadleaf mixed plantation after 34 years of afforestation As expected MBC MBNMBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands suggesting highercarbon and nitrogen availability in soil and higher soil quality for the growth of soil microorganisms inour coniferous-broadleaf mixed stand In addition coniferous-broadleaf mixture can increase carbonsource utilization ability of soil microbial community and accelerate the mineralization rates of soilorganic phosphorus and nitrogen by increasing acid phosphatase β-14-N-acetylglucosaminidaseand urease activities The higher soil microbial biomass and activities are likely the result of greateramount and more diverse organic matter input via litter and rhizodeposition products and the higheruse efficiency of soil organic carbon and nitrogen by microorganisms in mixed stands Howeverthe correlations between soil dominated microbial functional groups and the higher availabilityof soil organic carbon and nitrogen along with the quality of SOC in mixed stands merit furtherinvestigation Our results highlight that establishment of coniferous-broadleaf mixed forests maybe a good management practice as coniferous-broadleaf mixture could accumulate higher stand

Forests 2019 10 879 17 of 20

biomass return more litter and increase soil microbial biomass and functions over the long term insubtropical China

Supplementary Materials The following are available online at httpwwwmdpicom1999-49071010879s1Table S1 Effects of stand type season and their interactions on stand biomass Table S2 Effects of stand type seasonand their interactions on soil microbial structure Table S3 Effects of stand type season and their interactionson relative abundance of the soil microbial community PLFAs Table S4 Effects of stand type season and theirinteractions on soil enzyme activity Table S5 Marginal and conditional effects of biotic and abiotic factors on soilmicrobial composition obtained from the summary of forward selection in Redundancy analysis (RDA) Table S6Marginal and conditional effects of soil microbial functional groups on soil enzyme activity and soil microbialcarbon source utilization ability obtained from the summary of forward selection in Redundancy analysis (RDA)

Author Contributions WW collected data made experiment analysis formal data and drafted the manuscriptYW designed the work XZ revised the manuscript and participated in analyzing the experiment data HZ YYXH and XL ZQ YL LY HL participated in collecting the experiment data

Funding This research was funded by National Natural Science Foundation of China (31860171 3146012131560201) the Guangxi Key Research and Development Program (2018AB40007) Innovation Project of GuangxiGraduate Education (YCBZ2017011) and the Guangxi Universities Science and technology (201201ZD001)

Acknowledgments We gratefully acknowledge the supports in field sampling by the Experimental Center ofTropical Forestry Chinese Academy of Forestry A special acknowledgment is given to Patrick M Finnegan for hiswriting suggestions

Conflicts of Interest The authors declare no conflicts of interest

References

1 Zhang X Liu S Huang Y Fu S Wang J Ming A Li X Yao M Li H Tree species mixture inhibits soilorganic carbon mineralization accompanied by decreased r-selected bacteria Plant Soil 2018 431 203ndash216[CrossRef]

2 Pan Y Birdsey RA Fang J Houghton R Kauppi PE Kurz WA Phillips OL Shvidenko ALewis SL Canadell JG et al A large and persistent carbon sink in the worldrsquos forests Science 2011 333988ndash993 [CrossRef] [PubMed]

3 Chen X Ju Q Lin K Development status issues and countermeasures of Chinarsquo s plantation World For Res2014 27 54ndash59

4 Wang H Lencinas MV Ross Friedman C Wang X Qiu J Understory plant diversity assessment ofEucalyptus plantations over three vegetation types in Yunnan China New For 2011 42 101ndash116 [CrossRef]

5 Sang PM Lamb D Bonner M Schmidt S Carbon sequestration and soil fertility of tropical tree plantationsand secondary forest established on degraded land Plant Soil 2013 362 187ndash200 [CrossRef]

6 Li C Zhou X Wen Y Zhu H Qin Z Li X You Y Huang X Effects of high-generation ratations ofEucalyptus on undergrowth soil fertility and enzyme activities Guangxi Sci 2019 26 176ndash186

7 You Y Huang X Zhu H Liu S Liang H Wen Y Wang H Cai D Ye D Positive interactions betweenPinus massoniana and Castanopsis hystrix species in the uneven-aged mixed plantations can produce moreecosystem carbon in subtropical China For Ecol Manag 2018 410 193ndash200 [CrossRef]

8 Huang X Liu S You Y Wen Y Wang H Wang J Microbial community and associated enzymes activityinfluence soil carbon chemical composition in Eucalyptus urophylla plantation with mixing N2-fixing speciesin sub-tropical China Plant Soil 2017 414 199ndash212 [CrossRef]

9 Kelty MJ The role of species mixtures in plantation forestry For Ecol Manag 2006 233 195ndash204 [CrossRef]10 Bauhus J Khanna P Menden N Aboveground and belowground interactions in mixed plantations of

Eucalyptus globulus and Acacia mearnsii Can J For Res 2000 30 1886ndash1894 [CrossRef]11 Vandermeer J The Ecology of Intercropping Cambridge University Press New York NY USA 1989 p 23712 Forrester DI The spatial and temporal dynamics of species interactions in mixed-species forests From

pattern to process For Ecol Manag 2014 312 282ndash292 [CrossRef]13 Forrester DI Bauhus J A review of processes behind diversity-productivity relationships in forests

Curr For Rep 2016 2 45ndash61 [CrossRef]14 Wang H Liu S Wang J You Y Yang Y Shi Z Huang X Zheng L Li Z Ming A et al Mixed-species

plantation with Pinus massoniana and Castanopsis hystrix accelerates C loss in recalcitrant coniferous litter butslows C loss in labile broadleaf litter in southern China For Ecol Manag 2018 422 207ndash213 [CrossRef]

Forests 2019 10 879 11 of 20

Table 4 Soil microbial community PLFAs content and structure (0ndash10 cm soil depth) in pure Castanopsis hystrix (PCH) plantation pure Pinus massoniana (PPM)plantation and mixed C hystrix and P massoniana (MIX) plantation (mean plusmn standard error n = 5)

FactorsDry-Cool Season Wet-Warm Season Stand Type Season Stand Type times Season

PCH MIX PPM PCH MIX PPM F p F p F p

TPLFAs 3767 plusmn 271 a 3255 plusmn 124 a 3796 plusmn 128 a 4373 plusmn 208 a 4001 plusmn 07 a 3478 plusmn 111 b 464 0020 646 0018 607 0007B 1579 plusmn 081 a 1306 plusmn 043 b 1490 plusmn 051 ab 1764 plusmn 072 a 1723 plusmn 042 a 1276 plusmn 041 b 1274 lt0001 768 0011 1556 lt0001

GP 945 plusmn 043 a 794 plusmn 029 b 918 plusmn 031 a 1066 plusmn 051 a 1155 plusmn 031 a 810 plusmn 031 b 817 0002 1709 lt0001 2030 lt0001GN 591 plusmn 076 a 590 plusmn 016 a 553 plusmn 022 a 619 plusmn 025 a 455 plusmn 037 b 411 plusmn 020 b 526 0013 702 0014 313 0062

F 498 plusmn 034 a 375 plusmn 022 b 424 plusmn 028 ab 492 plusmn 035 a 296 plusmn 043 b 345 plusmn 021 b 1356 lt0001 448 0045 091 0416AMF 104 plusmn 003 a 075 plusmn 003 b 075 plusmn 004 b 085 plusmn 007 a 044 plusmn 008 b 048 plusmn 003 b 2822 lt0001 3483 lt0001 056 0577Acti 426 plusmn 037 ab 377 plusmn 021 b 486 plusmn 020 a 531 plusmn 019 a 487 plusmn 012 b 458 plusmn 010 b 273 0085 1236 0002 650 0006BF 319 plusmn 011 a 351 plusmn 013 a 356 plusmn 017 a 362 plusmn 014 b 635 plusmn 094 a 377 plusmn 033 b 750 0003 1131 0003 598 0008

GPGN 174 plusmn 029 b 135 plusmn 003 b 166 plusmn 004 a 173 plusmn 008 b 263 plusmn 028 a 198 plusmn 011 b 107 0359 1376 0001 733 0003

Different superscripts indicate significant differences among stand types in each season (p lt 005) TPLFAs (nmol gminus1 soil) total PLFAs B (nmol gminus1 soil) bacteria PLFAs GP (nmol gminus1

soil) gram-positive bacteria PLFAs GN (nmol gminus1 soil) gram-negative bacteria PLFAs F (nmol gminus1 soil) fungi PLFAs Acti (nmol gminus1 soil) actinobacteria PLFAs AMF (nmol gminus1 soil)arbuscular mycorrhizal fungi PLFAs BF bacteria-to-fungi PLFA ratio GPGN GP-to-GN ratio

Forests 2019 10 879 12 of 20

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleafplantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increasedAPH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increasedurease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg)Carbon source utilization ability of the soil microbial community indicated by average well colordevelopment (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation(Figure 6)Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in threeplantations in different seasons Error bars are standard error Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 13 of 20

Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in threeplantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in thedry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons(F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pHTK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-coolseason in which SOC and TBB were the major correlates of changes in soil microbial composition(Table S5) A total of 11 biotic and abiotic factors including NO3

minus-N TBB pH TKlitter HBB AP AKTP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warmseason in which NO3

minus-N TBB pH and TKlitter were the major correlates of changes in soil microbialcomposition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

37 Relationship between Soil Microbial Structure and Functions

Soil enzyme activity and soil microbial carbon source utilization ability are both important soilmicrobial functions The first two RDA axes explained 4948 of the variation in soil enzyme activityand carbon utilization ability in the dry-cool season (F = 24 p = 0006 Figure 7b) and 6891 of thevariation in the wet-warm season (F = 64 p = 0002 Figure 7d) AMF and GP were significantlycorrelated with soil microbial functions in the dry-cool season (Table S6) Total bacteria GP bacteriaGN bacteria and actinobacteria were significantly correlated with soil microbial functions in thewet-warm season in which total bacteria GP bacteria and GN bacteria were the major correlates ofchanges of soil enzyme activity (Table S6)

Forests 2019 10 879 14 of 20

Forests 2019 10 x FOR PEER REVIEW 15 of 22

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in the dry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons (F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pH TK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-cool season in which SOC and TBB were the major correlates of changes in soil microbial composition (Table S5) A total of 11 biotic and abiotic factors including NO3minus-N TBB pH TKlitter HBB AP AK TP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warm season in which NO3minus-N TBB pH and TKlitter were the major correlates of changes in soil microbial composition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160 were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant biotic and abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot of soil microbial community structure and soil microbial functions (bd) Above the label represents a significant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowground biomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soil total potassium NO3ndash-N soil nitrate nitrogen HBB herb belowground biomass AP soil available phosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil water

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant bioticand abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot ofsoil microbial community structure and soil microbial functions (bd) Above the label represents asignificant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowgroundbiomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soiltotal potassium NO3

minus-N soil nitrate nitrogen HBB herb belowground biomass AP soil availablephosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil watercontent LP litter production TKlitter litter total potassium GP gram-positive bacteria GN gramnegative bacteria AMF arbuscular mycorrhizal fungi AG α-Glucosidase BG β-14-glucosidaseCB β-Cellobiosidase APH acid phosphatase NAG β-14-N-acetylglucosaminidase PHO phenoloxidase PO peroxidase AWCD average well color development of carbon source metabolism at192 h incubation

4 Discussion

41 Coniferous-Broadleaf Mixture Significantly Increase Stand Biomass and Litter Production

The results of this study indicated that admixing of coniferous and broadleaf tree speciessignificantly increased stand biomass and litter production after 34 years of afforestation in bothdry-cool and wet-warm seasons (Figure 1 Table 2) This may be a consequence of a greater growth of

Forests 2019 10 879 15 of 20

C hystrix in the mixed stands These results confirmed previous findings conducted in our experimentsite measured when the plantations were 26-year-old and 28-year-old respectively [716] A bulkof studies revealed that mixed-species management could affect tree growth stand structure andenvironment [145253] Mixed forests can develop stratified canopies due to niche differences amongdifferent species In the present study trees of the faster growing broad-leaf species (C hystrix) weretaller had a larger DBH and thus formed an upper canopy stratum in the mixed stands (Table 1)While trees of the shorter species (P massoniana) formed a lower stratum with lower DBH and treeheight and the reduced amount of light transmitted through the upper canopy contributed to theirslower growth The dominant tree species within the stand can absorb more sunlight and use itmore efficiently for growth [54] and thus contribute to higher tree biomass and litter productionin mixed stands In addition stratified canopies in mixed plantations can help to maintain levelsof understory species richness and biomass compared with pure plantations due to relatively highlight availabilities [5556] This may be why the mixed stand had higher shrub and herb biomass inour study

Specifically in the present study the broad-leaf species attained greater heights and diameters inmixed stands while masson pine had reduced diameter and height growth in mixed-species comparedto mono-species stands (Table 1) The severe suppression of P massoniana by the faster growingC hystrix may be attributed to differences in the light requirements of the two species [7] The growthpattern of C hystrix and P massoniana in mixed stands in our study (34-year-old) were differed fromwhen they were 26-years-old in which the growth of C hystrix and P massoniana in mixed standswere both greater than in the corresponding mono-species stands [7] This suggests that effects of thetree species mixture could change over time and long-term investigation of mixed forests is of greatsignificance to reveal mixed effects

42 Coniferous-Broadleaf Mixture Significantly Increase Soil Microbial Biomass

The changes of tree species and composition may significantly affect the amount of organic matterinput in soil [57] As the most active part in soil organic matter (SOM) soil microbial biomass isimportant in nutrient cycling and release participating in the circulation of critical elements such ascarbon and nitrogen [23] Because of the quantity and quality of the bulk of SOM changes slowly andthe changes of SOM are difficult to measure due to the spatial heterogeneity of soils soil microbialbiomass and in particular MBC-to-SOC and MBN-to-TN ratio have been suggested as indicators ofthe state and modification of SOM [58ndash60] and as an indicator for carbon (nitrogen) availability insoil [6162] The concentrations of SOC TN and MBC and MBN measured in our study were affectedby stand type and seasons (Table 3 and Table S2 Figure 2) In the dry cool season although the SOCcontent in mixed stands did not significantly differ with that in PCH but significantly lower than thatin PPM the MBC concentration in mixed stands was markedly higher than that in the correspondingmono-species stands Likewise although the TN content in mixed stands was smaller than that in PCHand PPM stands the MBN content in mixed stands was markedly higher than that in the correspondingmono-species stands in the wet-warn season Previous studies found that higher soil microbial biomasscorrelated with higher SOC and TN content that sustain microbial metabolism [2163] However inour study the lower SOC and TN content in MIX was able to maintain a higher microbial biomass thansites where SOC was more abundant Presumably the quality of SOC could explain such a trend [64]and merit further investigation

In this study MBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands(Figure 2) suggesting higher carbon and nitrogen availability in soil and higher soil quality for thegrowth of soil microorganisms by coniferous-broadleaf mixture [21] In addition the effects of admixingon soil microbial biomass MBC-to-SOC and MBN-to-TN ratio were non-additive suggesting theinteraction of P massoniana and C hystrix can have a synergistic effect on improving soil carbon andnitrogen availability (Figure 3) These findings are consistent with many previous studies that havedetermined the effects of tree species mixture on soil microbial biomass [2165] These results may be

Forests 2019 10 879 16 of 20

contributed to (1) greater amount and more diverse organic matter input via litter and rhizodepositionproducts in the mixture of two contrasting tree species [21] (2) the higher use efficiency of soil organiccarbon and nitrogen by microorganisms in mixed stands [66]

We also sought to determine the admixing effects on composition and abundance of soil microbialcommunity with community-level PLFA profiles Surprisingly there were not significant admixingeffects on PLFA content of the major microbial functional groups such as bacteria fungi AMF andactinobacteria (Table 4) However tree species mixture significantly increased the abundance of GNbacteria in the dry-cool season while markedly increased the abundance of GP bacteria but decreasedthe abundance of fungi in the wet-warn season (Figure 4) It seemed that the dominated GN bacteria inthe dry-cool season and GP bacteria in the wet-warn season would correlate with the higher availabilityof soil organic carbon and nitrogen in mixed stands [6768] These correlations are complicated andfurther investigation is required to reveal the underlying mechanisms

43 Effects of Coniferous-Broadleaf Mixture on Soil Microbial Functions

Soil microorganisms excrete soil enzymes to drive nutrient mineralization and litterdecomposition [64] Therefore soil extracellular enzyme activities may be related to the carbonnitrogen and phosphorus cycling in soil [6970] In the present study we found positive admixingeffects on the activities of APH and NAG in the dry-cool season (Figure 5de) On the other handpositive admixing effects on the activities of urease and peroxidase while negative admixing effectson APH was detected in the wet-warn season (Figure 5dfg) This result suggests that the mixtureof C hystrix and P massoniana can accelerate the mineralization rates of soil organic phosphorus andnitrogen These findings were consistent with previous study [67]

The community-level physiological profiling (CLPP) is widely used to characterize microbialcommunities [71ndash73] The average well color development (AWCD) in the CLPP approach reflectsthe ability of soil microorganisms to utilize carbon sources and their metabolic activity The higherAWCD represents the stronger metabolic activity of soil microorganisms [71] In this present study wedetected a significantly positive admixing effect on carbon source utilization ability of the soil microbialcommunities in both dry-cool and wet-warn seasons after 144 h of incubation (Figure 6) Moreover theRDA analysis revealed that carbon source utilization ability of the soil microbial communities werepositive related with APH in the dry-cool season while related with urease and peroxidase activitiesin the wet-warn season (Figure 7) As a whole the higher soil microbial functions in mixed stands arelikely the results of the effects of AMF and GP bacteria in the dry-cool season and the effects of bacteriaincluding GP and GN bacteria the wet-warn season (Table S6)

5 Conclusions

In our study we measured significantly higher tree shrub and herb biomass and litter productionin the coniferous-broadleaf mixed plantation after 34 years of afforestation As expected MBC MBNMBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands suggesting highercarbon and nitrogen availability in soil and higher soil quality for the growth of soil microorganisms inour coniferous-broadleaf mixed stand In addition coniferous-broadleaf mixture can increase carbonsource utilization ability of soil microbial community and accelerate the mineralization rates of soilorganic phosphorus and nitrogen by increasing acid phosphatase β-14-N-acetylglucosaminidaseand urease activities The higher soil microbial biomass and activities are likely the result of greateramount and more diverse organic matter input via litter and rhizodeposition products and the higheruse efficiency of soil organic carbon and nitrogen by microorganisms in mixed stands Howeverthe correlations between soil dominated microbial functional groups and the higher availabilityof soil organic carbon and nitrogen along with the quality of SOC in mixed stands merit furtherinvestigation Our results highlight that establishment of coniferous-broadleaf mixed forests maybe a good management practice as coniferous-broadleaf mixture could accumulate higher stand

Forests 2019 10 879 17 of 20

biomass return more litter and increase soil microbial biomass and functions over the long term insubtropical China

Supplementary Materials The following are available online at httpwwwmdpicom1999-49071010879s1Table S1 Effects of stand type season and their interactions on stand biomass Table S2 Effects of stand type seasonand their interactions on soil microbial structure Table S3 Effects of stand type season and their interactionson relative abundance of the soil microbial community PLFAs Table S4 Effects of stand type season and theirinteractions on soil enzyme activity Table S5 Marginal and conditional effects of biotic and abiotic factors on soilmicrobial composition obtained from the summary of forward selection in Redundancy analysis (RDA) Table S6Marginal and conditional effects of soil microbial functional groups on soil enzyme activity and soil microbialcarbon source utilization ability obtained from the summary of forward selection in Redundancy analysis (RDA)

Author Contributions WW collected data made experiment analysis formal data and drafted the manuscriptYW designed the work XZ revised the manuscript and participated in analyzing the experiment data HZ YYXH and XL ZQ YL LY HL participated in collecting the experiment data

Funding This research was funded by National Natural Science Foundation of China (31860171 3146012131560201) the Guangxi Key Research and Development Program (2018AB40007) Innovation Project of GuangxiGraduate Education (YCBZ2017011) and the Guangxi Universities Science and technology (201201ZD001)

Acknowledgments We gratefully acknowledge the supports in field sampling by the Experimental Center ofTropical Forestry Chinese Academy of Forestry A special acknowledgment is given to Patrick M Finnegan for hiswriting suggestions

Conflicts of Interest The authors declare no conflicts of interest

References

1 Zhang X Liu S Huang Y Fu S Wang J Ming A Li X Yao M Li H Tree species mixture inhibits soilorganic carbon mineralization accompanied by decreased r-selected bacteria Plant Soil 2018 431 203ndash216[CrossRef]

2 Pan Y Birdsey RA Fang J Houghton R Kauppi PE Kurz WA Phillips OL Shvidenko ALewis SL Canadell JG et al A large and persistent carbon sink in the worldrsquos forests Science 2011 333988ndash993 [CrossRef] [PubMed]

3 Chen X Ju Q Lin K Development status issues and countermeasures of Chinarsquo s plantation World For Res2014 27 54ndash59

4 Wang H Lencinas MV Ross Friedman C Wang X Qiu J Understory plant diversity assessment ofEucalyptus plantations over three vegetation types in Yunnan China New For 2011 42 101ndash116 [CrossRef]

5 Sang PM Lamb D Bonner M Schmidt S Carbon sequestration and soil fertility of tropical tree plantationsand secondary forest established on degraded land Plant Soil 2013 362 187ndash200 [CrossRef]

6 Li C Zhou X Wen Y Zhu H Qin Z Li X You Y Huang X Effects of high-generation ratations ofEucalyptus on undergrowth soil fertility and enzyme activities Guangxi Sci 2019 26 176ndash186

7 You Y Huang X Zhu H Liu S Liang H Wen Y Wang H Cai D Ye D Positive interactions betweenPinus massoniana and Castanopsis hystrix species in the uneven-aged mixed plantations can produce moreecosystem carbon in subtropical China For Ecol Manag 2018 410 193ndash200 [CrossRef]

8 Huang X Liu S You Y Wen Y Wang H Wang J Microbial community and associated enzymes activityinfluence soil carbon chemical composition in Eucalyptus urophylla plantation with mixing N2-fixing speciesin sub-tropical China Plant Soil 2017 414 199ndash212 [CrossRef]

9 Kelty MJ The role of species mixtures in plantation forestry For Ecol Manag 2006 233 195ndash204 [CrossRef]10 Bauhus J Khanna P Menden N Aboveground and belowground interactions in mixed plantations of

Eucalyptus globulus and Acacia mearnsii Can J For Res 2000 30 1886ndash1894 [CrossRef]11 Vandermeer J The Ecology of Intercropping Cambridge University Press New York NY USA 1989 p 23712 Forrester DI The spatial and temporal dynamics of species interactions in mixed-species forests From

pattern to process For Ecol Manag 2014 312 282ndash292 [CrossRef]13 Forrester DI Bauhus J A review of processes behind diversity-productivity relationships in forests

Curr For Rep 2016 2 45ndash61 [CrossRef]14 Wang H Liu S Wang J You Y Yang Y Shi Z Huang X Zheng L Li Z Ming A et al Mixed-species

plantation with Pinus massoniana and Castanopsis hystrix accelerates C loss in recalcitrant coniferous litter butslows C loss in labile broadleaf litter in southern China For Ecol Manag 2018 422 207ndash213 [CrossRef]

Forests 2019 10 879 12 of 20

35 Soil Microbial Functions

Soil enzyme activity was lower in the pure coniferous plantation (PPM) than in the pure broadleafplantation in both seasons (Figure 5 and Table S4) The mixture of tree species significantly increasedAPH and NAG activity in the dry-cool season Furthermore mixed tree species significantly increasedurease and PO activity but significantly decreased APH activity in the wet-warm season (Figure 5dfg)Carbon source utilization ability of the soil microbial community indicated by average well colordevelopment (AWCD) was significantly higher in MIX than in PCH and PPM after 144 h of incubation(Figure 6)Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in threeplantations in different seasons Error bars are standard error Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Forests 2019 10 879 13 of 20

Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in threeplantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in thedry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons(F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pHTK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-coolseason in which SOC and TBB were the major correlates of changes in soil microbial composition(Table S5) A total of 11 biotic and abiotic factors including NO3

minus-N TBB pH TKlitter HBB AP AKTP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warmseason in which NO3

minus-N TBB pH and TKlitter were the major correlates of changes in soil microbialcomposition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

37 Relationship between Soil Microbial Structure and Functions

Soil enzyme activity and soil microbial carbon source utilization ability are both important soilmicrobial functions The first two RDA axes explained 4948 of the variation in soil enzyme activityand carbon utilization ability in the dry-cool season (F = 24 p = 0006 Figure 7b) and 6891 of thevariation in the wet-warm season (F = 64 p = 0002 Figure 7d) AMF and GP were significantlycorrelated with soil microbial functions in the dry-cool season (Table S6) Total bacteria GP bacteriaGN bacteria and actinobacteria were significantly correlated with soil microbial functions in thewet-warm season in which total bacteria GP bacteria and GN bacteria were the major correlates ofchanges of soil enzyme activity (Table S6)

Forests 2019 10 879 14 of 20

Forests 2019 10 x FOR PEER REVIEW 15 of 22

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in the dry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons (F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pH TK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-cool season in which SOC and TBB were the major correlates of changes in soil microbial composition (Table S5) A total of 11 biotic and abiotic factors including NO3minus-N TBB pH TKlitter HBB AP AK TP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warm season in which NO3minus-N TBB pH and TKlitter were the major correlates of changes in soil microbial composition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160 were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant biotic and abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot of soil microbial community structure and soil microbial functions (bd) Above the label represents a significant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowground biomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soil total potassium NO3ndash-N soil nitrate nitrogen HBB herb belowground biomass AP soil available phosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil water

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant bioticand abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot ofsoil microbial community structure and soil microbial functions (bd) Above the label represents asignificant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowgroundbiomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soiltotal potassium NO3

minus-N soil nitrate nitrogen HBB herb belowground biomass AP soil availablephosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil watercontent LP litter production TKlitter litter total potassium GP gram-positive bacteria GN gramnegative bacteria AMF arbuscular mycorrhizal fungi AG α-Glucosidase BG β-14-glucosidaseCB β-Cellobiosidase APH acid phosphatase NAG β-14-N-acetylglucosaminidase PHO phenoloxidase PO peroxidase AWCD average well color development of carbon source metabolism at192 h incubation

4 Discussion

41 Coniferous-Broadleaf Mixture Significantly Increase Stand Biomass and Litter Production

The results of this study indicated that admixing of coniferous and broadleaf tree speciessignificantly increased stand biomass and litter production after 34 years of afforestation in bothdry-cool and wet-warm seasons (Figure 1 Table 2) This may be a consequence of a greater growth of

Forests 2019 10 879 15 of 20

C hystrix in the mixed stands These results confirmed previous findings conducted in our experimentsite measured when the plantations were 26-year-old and 28-year-old respectively [716] A bulkof studies revealed that mixed-species management could affect tree growth stand structure andenvironment [145253] Mixed forests can develop stratified canopies due to niche differences amongdifferent species In the present study trees of the faster growing broad-leaf species (C hystrix) weretaller had a larger DBH and thus formed an upper canopy stratum in the mixed stands (Table 1)While trees of the shorter species (P massoniana) formed a lower stratum with lower DBH and treeheight and the reduced amount of light transmitted through the upper canopy contributed to theirslower growth The dominant tree species within the stand can absorb more sunlight and use itmore efficiently for growth [54] and thus contribute to higher tree biomass and litter productionin mixed stands In addition stratified canopies in mixed plantations can help to maintain levelsof understory species richness and biomass compared with pure plantations due to relatively highlight availabilities [5556] This may be why the mixed stand had higher shrub and herb biomass inour study

Specifically in the present study the broad-leaf species attained greater heights and diameters inmixed stands while masson pine had reduced diameter and height growth in mixed-species comparedto mono-species stands (Table 1) The severe suppression of P massoniana by the faster growingC hystrix may be attributed to differences in the light requirements of the two species [7] The growthpattern of C hystrix and P massoniana in mixed stands in our study (34-year-old) were differed fromwhen they were 26-years-old in which the growth of C hystrix and P massoniana in mixed standswere both greater than in the corresponding mono-species stands [7] This suggests that effects of thetree species mixture could change over time and long-term investigation of mixed forests is of greatsignificance to reveal mixed effects

42 Coniferous-Broadleaf Mixture Significantly Increase Soil Microbial Biomass

The changes of tree species and composition may significantly affect the amount of organic matterinput in soil [57] As the most active part in soil organic matter (SOM) soil microbial biomass isimportant in nutrient cycling and release participating in the circulation of critical elements such ascarbon and nitrogen [23] Because of the quantity and quality of the bulk of SOM changes slowly andthe changes of SOM are difficult to measure due to the spatial heterogeneity of soils soil microbialbiomass and in particular MBC-to-SOC and MBN-to-TN ratio have been suggested as indicators ofthe state and modification of SOM [58ndash60] and as an indicator for carbon (nitrogen) availability insoil [6162] The concentrations of SOC TN and MBC and MBN measured in our study were affectedby stand type and seasons (Table 3 and Table S2 Figure 2) In the dry cool season although the SOCcontent in mixed stands did not significantly differ with that in PCH but significantly lower than thatin PPM the MBC concentration in mixed stands was markedly higher than that in the correspondingmono-species stands Likewise although the TN content in mixed stands was smaller than that in PCHand PPM stands the MBN content in mixed stands was markedly higher than that in the correspondingmono-species stands in the wet-warn season Previous studies found that higher soil microbial biomasscorrelated with higher SOC and TN content that sustain microbial metabolism [2163] However inour study the lower SOC and TN content in MIX was able to maintain a higher microbial biomass thansites where SOC was more abundant Presumably the quality of SOC could explain such a trend [64]and merit further investigation

In this study MBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands(Figure 2) suggesting higher carbon and nitrogen availability in soil and higher soil quality for thegrowth of soil microorganisms by coniferous-broadleaf mixture [21] In addition the effects of admixingon soil microbial biomass MBC-to-SOC and MBN-to-TN ratio were non-additive suggesting theinteraction of P massoniana and C hystrix can have a synergistic effect on improving soil carbon andnitrogen availability (Figure 3) These findings are consistent with many previous studies that havedetermined the effects of tree species mixture on soil microbial biomass [2165] These results may be

Forests 2019 10 879 16 of 20

contributed to (1) greater amount and more diverse organic matter input via litter and rhizodepositionproducts in the mixture of two contrasting tree species [21] (2) the higher use efficiency of soil organiccarbon and nitrogen by microorganisms in mixed stands [66]

We also sought to determine the admixing effects on composition and abundance of soil microbialcommunity with community-level PLFA profiles Surprisingly there were not significant admixingeffects on PLFA content of the major microbial functional groups such as bacteria fungi AMF andactinobacteria (Table 4) However tree species mixture significantly increased the abundance of GNbacteria in the dry-cool season while markedly increased the abundance of GP bacteria but decreasedthe abundance of fungi in the wet-warn season (Figure 4) It seemed that the dominated GN bacteria inthe dry-cool season and GP bacteria in the wet-warn season would correlate with the higher availabilityof soil organic carbon and nitrogen in mixed stands [6768] These correlations are complicated andfurther investigation is required to reveal the underlying mechanisms

43 Effects of Coniferous-Broadleaf Mixture on Soil Microbial Functions

Soil microorganisms excrete soil enzymes to drive nutrient mineralization and litterdecomposition [64] Therefore soil extracellular enzyme activities may be related to the carbonnitrogen and phosphorus cycling in soil [6970] In the present study we found positive admixingeffects on the activities of APH and NAG in the dry-cool season (Figure 5de) On the other handpositive admixing effects on the activities of urease and peroxidase while negative admixing effectson APH was detected in the wet-warn season (Figure 5dfg) This result suggests that the mixtureof C hystrix and P massoniana can accelerate the mineralization rates of soil organic phosphorus andnitrogen These findings were consistent with previous study [67]

The community-level physiological profiling (CLPP) is widely used to characterize microbialcommunities [71ndash73] The average well color development (AWCD) in the CLPP approach reflectsthe ability of soil microorganisms to utilize carbon sources and their metabolic activity The higherAWCD represents the stronger metabolic activity of soil microorganisms [71] In this present study wedetected a significantly positive admixing effect on carbon source utilization ability of the soil microbialcommunities in both dry-cool and wet-warn seasons after 144 h of incubation (Figure 6) Moreover theRDA analysis revealed that carbon source utilization ability of the soil microbial communities werepositive related with APH in the dry-cool season while related with urease and peroxidase activitiesin the wet-warn season (Figure 7) As a whole the higher soil microbial functions in mixed stands arelikely the results of the effects of AMF and GP bacteria in the dry-cool season and the effects of bacteriaincluding GP and GN bacteria the wet-warn season (Table S6)

5 Conclusions

In our study we measured significantly higher tree shrub and herb biomass and litter productionin the coniferous-broadleaf mixed plantation after 34 years of afforestation As expected MBC MBNMBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands suggesting highercarbon and nitrogen availability in soil and higher soil quality for the growth of soil microorganisms inour coniferous-broadleaf mixed stand In addition coniferous-broadleaf mixture can increase carbonsource utilization ability of soil microbial community and accelerate the mineralization rates of soilorganic phosphorus and nitrogen by increasing acid phosphatase β-14-N-acetylglucosaminidaseand urease activities The higher soil microbial biomass and activities are likely the result of greateramount and more diverse organic matter input via litter and rhizodeposition products and the higheruse efficiency of soil organic carbon and nitrogen by microorganisms in mixed stands Howeverthe correlations between soil dominated microbial functional groups and the higher availabilityof soil organic carbon and nitrogen along with the quality of SOC in mixed stands merit furtherinvestigation Our results highlight that establishment of coniferous-broadleaf mixed forests maybe a good management practice as coniferous-broadleaf mixture could accumulate higher stand

Forests 2019 10 879 17 of 20

biomass return more litter and increase soil microbial biomass and functions over the long term insubtropical China

Supplementary Materials The following are available online at httpwwwmdpicom1999-49071010879s1Table S1 Effects of stand type season and their interactions on stand biomass Table S2 Effects of stand type seasonand their interactions on soil microbial structure Table S3 Effects of stand type season and their interactionson relative abundance of the soil microbial community PLFAs Table S4 Effects of stand type season and theirinteractions on soil enzyme activity Table S5 Marginal and conditional effects of biotic and abiotic factors on soilmicrobial composition obtained from the summary of forward selection in Redundancy analysis (RDA) Table S6Marginal and conditional effects of soil microbial functional groups on soil enzyme activity and soil microbialcarbon source utilization ability obtained from the summary of forward selection in Redundancy analysis (RDA)

Author Contributions WW collected data made experiment analysis formal data and drafted the manuscriptYW designed the work XZ revised the manuscript and participated in analyzing the experiment data HZ YYXH and XL ZQ YL LY HL participated in collecting the experiment data

Funding This research was funded by National Natural Science Foundation of China (31860171 3146012131560201) the Guangxi Key Research and Development Program (2018AB40007) Innovation Project of GuangxiGraduate Education (YCBZ2017011) and the Guangxi Universities Science and technology (201201ZD001)

Acknowledgments We gratefully acknowledge the supports in field sampling by the Experimental Center ofTropical Forestry Chinese Academy of Forestry A special acknowledgment is given to Patrick M Finnegan for hiswriting suggestions

Conflicts of Interest The authors declare no conflicts of interest

References

1 Zhang X Liu S Huang Y Fu S Wang J Ming A Li X Yao M Li H Tree species mixture inhibits soilorganic carbon mineralization accompanied by decreased r-selected bacteria Plant Soil 2018 431 203ndash216[CrossRef]

2 Pan Y Birdsey RA Fang J Houghton R Kauppi PE Kurz WA Phillips OL Shvidenko ALewis SL Canadell JG et al A large and persistent carbon sink in the worldrsquos forests Science 2011 333988ndash993 [CrossRef] [PubMed]

3 Chen X Ju Q Lin K Development status issues and countermeasures of Chinarsquo s plantation World For Res2014 27 54ndash59

4 Wang H Lencinas MV Ross Friedman C Wang X Qiu J Understory plant diversity assessment ofEucalyptus plantations over three vegetation types in Yunnan China New For 2011 42 101ndash116 [CrossRef]

5 Sang PM Lamb D Bonner M Schmidt S Carbon sequestration and soil fertility of tropical tree plantationsand secondary forest established on degraded land Plant Soil 2013 362 187ndash200 [CrossRef]

6 Li C Zhou X Wen Y Zhu H Qin Z Li X You Y Huang X Effects of high-generation ratations ofEucalyptus on undergrowth soil fertility and enzyme activities Guangxi Sci 2019 26 176ndash186

7 You Y Huang X Zhu H Liu S Liang H Wen Y Wang H Cai D Ye D Positive interactions betweenPinus massoniana and Castanopsis hystrix species in the uneven-aged mixed plantations can produce moreecosystem carbon in subtropical China For Ecol Manag 2018 410 193ndash200 [CrossRef]

8 Huang X Liu S You Y Wen Y Wang H Wang J Microbial community and associated enzymes activityinfluence soil carbon chemical composition in Eucalyptus urophylla plantation with mixing N2-fixing speciesin sub-tropical China Plant Soil 2017 414 199ndash212 [CrossRef]

9 Kelty MJ The role of species mixtures in plantation forestry For Ecol Manag 2006 233 195ndash204 [CrossRef]10 Bauhus J Khanna P Menden N Aboveground and belowground interactions in mixed plantations of

Eucalyptus globulus and Acacia mearnsii Can J For Res 2000 30 1886ndash1894 [CrossRef]11 Vandermeer J The Ecology of Intercropping Cambridge University Press New York NY USA 1989 p 23712 Forrester DI The spatial and temporal dynamics of species interactions in mixed-species forests From

pattern to process For Ecol Manag 2014 312 282ndash292 [CrossRef]13 Forrester DI Bauhus J A review of processes behind diversity-productivity relationships in forests

Curr For Rep 2016 2 45ndash61 [CrossRef]14 Wang H Liu S Wang J You Y Yang Y Shi Z Huang X Zheng L Li Z Ming A et al Mixed-species

plantation with Pinus massoniana and Castanopsis hystrix accelerates C loss in recalcitrant coniferous litter butslows C loss in labile broadleaf litter in southern China For Ecol Manag 2018 422 207ndash213 [CrossRef]

Forests 2019 10 879 13 of 20

Forests 2019 10 x FOR PEER REVIEW 14 of 22

Figure 5 Soil extracellular enzyme activities ((andashf) refer to hydrolases (gh) refer to oxidases) in three plantations in different seasons Error bars are standard error Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in three plantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denote significant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

Figure 6 Soil microbial activity represented by average well color development (AWCD) in threeplantations in the dry-cool season (a) and wet-warm season (b) Different letters above the bars denotesignificant differences at p lt 005 level by LSD multiple comparisons among stand types (n = 5)

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in thedry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons(F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pHTK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-coolseason in which SOC and TBB were the major correlates of changes in soil microbial composition(Table S5) A total of 11 biotic and abiotic factors including NO3

minus-N TBB pH TKlitter HBB AP AKTP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warmseason in which NO3

minus-N TBB pH and TKlitter were the major correlates of changes in soil microbialcomposition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

37 Relationship between Soil Microbial Structure and Functions

Soil enzyme activity and soil microbial carbon source utilization ability are both important soilmicrobial functions The first two RDA axes explained 4948 of the variation in soil enzyme activityand carbon utilization ability in the dry-cool season (F = 24 p = 0006 Figure 7b) and 6891 of thevariation in the wet-warm season (F = 64 p = 0002 Figure 7d) AMF and GP were significantlycorrelated with soil microbial functions in the dry-cool season (Table S6) Total bacteria GP bacteriaGN bacteria and actinobacteria were significantly correlated with soil microbial functions in thewet-warm season in which total bacteria GP bacteria and GN bacteria were the major correlates ofchanges of soil enzyme activity (Table S6)

Forests 2019 10 879 14 of 20

Forests 2019 10 x FOR PEER REVIEW 15 of 22

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in the dry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons (F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pH TK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-cool season in which SOC and TBB were the major correlates of changes in soil microbial composition (Table S5) A total of 11 biotic and abiotic factors including NO3minus-N TBB pH TKlitter HBB AP AK TP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warm season in which NO3minus-N TBB pH and TKlitter were the major correlates of changes in soil microbial composition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160 were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant biotic and abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot of soil microbial community structure and soil microbial functions (bd) Above the label represents a significant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowground biomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soil total potassium NO3ndash-N soil nitrate nitrogen HBB herb belowground biomass AP soil available phosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil water

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant bioticand abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot ofsoil microbial community structure and soil microbial functions (bd) Above the label represents asignificant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowgroundbiomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soiltotal potassium NO3

minus-N soil nitrate nitrogen HBB herb belowground biomass AP soil availablephosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil watercontent LP litter production TKlitter litter total potassium GP gram-positive bacteria GN gramnegative bacteria AMF arbuscular mycorrhizal fungi AG α-Glucosidase BG β-14-glucosidaseCB β-Cellobiosidase APH acid phosphatase NAG β-14-N-acetylglucosaminidase PHO phenoloxidase PO peroxidase AWCD average well color development of carbon source metabolism at192 h incubation

4 Discussion

41 Coniferous-Broadleaf Mixture Significantly Increase Stand Biomass and Litter Production

The results of this study indicated that admixing of coniferous and broadleaf tree speciessignificantly increased stand biomass and litter production after 34 years of afforestation in bothdry-cool and wet-warm seasons (Figure 1 Table 2) This may be a consequence of a greater growth of

Forests 2019 10 879 15 of 20

C hystrix in the mixed stands These results confirmed previous findings conducted in our experimentsite measured when the plantations were 26-year-old and 28-year-old respectively [716] A bulkof studies revealed that mixed-species management could affect tree growth stand structure andenvironment [145253] Mixed forests can develop stratified canopies due to niche differences amongdifferent species In the present study trees of the faster growing broad-leaf species (C hystrix) weretaller had a larger DBH and thus formed an upper canopy stratum in the mixed stands (Table 1)While trees of the shorter species (P massoniana) formed a lower stratum with lower DBH and treeheight and the reduced amount of light transmitted through the upper canopy contributed to theirslower growth The dominant tree species within the stand can absorb more sunlight and use itmore efficiently for growth [54] and thus contribute to higher tree biomass and litter productionin mixed stands In addition stratified canopies in mixed plantations can help to maintain levelsof understory species richness and biomass compared with pure plantations due to relatively highlight availabilities [5556] This may be why the mixed stand had higher shrub and herb biomass inour study

Specifically in the present study the broad-leaf species attained greater heights and diameters inmixed stands while masson pine had reduced diameter and height growth in mixed-species comparedto mono-species stands (Table 1) The severe suppression of P massoniana by the faster growingC hystrix may be attributed to differences in the light requirements of the two species [7] The growthpattern of C hystrix and P massoniana in mixed stands in our study (34-year-old) were differed fromwhen they were 26-years-old in which the growth of C hystrix and P massoniana in mixed standswere both greater than in the corresponding mono-species stands [7] This suggests that effects of thetree species mixture could change over time and long-term investigation of mixed forests is of greatsignificance to reveal mixed effects

42 Coniferous-Broadleaf Mixture Significantly Increase Soil Microbial Biomass

The changes of tree species and composition may significantly affect the amount of organic matterinput in soil [57] As the most active part in soil organic matter (SOM) soil microbial biomass isimportant in nutrient cycling and release participating in the circulation of critical elements such ascarbon and nitrogen [23] Because of the quantity and quality of the bulk of SOM changes slowly andthe changes of SOM are difficult to measure due to the spatial heterogeneity of soils soil microbialbiomass and in particular MBC-to-SOC and MBN-to-TN ratio have been suggested as indicators ofthe state and modification of SOM [58ndash60] and as an indicator for carbon (nitrogen) availability insoil [6162] The concentrations of SOC TN and MBC and MBN measured in our study were affectedby stand type and seasons (Table 3 and Table S2 Figure 2) In the dry cool season although the SOCcontent in mixed stands did not significantly differ with that in PCH but significantly lower than thatin PPM the MBC concentration in mixed stands was markedly higher than that in the correspondingmono-species stands Likewise although the TN content in mixed stands was smaller than that in PCHand PPM stands the MBN content in mixed stands was markedly higher than that in the correspondingmono-species stands in the wet-warn season Previous studies found that higher soil microbial biomasscorrelated with higher SOC and TN content that sustain microbial metabolism [2163] However inour study the lower SOC and TN content in MIX was able to maintain a higher microbial biomass thansites where SOC was more abundant Presumably the quality of SOC could explain such a trend [64]and merit further investigation

In this study MBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands(Figure 2) suggesting higher carbon and nitrogen availability in soil and higher soil quality for thegrowth of soil microorganisms by coniferous-broadleaf mixture [21] In addition the effects of admixingon soil microbial biomass MBC-to-SOC and MBN-to-TN ratio were non-additive suggesting theinteraction of P massoniana and C hystrix can have a synergistic effect on improving soil carbon andnitrogen availability (Figure 3) These findings are consistent with many previous studies that havedetermined the effects of tree species mixture on soil microbial biomass [2165] These results may be

Forests 2019 10 879 16 of 20

contributed to (1) greater amount and more diverse organic matter input via litter and rhizodepositionproducts in the mixture of two contrasting tree species [21] (2) the higher use efficiency of soil organiccarbon and nitrogen by microorganisms in mixed stands [66]

We also sought to determine the admixing effects on composition and abundance of soil microbialcommunity with community-level PLFA profiles Surprisingly there were not significant admixingeffects on PLFA content of the major microbial functional groups such as bacteria fungi AMF andactinobacteria (Table 4) However tree species mixture significantly increased the abundance of GNbacteria in the dry-cool season while markedly increased the abundance of GP bacteria but decreasedthe abundance of fungi in the wet-warn season (Figure 4) It seemed that the dominated GN bacteria inthe dry-cool season and GP bacteria in the wet-warn season would correlate with the higher availabilityof soil organic carbon and nitrogen in mixed stands [6768] These correlations are complicated andfurther investigation is required to reveal the underlying mechanisms

43 Effects of Coniferous-Broadleaf Mixture on Soil Microbial Functions

Soil microorganisms excrete soil enzymes to drive nutrient mineralization and litterdecomposition [64] Therefore soil extracellular enzyme activities may be related to the carbonnitrogen and phosphorus cycling in soil [6970] In the present study we found positive admixingeffects on the activities of APH and NAG in the dry-cool season (Figure 5de) On the other handpositive admixing effects on the activities of urease and peroxidase while negative admixing effectson APH was detected in the wet-warn season (Figure 5dfg) This result suggests that the mixtureof C hystrix and P massoniana can accelerate the mineralization rates of soil organic phosphorus andnitrogen These findings were consistent with previous study [67]

The community-level physiological profiling (CLPP) is widely used to characterize microbialcommunities [71ndash73] The average well color development (AWCD) in the CLPP approach reflectsthe ability of soil microorganisms to utilize carbon sources and their metabolic activity The higherAWCD represents the stronger metabolic activity of soil microorganisms [71] In this present study wedetected a significantly positive admixing effect on carbon source utilization ability of the soil microbialcommunities in both dry-cool and wet-warn seasons after 144 h of incubation (Figure 6) Moreover theRDA analysis revealed that carbon source utilization ability of the soil microbial communities werepositive related with APH in the dry-cool season while related with urease and peroxidase activitiesin the wet-warn season (Figure 7) As a whole the higher soil microbial functions in mixed stands arelikely the results of the effects of AMF and GP bacteria in the dry-cool season and the effects of bacteriaincluding GP and GN bacteria the wet-warn season (Table S6)

5 Conclusions

In our study we measured significantly higher tree shrub and herb biomass and litter productionin the coniferous-broadleaf mixed plantation after 34 years of afforestation As expected MBC MBNMBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands suggesting highercarbon and nitrogen availability in soil and higher soil quality for the growth of soil microorganisms inour coniferous-broadleaf mixed stand In addition coniferous-broadleaf mixture can increase carbonsource utilization ability of soil microbial community and accelerate the mineralization rates of soilorganic phosphorus and nitrogen by increasing acid phosphatase β-14-N-acetylglucosaminidaseand urease activities The higher soil microbial biomass and activities are likely the result of greateramount and more diverse organic matter input via litter and rhizodeposition products and the higheruse efficiency of soil organic carbon and nitrogen by microorganisms in mixed stands Howeverthe correlations between soil dominated microbial functional groups and the higher availabilityof soil organic carbon and nitrogen along with the quality of SOC in mixed stands merit furtherinvestigation Our results highlight that establishment of coniferous-broadleaf mixed forests maybe a good management practice as coniferous-broadleaf mixture could accumulate higher stand

Forests 2019 10 879 17 of 20

biomass return more litter and increase soil microbial biomass and functions over the long term insubtropical China

Supplementary Materials The following are available online at httpwwwmdpicom1999-49071010879s1Table S1 Effects of stand type season and their interactions on stand biomass Table S2 Effects of stand type seasonand their interactions on soil microbial structure Table S3 Effects of stand type season and their interactionson relative abundance of the soil microbial community PLFAs Table S4 Effects of stand type season and theirinteractions on soil enzyme activity Table S5 Marginal and conditional effects of biotic and abiotic factors on soilmicrobial composition obtained from the summary of forward selection in Redundancy analysis (RDA) Table S6Marginal and conditional effects of soil microbial functional groups on soil enzyme activity and soil microbialcarbon source utilization ability obtained from the summary of forward selection in Redundancy analysis (RDA)

Author Contributions WW collected data made experiment analysis formal data and drafted the manuscriptYW designed the work XZ revised the manuscript and participated in analyzing the experiment data HZ YYXH and XL ZQ YL LY HL participated in collecting the experiment data

Funding This research was funded by National Natural Science Foundation of China (31860171 3146012131560201) the Guangxi Key Research and Development Program (2018AB40007) Innovation Project of GuangxiGraduate Education (YCBZ2017011) and the Guangxi Universities Science and technology (201201ZD001)

Acknowledgments We gratefully acknowledge the supports in field sampling by the Experimental Center ofTropical Forestry Chinese Academy of Forestry A special acknowledgment is given to Patrick M Finnegan for hiswriting suggestions

Conflicts of Interest The authors declare no conflicts of interest

References

1 Zhang X Liu S Huang Y Fu S Wang J Ming A Li X Yao M Li H Tree species mixture inhibits soilorganic carbon mineralization accompanied by decreased r-selected bacteria Plant Soil 2018 431 203ndash216[CrossRef]

2 Pan Y Birdsey RA Fang J Houghton R Kauppi PE Kurz WA Phillips OL Shvidenko ALewis SL Canadell JG et al A large and persistent carbon sink in the worldrsquos forests Science 2011 333988ndash993 [CrossRef] [PubMed]

3 Chen X Ju Q Lin K Development status issues and countermeasures of Chinarsquo s plantation World For Res2014 27 54ndash59

4 Wang H Lencinas MV Ross Friedman C Wang X Qiu J Understory plant diversity assessment ofEucalyptus plantations over three vegetation types in Yunnan China New For 2011 42 101ndash116 [CrossRef]

5 Sang PM Lamb D Bonner M Schmidt S Carbon sequestration and soil fertility of tropical tree plantationsand secondary forest established on degraded land Plant Soil 2013 362 187ndash200 [CrossRef]

6 Li C Zhou X Wen Y Zhu H Qin Z Li X You Y Huang X Effects of high-generation ratations ofEucalyptus on undergrowth soil fertility and enzyme activities Guangxi Sci 2019 26 176ndash186

7 You Y Huang X Zhu H Liu S Liang H Wen Y Wang H Cai D Ye D Positive interactions betweenPinus massoniana and Castanopsis hystrix species in the uneven-aged mixed plantations can produce moreecosystem carbon in subtropical China For Ecol Manag 2018 410 193ndash200 [CrossRef]

8 Huang X Liu S You Y Wen Y Wang H Wang J Microbial community and associated enzymes activityinfluence soil carbon chemical composition in Eucalyptus urophylla plantation with mixing N2-fixing speciesin sub-tropical China Plant Soil 2017 414 199ndash212 [CrossRef]

9 Kelty MJ The role of species mixtures in plantation forestry For Ecol Manag 2006 233 195ndash204 [CrossRef]10 Bauhus J Khanna P Menden N Aboveground and belowground interactions in mixed plantations of

Eucalyptus globulus and Acacia mearnsii Can J For Res 2000 30 1886ndash1894 [CrossRef]11 Vandermeer J The Ecology of Intercropping Cambridge University Press New York NY USA 1989 p 23712 Forrester DI The spatial and temporal dynamics of species interactions in mixed-species forests From

pattern to process For Ecol Manag 2014 312 282ndash292 [CrossRef]13 Forrester DI Bauhus J A review of processes behind diversity-productivity relationships in forests

Curr For Rep 2016 2 45ndash61 [CrossRef]14 Wang H Liu S Wang J You Y Yang Y Shi Z Huang X Zheng L Li Z Ming A et al Mixed-species

plantation with Pinus massoniana and Castanopsis hystrix accelerates C loss in recalcitrant coniferous litter butslows C loss in labile broadleaf litter in southern China For Ecol Manag 2018 422 207ndash213 [CrossRef]

Forests 2019 10 879 14 of 20

Forests 2019 10 x FOR PEER REVIEW 15 of 22

36 Relationship between Biotic and Abiotic Factors and Soil Microbial Composition

The first two RDA axes explained 6460 of the variation in soil microbial composition in the dry-cool season (F = 20 p = 0044 Figure 7a) and 6587 of the variation in the wet-warm seasons (F = 23 p = 0036 Figure 7c) Nine biotic and abiotic factors including SOC TBB TN CEC pH TK LP TP and TKlitter were significantly related to the soil microbial composition in the dry-cool season in which SOC and TBB were the major correlates of changes in soil microbial composition (Table S5) A total of 11 biotic and abiotic factors including NO3minus-N TBB pH TKlitter HBB AP AK TP SAB LP and SWC were significantly related to the soil microbial composition in the wet-warm season in which NO3minus-N TBB pH and TKlitter were the major correlates of changes in soil microbial composition (Table S5) Specifically the Gram-positive bacteria a150 a170 i170 i140 i150 and i160 were positively related to TBB pH and TKlitter in MIX during the wet-warm season (Figure 7c)

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant biotic and abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot of soil microbial community structure and soil microbial functions (bd) Above the label represents a significant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowground biomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soil total potassium NO3ndash-N soil nitrate nitrogen HBB herb belowground biomass AP soil available phosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil water

Figure 7 Redundancy analysis (RDA) ordination biplot of individual PLFAs and dominant bioticand abiotic factors in dry-cool season and wet-warm season (ac) And RDA ordination biplot ofsoil microbial community structure and soil microbial functions (bd) Above the label represents asignificant conditional effect of this factor (p lt 005) SOC soil organic carbon TBB tree belowgroundbiomass TP soil total phosphorus TN soil total nitrogen CEC soil cation exchange capacity TK soiltotal potassium NO3

minus-N soil nitrate nitrogen HBB herb belowground biomass AP soil availablephosphorus AK soil available potassium SAB shrubs aboveground biomass SWC soil watercontent LP litter production TKlitter litter total potassium GP gram-positive bacteria GN gramnegative bacteria AMF arbuscular mycorrhizal fungi AG α-Glucosidase BG β-14-glucosidaseCB β-Cellobiosidase APH acid phosphatase NAG β-14-N-acetylglucosaminidase PHO phenoloxidase PO peroxidase AWCD average well color development of carbon source metabolism at192 h incubation

4 Discussion

41 Coniferous-Broadleaf Mixture Significantly Increase Stand Biomass and Litter Production

The results of this study indicated that admixing of coniferous and broadleaf tree speciessignificantly increased stand biomass and litter production after 34 years of afforestation in bothdry-cool and wet-warm seasons (Figure 1 Table 2) This may be a consequence of a greater growth of

Forests 2019 10 879 15 of 20

C hystrix in the mixed stands These results confirmed previous findings conducted in our experimentsite measured when the plantations were 26-year-old and 28-year-old respectively [716] A bulkof studies revealed that mixed-species management could affect tree growth stand structure andenvironment [145253] Mixed forests can develop stratified canopies due to niche differences amongdifferent species In the present study trees of the faster growing broad-leaf species (C hystrix) weretaller had a larger DBH and thus formed an upper canopy stratum in the mixed stands (Table 1)While trees of the shorter species (P massoniana) formed a lower stratum with lower DBH and treeheight and the reduced amount of light transmitted through the upper canopy contributed to theirslower growth The dominant tree species within the stand can absorb more sunlight and use itmore efficiently for growth [54] and thus contribute to higher tree biomass and litter productionin mixed stands In addition stratified canopies in mixed plantations can help to maintain levelsof understory species richness and biomass compared with pure plantations due to relatively highlight availabilities [5556] This may be why the mixed stand had higher shrub and herb biomass inour study

Specifically in the present study the broad-leaf species attained greater heights and diameters inmixed stands while masson pine had reduced diameter and height growth in mixed-species comparedto mono-species stands (Table 1) The severe suppression of P massoniana by the faster growingC hystrix may be attributed to differences in the light requirements of the two species [7] The growthpattern of C hystrix and P massoniana in mixed stands in our study (34-year-old) were differed fromwhen they were 26-years-old in which the growth of C hystrix and P massoniana in mixed standswere both greater than in the corresponding mono-species stands [7] This suggests that effects of thetree species mixture could change over time and long-term investigation of mixed forests is of greatsignificance to reveal mixed effects

42 Coniferous-Broadleaf Mixture Significantly Increase Soil Microbial Biomass

The changes of tree species and composition may significantly affect the amount of organic matterinput in soil [57] As the most active part in soil organic matter (SOM) soil microbial biomass isimportant in nutrient cycling and release participating in the circulation of critical elements such ascarbon and nitrogen [23] Because of the quantity and quality of the bulk of SOM changes slowly andthe changes of SOM are difficult to measure due to the spatial heterogeneity of soils soil microbialbiomass and in particular MBC-to-SOC and MBN-to-TN ratio have been suggested as indicators ofthe state and modification of SOM [58ndash60] and as an indicator for carbon (nitrogen) availability insoil [6162] The concentrations of SOC TN and MBC and MBN measured in our study were affectedby stand type and seasons (Table 3 and Table S2 Figure 2) In the dry cool season although the SOCcontent in mixed stands did not significantly differ with that in PCH but significantly lower than thatin PPM the MBC concentration in mixed stands was markedly higher than that in the correspondingmono-species stands Likewise although the TN content in mixed stands was smaller than that in PCHand PPM stands the MBN content in mixed stands was markedly higher than that in the correspondingmono-species stands in the wet-warn season Previous studies found that higher soil microbial biomasscorrelated with higher SOC and TN content that sustain microbial metabolism [2163] However inour study the lower SOC and TN content in MIX was able to maintain a higher microbial biomass thansites where SOC was more abundant Presumably the quality of SOC could explain such a trend [64]and merit further investigation

In this study MBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands(Figure 2) suggesting higher carbon and nitrogen availability in soil and higher soil quality for thegrowth of soil microorganisms by coniferous-broadleaf mixture [21] In addition the effects of admixingon soil microbial biomass MBC-to-SOC and MBN-to-TN ratio were non-additive suggesting theinteraction of P massoniana and C hystrix can have a synergistic effect on improving soil carbon andnitrogen availability (Figure 3) These findings are consistent with many previous studies that havedetermined the effects of tree species mixture on soil microbial biomass [2165] These results may be

Forests 2019 10 879 16 of 20

contributed to (1) greater amount and more diverse organic matter input via litter and rhizodepositionproducts in the mixture of two contrasting tree species [21] (2) the higher use efficiency of soil organiccarbon and nitrogen by microorganisms in mixed stands [66]

We also sought to determine the admixing effects on composition and abundance of soil microbialcommunity with community-level PLFA profiles Surprisingly there were not significant admixingeffects on PLFA content of the major microbial functional groups such as bacteria fungi AMF andactinobacteria (Table 4) However tree species mixture significantly increased the abundance of GNbacteria in the dry-cool season while markedly increased the abundance of GP bacteria but decreasedthe abundance of fungi in the wet-warn season (Figure 4) It seemed that the dominated GN bacteria inthe dry-cool season and GP bacteria in the wet-warn season would correlate with the higher availabilityof soil organic carbon and nitrogen in mixed stands [6768] These correlations are complicated andfurther investigation is required to reveal the underlying mechanisms

43 Effects of Coniferous-Broadleaf Mixture on Soil Microbial Functions

Soil microorganisms excrete soil enzymes to drive nutrient mineralization and litterdecomposition [64] Therefore soil extracellular enzyme activities may be related to the carbonnitrogen and phosphorus cycling in soil [6970] In the present study we found positive admixingeffects on the activities of APH and NAG in the dry-cool season (Figure 5de) On the other handpositive admixing effects on the activities of urease and peroxidase while negative admixing effectson APH was detected in the wet-warn season (Figure 5dfg) This result suggests that the mixtureof C hystrix and P massoniana can accelerate the mineralization rates of soil organic phosphorus andnitrogen These findings were consistent with previous study [67]

The community-level physiological profiling (CLPP) is widely used to characterize microbialcommunities [71ndash73] The average well color development (AWCD) in the CLPP approach reflectsthe ability of soil microorganisms to utilize carbon sources and their metabolic activity The higherAWCD represents the stronger metabolic activity of soil microorganisms [71] In this present study wedetected a significantly positive admixing effect on carbon source utilization ability of the soil microbialcommunities in both dry-cool and wet-warn seasons after 144 h of incubation (Figure 6) Moreover theRDA analysis revealed that carbon source utilization ability of the soil microbial communities werepositive related with APH in the dry-cool season while related with urease and peroxidase activitiesin the wet-warn season (Figure 7) As a whole the higher soil microbial functions in mixed stands arelikely the results of the effects of AMF and GP bacteria in the dry-cool season and the effects of bacteriaincluding GP and GN bacteria the wet-warn season (Table S6)

5 Conclusions

In our study we measured significantly higher tree shrub and herb biomass and litter productionin the coniferous-broadleaf mixed plantation after 34 years of afforestation As expected MBC MBNMBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands suggesting highercarbon and nitrogen availability in soil and higher soil quality for the growth of soil microorganisms inour coniferous-broadleaf mixed stand In addition coniferous-broadleaf mixture can increase carbonsource utilization ability of soil microbial community and accelerate the mineralization rates of soilorganic phosphorus and nitrogen by increasing acid phosphatase β-14-N-acetylglucosaminidaseand urease activities The higher soil microbial biomass and activities are likely the result of greateramount and more diverse organic matter input via litter and rhizodeposition products and the higheruse efficiency of soil organic carbon and nitrogen by microorganisms in mixed stands Howeverthe correlations between soil dominated microbial functional groups and the higher availabilityof soil organic carbon and nitrogen along with the quality of SOC in mixed stands merit furtherinvestigation Our results highlight that establishment of coniferous-broadleaf mixed forests maybe a good management practice as coniferous-broadleaf mixture could accumulate higher stand

Forests 2019 10 879 17 of 20

biomass return more litter and increase soil microbial biomass and functions over the long term insubtropical China

Supplementary Materials The following are available online at httpwwwmdpicom1999-49071010879s1Table S1 Effects of stand type season and their interactions on stand biomass Table S2 Effects of stand type seasonand their interactions on soil microbial structure Table S3 Effects of stand type season and their interactionson relative abundance of the soil microbial community PLFAs Table S4 Effects of stand type season and theirinteractions on soil enzyme activity Table S5 Marginal and conditional effects of biotic and abiotic factors on soilmicrobial composition obtained from the summary of forward selection in Redundancy analysis (RDA) Table S6Marginal and conditional effects of soil microbial functional groups on soil enzyme activity and soil microbialcarbon source utilization ability obtained from the summary of forward selection in Redundancy analysis (RDA)

Author Contributions WW collected data made experiment analysis formal data and drafted the manuscriptYW designed the work XZ revised the manuscript and participated in analyzing the experiment data HZ YYXH and XL ZQ YL LY HL participated in collecting the experiment data

Funding This research was funded by National Natural Science Foundation of China (31860171 3146012131560201) the Guangxi Key Research and Development Program (2018AB40007) Innovation Project of GuangxiGraduate Education (YCBZ2017011) and the Guangxi Universities Science and technology (201201ZD001)

Acknowledgments We gratefully acknowledge the supports in field sampling by the Experimental Center ofTropical Forestry Chinese Academy of Forestry A special acknowledgment is given to Patrick M Finnegan for hiswriting suggestions

Conflicts of Interest The authors declare no conflicts of interest

References

1 Zhang X Liu S Huang Y Fu S Wang J Ming A Li X Yao M Li H Tree species mixture inhibits soilorganic carbon mineralization accompanied by decreased r-selected bacteria Plant Soil 2018 431 203ndash216[CrossRef]

2 Pan Y Birdsey RA Fang J Houghton R Kauppi PE Kurz WA Phillips OL Shvidenko ALewis SL Canadell JG et al A large and persistent carbon sink in the worldrsquos forests Science 2011 333988ndash993 [CrossRef] [PubMed]

3 Chen X Ju Q Lin K Development status issues and countermeasures of Chinarsquo s plantation World For Res2014 27 54ndash59

4 Wang H Lencinas MV Ross Friedman C Wang X Qiu J Understory plant diversity assessment ofEucalyptus plantations over three vegetation types in Yunnan China New For 2011 42 101ndash116 [CrossRef]

5 Sang PM Lamb D Bonner M Schmidt S Carbon sequestration and soil fertility of tropical tree plantationsand secondary forest established on degraded land Plant Soil 2013 362 187ndash200 [CrossRef]

6 Li C Zhou X Wen Y Zhu H Qin Z Li X You Y Huang X Effects of high-generation ratations ofEucalyptus on undergrowth soil fertility and enzyme activities Guangxi Sci 2019 26 176ndash186

7 You Y Huang X Zhu H Liu S Liang H Wen Y Wang H Cai D Ye D Positive interactions betweenPinus massoniana and Castanopsis hystrix species in the uneven-aged mixed plantations can produce moreecosystem carbon in subtropical China For Ecol Manag 2018 410 193ndash200 [CrossRef]

8 Huang X Liu S You Y Wen Y Wang H Wang J Microbial community and associated enzymes activityinfluence soil carbon chemical composition in Eucalyptus urophylla plantation with mixing N2-fixing speciesin sub-tropical China Plant Soil 2017 414 199ndash212 [CrossRef]

9 Kelty MJ The role of species mixtures in plantation forestry For Ecol Manag 2006 233 195ndash204 [CrossRef]10 Bauhus J Khanna P Menden N Aboveground and belowground interactions in mixed plantations of

Eucalyptus globulus and Acacia mearnsii Can J For Res 2000 30 1886ndash1894 [CrossRef]11 Vandermeer J The Ecology of Intercropping Cambridge University Press New York NY USA 1989 p 23712 Forrester DI The spatial and temporal dynamics of species interactions in mixed-species forests From

pattern to process For Ecol Manag 2014 312 282ndash292 [CrossRef]13 Forrester DI Bauhus J A review of processes behind diversity-productivity relationships in forests

Curr For Rep 2016 2 45ndash61 [CrossRef]14 Wang H Liu S Wang J You Y Yang Y Shi Z Huang X Zheng L Li Z Ming A et al Mixed-species

plantation with Pinus massoniana and Castanopsis hystrix accelerates C loss in recalcitrant coniferous litter butslows C loss in labile broadleaf litter in southern China For Ecol Manag 2018 422 207ndash213 [CrossRef]

Forests 2019 10 879 15 of 20

C hystrix in the mixed stands These results confirmed previous findings conducted in our experimentsite measured when the plantations were 26-year-old and 28-year-old respectively [716] A bulkof studies revealed that mixed-species management could affect tree growth stand structure andenvironment [145253] Mixed forests can develop stratified canopies due to niche differences amongdifferent species In the present study trees of the faster growing broad-leaf species (C hystrix) weretaller had a larger DBH and thus formed an upper canopy stratum in the mixed stands (Table 1)While trees of the shorter species (P massoniana) formed a lower stratum with lower DBH and treeheight and the reduced amount of light transmitted through the upper canopy contributed to theirslower growth The dominant tree species within the stand can absorb more sunlight and use itmore efficiently for growth [54] and thus contribute to higher tree biomass and litter productionin mixed stands In addition stratified canopies in mixed plantations can help to maintain levelsof understory species richness and biomass compared with pure plantations due to relatively highlight availabilities [5556] This may be why the mixed stand had higher shrub and herb biomass inour study

Specifically in the present study the broad-leaf species attained greater heights and diameters inmixed stands while masson pine had reduced diameter and height growth in mixed-species comparedto mono-species stands (Table 1) The severe suppression of P massoniana by the faster growingC hystrix may be attributed to differences in the light requirements of the two species [7] The growthpattern of C hystrix and P massoniana in mixed stands in our study (34-year-old) were differed fromwhen they were 26-years-old in which the growth of C hystrix and P massoniana in mixed standswere both greater than in the corresponding mono-species stands [7] This suggests that effects of thetree species mixture could change over time and long-term investigation of mixed forests is of greatsignificance to reveal mixed effects

42 Coniferous-Broadleaf Mixture Significantly Increase Soil Microbial Biomass

The changes of tree species and composition may significantly affect the amount of organic matterinput in soil [57] As the most active part in soil organic matter (SOM) soil microbial biomass isimportant in nutrient cycling and release participating in the circulation of critical elements such ascarbon and nitrogen [23] Because of the quantity and quality of the bulk of SOM changes slowly andthe changes of SOM are difficult to measure due to the spatial heterogeneity of soils soil microbialbiomass and in particular MBC-to-SOC and MBN-to-TN ratio have been suggested as indicators ofthe state and modification of SOM [58ndash60] and as an indicator for carbon (nitrogen) availability insoil [6162] The concentrations of SOC TN and MBC and MBN measured in our study were affectedby stand type and seasons (Table 3 and Table S2 Figure 2) In the dry cool season although the SOCcontent in mixed stands did not significantly differ with that in PCH but significantly lower than thatin PPM the MBC concentration in mixed stands was markedly higher than that in the correspondingmono-species stands Likewise although the TN content in mixed stands was smaller than that in PCHand PPM stands the MBN content in mixed stands was markedly higher than that in the correspondingmono-species stands in the wet-warn season Previous studies found that higher soil microbial biomasscorrelated with higher SOC and TN content that sustain microbial metabolism [2163] However inour study the lower SOC and TN content in MIX was able to maintain a higher microbial biomass thansites where SOC was more abundant Presumably the quality of SOC could explain such a trend [64]and merit further investigation

In this study MBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands(Figure 2) suggesting higher carbon and nitrogen availability in soil and higher soil quality for thegrowth of soil microorganisms by coniferous-broadleaf mixture [21] In addition the effects of admixingon soil microbial biomass MBC-to-SOC and MBN-to-TN ratio were non-additive suggesting theinteraction of P massoniana and C hystrix can have a synergistic effect on improving soil carbon andnitrogen availability (Figure 3) These findings are consistent with many previous studies that havedetermined the effects of tree species mixture on soil microbial biomass [2165] These results may be

Forests 2019 10 879 16 of 20

contributed to (1) greater amount and more diverse organic matter input via litter and rhizodepositionproducts in the mixture of two contrasting tree species [21] (2) the higher use efficiency of soil organiccarbon and nitrogen by microorganisms in mixed stands [66]

We also sought to determine the admixing effects on composition and abundance of soil microbialcommunity with community-level PLFA profiles Surprisingly there were not significant admixingeffects on PLFA content of the major microbial functional groups such as bacteria fungi AMF andactinobacteria (Table 4) However tree species mixture significantly increased the abundance of GNbacteria in the dry-cool season while markedly increased the abundance of GP bacteria but decreasedthe abundance of fungi in the wet-warn season (Figure 4) It seemed that the dominated GN bacteria inthe dry-cool season and GP bacteria in the wet-warn season would correlate with the higher availabilityof soil organic carbon and nitrogen in mixed stands [6768] These correlations are complicated andfurther investigation is required to reveal the underlying mechanisms

43 Effects of Coniferous-Broadleaf Mixture on Soil Microbial Functions

Soil microorganisms excrete soil enzymes to drive nutrient mineralization and litterdecomposition [64] Therefore soil extracellular enzyme activities may be related to the carbonnitrogen and phosphorus cycling in soil [6970] In the present study we found positive admixingeffects on the activities of APH and NAG in the dry-cool season (Figure 5de) On the other handpositive admixing effects on the activities of urease and peroxidase while negative admixing effectson APH was detected in the wet-warn season (Figure 5dfg) This result suggests that the mixtureof C hystrix and P massoniana can accelerate the mineralization rates of soil organic phosphorus andnitrogen These findings were consistent with previous study [67]

The community-level physiological profiling (CLPP) is widely used to characterize microbialcommunities [71ndash73] The average well color development (AWCD) in the CLPP approach reflectsthe ability of soil microorganisms to utilize carbon sources and their metabolic activity The higherAWCD represents the stronger metabolic activity of soil microorganisms [71] In this present study wedetected a significantly positive admixing effect on carbon source utilization ability of the soil microbialcommunities in both dry-cool and wet-warn seasons after 144 h of incubation (Figure 6) Moreover theRDA analysis revealed that carbon source utilization ability of the soil microbial communities werepositive related with APH in the dry-cool season while related with urease and peroxidase activitiesin the wet-warn season (Figure 7) As a whole the higher soil microbial functions in mixed stands arelikely the results of the effects of AMF and GP bacteria in the dry-cool season and the effects of bacteriaincluding GP and GN bacteria the wet-warn season (Table S6)

5 Conclusions

In our study we measured significantly higher tree shrub and herb biomass and litter productionin the coniferous-broadleaf mixed plantation after 34 years of afforestation As expected MBC MBNMBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands suggesting highercarbon and nitrogen availability in soil and higher soil quality for the growth of soil microorganisms inour coniferous-broadleaf mixed stand In addition coniferous-broadleaf mixture can increase carbonsource utilization ability of soil microbial community and accelerate the mineralization rates of soilorganic phosphorus and nitrogen by increasing acid phosphatase β-14-N-acetylglucosaminidaseand urease activities The higher soil microbial biomass and activities are likely the result of greateramount and more diverse organic matter input via litter and rhizodeposition products and the higheruse efficiency of soil organic carbon and nitrogen by microorganisms in mixed stands Howeverthe correlations between soil dominated microbial functional groups and the higher availabilityof soil organic carbon and nitrogen along with the quality of SOC in mixed stands merit furtherinvestigation Our results highlight that establishment of coniferous-broadleaf mixed forests maybe a good management practice as coniferous-broadleaf mixture could accumulate higher stand

Forests 2019 10 879 17 of 20

biomass return more litter and increase soil microbial biomass and functions over the long term insubtropical China

Supplementary Materials The following are available online at httpwwwmdpicom1999-49071010879s1Table S1 Effects of stand type season and their interactions on stand biomass Table S2 Effects of stand type seasonand their interactions on soil microbial structure Table S3 Effects of stand type season and their interactionson relative abundance of the soil microbial community PLFAs Table S4 Effects of stand type season and theirinteractions on soil enzyme activity Table S5 Marginal and conditional effects of biotic and abiotic factors on soilmicrobial composition obtained from the summary of forward selection in Redundancy analysis (RDA) Table S6Marginal and conditional effects of soil microbial functional groups on soil enzyme activity and soil microbialcarbon source utilization ability obtained from the summary of forward selection in Redundancy analysis (RDA)

Author Contributions WW collected data made experiment analysis formal data and drafted the manuscriptYW designed the work XZ revised the manuscript and participated in analyzing the experiment data HZ YYXH and XL ZQ YL LY HL participated in collecting the experiment data

Funding This research was funded by National Natural Science Foundation of China (31860171 3146012131560201) the Guangxi Key Research and Development Program (2018AB40007) Innovation Project of GuangxiGraduate Education (YCBZ2017011) and the Guangxi Universities Science and technology (201201ZD001)

Acknowledgments We gratefully acknowledge the supports in field sampling by the Experimental Center ofTropical Forestry Chinese Academy of Forestry A special acknowledgment is given to Patrick M Finnegan for hiswriting suggestions

Conflicts of Interest The authors declare no conflicts of interest

References

1 Zhang X Liu S Huang Y Fu S Wang J Ming A Li X Yao M Li H Tree species mixture inhibits soilorganic carbon mineralization accompanied by decreased r-selected bacteria Plant Soil 2018 431 203ndash216[CrossRef]

2 Pan Y Birdsey RA Fang J Houghton R Kauppi PE Kurz WA Phillips OL Shvidenko ALewis SL Canadell JG et al A large and persistent carbon sink in the worldrsquos forests Science 2011 333988ndash993 [CrossRef] [PubMed]

3 Chen X Ju Q Lin K Development status issues and countermeasures of Chinarsquo s plantation World For Res2014 27 54ndash59

4 Wang H Lencinas MV Ross Friedman C Wang X Qiu J Understory plant diversity assessment ofEucalyptus plantations over three vegetation types in Yunnan China New For 2011 42 101ndash116 [CrossRef]

5 Sang PM Lamb D Bonner M Schmidt S Carbon sequestration and soil fertility of tropical tree plantationsand secondary forest established on degraded land Plant Soil 2013 362 187ndash200 [CrossRef]

6 Li C Zhou X Wen Y Zhu H Qin Z Li X You Y Huang X Effects of high-generation ratations ofEucalyptus on undergrowth soil fertility and enzyme activities Guangxi Sci 2019 26 176ndash186

7 You Y Huang X Zhu H Liu S Liang H Wen Y Wang H Cai D Ye D Positive interactions betweenPinus massoniana and Castanopsis hystrix species in the uneven-aged mixed plantations can produce moreecosystem carbon in subtropical China For Ecol Manag 2018 410 193ndash200 [CrossRef]

8 Huang X Liu S You Y Wen Y Wang H Wang J Microbial community and associated enzymes activityinfluence soil carbon chemical composition in Eucalyptus urophylla plantation with mixing N2-fixing speciesin sub-tropical China Plant Soil 2017 414 199ndash212 [CrossRef]

9 Kelty MJ The role of species mixtures in plantation forestry For Ecol Manag 2006 233 195ndash204 [CrossRef]10 Bauhus J Khanna P Menden N Aboveground and belowground interactions in mixed plantations of

Eucalyptus globulus and Acacia mearnsii Can J For Res 2000 30 1886ndash1894 [CrossRef]11 Vandermeer J The Ecology of Intercropping Cambridge University Press New York NY USA 1989 p 23712 Forrester DI The spatial and temporal dynamics of species interactions in mixed-species forests From

pattern to process For Ecol Manag 2014 312 282ndash292 [CrossRef]13 Forrester DI Bauhus J A review of processes behind diversity-productivity relationships in forests

Curr For Rep 2016 2 45ndash61 [CrossRef]14 Wang H Liu S Wang J You Y Yang Y Shi Z Huang X Zheng L Li Z Ming A et al Mixed-species

plantation with Pinus massoniana and Castanopsis hystrix accelerates C loss in recalcitrant coniferous litter butslows C loss in labile broadleaf litter in southern China For Ecol Manag 2018 422 207ndash213 [CrossRef]

Forests 2019 10 879 16 of 20

contributed to (1) greater amount and more diverse organic matter input via litter and rhizodepositionproducts in the mixture of two contrasting tree species [21] (2) the higher use efficiency of soil organiccarbon and nitrogen by microorganisms in mixed stands [66]

We also sought to determine the admixing effects on composition and abundance of soil microbialcommunity with community-level PLFA profiles Surprisingly there were not significant admixingeffects on PLFA content of the major microbial functional groups such as bacteria fungi AMF andactinobacteria (Table 4) However tree species mixture significantly increased the abundance of GNbacteria in the dry-cool season while markedly increased the abundance of GP bacteria but decreasedthe abundance of fungi in the wet-warn season (Figure 4) It seemed that the dominated GN bacteria inthe dry-cool season and GP bacteria in the wet-warn season would correlate with the higher availabilityof soil organic carbon and nitrogen in mixed stands [6768] These correlations are complicated andfurther investigation is required to reveal the underlying mechanisms

43 Effects of Coniferous-Broadleaf Mixture on Soil Microbial Functions

Soil microorganisms excrete soil enzymes to drive nutrient mineralization and litterdecomposition [64] Therefore soil extracellular enzyme activities may be related to the carbonnitrogen and phosphorus cycling in soil [6970] In the present study we found positive admixingeffects on the activities of APH and NAG in the dry-cool season (Figure 5de) On the other handpositive admixing effects on the activities of urease and peroxidase while negative admixing effectson APH was detected in the wet-warn season (Figure 5dfg) This result suggests that the mixtureof C hystrix and P massoniana can accelerate the mineralization rates of soil organic phosphorus andnitrogen These findings were consistent with previous study [67]

The community-level physiological profiling (CLPP) is widely used to characterize microbialcommunities [71ndash73] The average well color development (AWCD) in the CLPP approach reflectsthe ability of soil microorganisms to utilize carbon sources and their metabolic activity The higherAWCD represents the stronger metabolic activity of soil microorganisms [71] In this present study wedetected a significantly positive admixing effect on carbon source utilization ability of the soil microbialcommunities in both dry-cool and wet-warn seasons after 144 h of incubation (Figure 6) Moreover theRDA analysis revealed that carbon source utilization ability of the soil microbial communities werepositive related with APH in the dry-cool season while related with urease and peroxidase activitiesin the wet-warn season (Figure 7) As a whole the higher soil microbial functions in mixed stands arelikely the results of the effects of AMF and GP bacteria in the dry-cool season and the effects of bacteriaincluding GP and GN bacteria the wet-warn season (Table S6)

5 Conclusions

In our study we measured significantly higher tree shrub and herb biomass and litter productionin the coniferous-broadleaf mixed plantation after 34 years of afforestation As expected MBC MBNMBC-to-SOC and MBN-to-TN ratio were significantly higher in mixed stands suggesting highercarbon and nitrogen availability in soil and higher soil quality for the growth of soil microorganisms inour coniferous-broadleaf mixed stand In addition coniferous-broadleaf mixture can increase carbonsource utilization ability of soil microbial community and accelerate the mineralization rates of soilorganic phosphorus and nitrogen by increasing acid phosphatase β-14-N-acetylglucosaminidaseand urease activities The higher soil microbial biomass and activities are likely the result of greateramount and more diverse organic matter input via litter and rhizodeposition products and the higheruse efficiency of soil organic carbon and nitrogen by microorganisms in mixed stands Howeverthe correlations between soil dominated microbial functional groups and the higher availabilityof soil organic carbon and nitrogen along with the quality of SOC in mixed stands merit furtherinvestigation Our results highlight that establishment of coniferous-broadleaf mixed forests maybe a good management practice as coniferous-broadleaf mixture could accumulate higher stand

Forests 2019 10 879 17 of 20

biomass return more litter and increase soil microbial biomass and functions over the long term insubtropical China

Supplementary Materials The following are available online at httpwwwmdpicom1999-49071010879s1Table S1 Effects of stand type season and their interactions on stand biomass Table S2 Effects of stand type seasonand their interactions on soil microbial structure Table S3 Effects of stand type season and their interactionson relative abundance of the soil microbial community PLFAs Table S4 Effects of stand type season and theirinteractions on soil enzyme activity Table S5 Marginal and conditional effects of biotic and abiotic factors on soilmicrobial composition obtained from the summary of forward selection in Redundancy analysis (RDA) Table S6Marginal and conditional effects of soil microbial functional groups on soil enzyme activity and soil microbialcarbon source utilization ability obtained from the summary of forward selection in Redundancy analysis (RDA)

Author Contributions WW collected data made experiment analysis formal data and drafted the manuscriptYW designed the work XZ revised the manuscript and participated in analyzing the experiment data HZ YYXH and XL ZQ YL LY HL participated in collecting the experiment data

Funding This research was funded by National Natural Science Foundation of China (31860171 3146012131560201) the Guangxi Key Research and Development Program (2018AB40007) Innovation Project of GuangxiGraduate Education (YCBZ2017011) and the Guangxi Universities Science and technology (201201ZD001)

Acknowledgments We gratefully acknowledge the supports in field sampling by the Experimental Center ofTropical Forestry Chinese Academy of Forestry A special acknowledgment is given to Patrick M Finnegan for hiswriting suggestions

Conflicts of Interest The authors declare no conflicts of interest

References

1 Zhang X Liu S Huang Y Fu S Wang J Ming A Li X Yao M Li H Tree species mixture inhibits soilorganic carbon mineralization accompanied by decreased r-selected bacteria Plant Soil 2018 431 203ndash216[CrossRef]

2 Pan Y Birdsey RA Fang J Houghton R Kauppi PE Kurz WA Phillips OL Shvidenko ALewis SL Canadell JG et al A large and persistent carbon sink in the worldrsquos forests Science 2011 333988ndash993 [CrossRef] [PubMed]

3 Chen X Ju Q Lin K Development status issues and countermeasures of Chinarsquo s plantation World For Res2014 27 54ndash59

4 Wang H Lencinas MV Ross Friedman C Wang X Qiu J Understory plant diversity assessment ofEucalyptus plantations over three vegetation types in Yunnan China New For 2011 42 101ndash116 [CrossRef]

5 Sang PM Lamb D Bonner M Schmidt S Carbon sequestration and soil fertility of tropical tree plantationsand secondary forest established on degraded land Plant Soil 2013 362 187ndash200 [CrossRef]

6 Li C Zhou X Wen Y Zhu H Qin Z Li X You Y Huang X Effects of high-generation ratations ofEucalyptus on undergrowth soil fertility and enzyme activities Guangxi Sci 2019 26 176ndash186

7 You Y Huang X Zhu H Liu S Liang H Wen Y Wang H Cai D Ye D Positive interactions betweenPinus massoniana and Castanopsis hystrix species in the uneven-aged mixed plantations can produce moreecosystem carbon in subtropical China For Ecol Manag 2018 410 193ndash200 [CrossRef]

8 Huang X Liu S You Y Wen Y Wang H Wang J Microbial community and associated enzymes activityinfluence soil carbon chemical composition in Eucalyptus urophylla plantation with mixing N2-fixing speciesin sub-tropical China Plant Soil 2017 414 199ndash212 [CrossRef]

9 Kelty MJ The role of species mixtures in plantation forestry For Ecol Manag 2006 233 195ndash204 [CrossRef]10 Bauhus J Khanna P Menden N Aboveground and belowground interactions in mixed plantations of

Eucalyptus globulus and Acacia mearnsii Can J For Res 2000 30 1886ndash1894 [CrossRef]11 Vandermeer J The Ecology of Intercropping Cambridge University Press New York NY USA 1989 p 23712 Forrester DI The spatial and temporal dynamics of species interactions in mixed-species forests From

pattern to process For Ecol Manag 2014 312 282ndash292 [CrossRef]13 Forrester DI Bauhus J A review of processes behind diversity-productivity relationships in forests

Curr For Rep 2016 2 45ndash61 [CrossRef]14 Wang H Liu S Wang J You Y Yang Y Shi Z Huang X Zheng L Li Z Ming A et al Mixed-species

plantation with Pinus massoniana and Castanopsis hystrix accelerates C loss in recalcitrant coniferous litter butslows C loss in labile broadleaf litter in southern China For Ecol Manag 2018 422 207ndash213 [CrossRef]

Forests 2019 10 879 17 of 20

biomass return more litter and increase soil microbial biomass and functions over the long term insubtropical China

Supplementary Materials The following are available online at httpwwwmdpicom1999-49071010879s1Table S1 Effects of stand type season and their interactions on stand biomass Table S2 Effects of stand type seasonand their interactions on soil microbial structure Table S3 Effects of stand type season and their interactionson relative abundance of the soil microbial community PLFAs Table S4 Effects of stand type season and theirinteractions on soil enzyme activity Table S5 Marginal and conditional effects of biotic and abiotic factors on soilmicrobial composition obtained from the summary of forward selection in Redundancy analysis (RDA) Table S6Marginal and conditional effects of soil microbial functional groups on soil enzyme activity and soil microbialcarbon source utilization ability obtained from the summary of forward selection in Redundancy analysis (RDA)

Author Contributions WW collected data made experiment analysis formal data and drafted the manuscriptYW designed the work XZ revised the manuscript and participated in analyzing the experiment data HZ YYXH and XL ZQ YL LY HL participated in collecting the experiment data

Funding This research was funded by National Natural Science Foundation of China (31860171 3146012131560201) the Guangxi Key Research and Development Program (2018AB40007) Innovation Project of GuangxiGraduate Education (YCBZ2017011) and the Guangxi Universities Science and technology (201201ZD001)

Acknowledgments We gratefully acknowledge the supports in field sampling by the Experimental Center ofTropical Forestry Chinese Academy of Forestry A special acknowledgment is given to Patrick M Finnegan for hiswriting suggestions

Conflicts of Interest The authors declare no conflicts of interest

References

1 Zhang X Liu S Huang Y Fu S Wang J Ming A Li X Yao M Li H Tree species mixture inhibits soilorganic carbon mineralization accompanied by decreased r-selected bacteria Plant Soil 2018 431 203ndash216[CrossRef]

2 Pan Y Birdsey RA Fang J Houghton R Kauppi PE Kurz WA Phillips OL Shvidenko ALewis SL Canadell JG et al A large and persistent carbon sink in the worldrsquos forests Science 2011 333988ndash993 [CrossRef] [PubMed]

3 Chen X Ju Q Lin K Development status issues and countermeasures of Chinarsquo s plantation World For Res2014 27 54ndash59

4 Wang H Lencinas MV Ross Friedman C Wang X Qiu J Understory plant diversity assessment ofEucalyptus plantations over three vegetation types in Yunnan China New For 2011 42 101ndash116 [CrossRef]

5 Sang PM Lamb D Bonner M Schmidt S Carbon sequestration and soil fertility of tropical tree plantationsand secondary forest established on degraded land Plant Soil 2013 362 187ndash200 [CrossRef]

6 Li C Zhou X Wen Y Zhu H Qin Z Li X You Y Huang X Effects of high-generation ratations ofEucalyptus on undergrowth soil fertility and enzyme activities Guangxi Sci 2019 26 176ndash186

7 You Y Huang X Zhu H Liu S Liang H Wen Y Wang H Cai D Ye D Positive interactions betweenPinus massoniana and Castanopsis hystrix species in the uneven-aged mixed plantations can produce moreecosystem carbon in subtropical China For Ecol Manag 2018 410 193ndash200 [CrossRef]

8 Huang X Liu S You Y Wen Y Wang H Wang J Microbial community and associated enzymes activityinfluence soil carbon chemical composition in Eucalyptus urophylla plantation with mixing N2-fixing speciesin sub-tropical China Plant Soil 2017 414 199ndash212 [CrossRef]

9 Kelty MJ The role of species mixtures in plantation forestry For Ecol Manag 2006 233 195ndash204 [CrossRef]10 Bauhus J Khanna P Menden N Aboveground and belowground interactions in mixed plantations of

Eucalyptus globulus and Acacia mearnsii Can J For Res 2000 30 1886ndash1894 [CrossRef]11 Vandermeer J The Ecology of Intercropping Cambridge University Press New York NY USA 1989 p 23712 Forrester DI The spatial and temporal dynamics of species interactions in mixed-species forests From

pattern to process For Ecol Manag 2014 312 282ndash292 [CrossRef]13 Forrester DI Bauhus J A review of processes behind diversity-productivity relationships in forests

Curr For Rep 2016 2 45ndash61 [CrossRef]14 Wang H Liu S Wang J You Y Yang Y Shi Z Huang X Zheng L Li Z Ming A et al Mixed-species

plantation with Pinus massoniana and Castanopsis hystrix accelerates C loss in recalcitrant coniferous litter butslows C loss in labile broadleaf litter in southern China For Ecol Manag 2018 422 207ndash213 [CrossRef]

Forests 2019 10 879 18 of 20

15 Zhao Z Jia H Cai D Pang S Ang N Liu Y Natural regeneration and its influencing factors ofCastanopsis hystrix J Beijing For Univ 2018 40 76ndash83

16 He Y Qin L Li Z Liang X Shao M Tan L Carbon storage capacity of monoculture and mixed-speciesplantations in subtropical China For Ecol Manag 2013 295 193ndash198 [CrossRef]

17 He Y Liang X Qin L Li Z Tan L Shao M Community structure species diversity of Pinus massonianaand Castanopsis hystrix plantation and the nature-based forest management in the southern subtropical ChinaSci Silvae Sin 2013 49 24ndash33

18 Cline LC Zak DR Soil microbial communities are shaped by plant-driven changes in resource availabilityduring secondary succession Ecology 2015 96 3374ndash3385 [CrossRef]

19 Malchair S Carnol M Microbial biomass and C and N transformations in forest floors under Europeanbeech Sessile oak Norway spruce and Douglas-fir at four temperate forest sites Soil Biol Biochem 2009 41831ndash839 [CrossRef]

20 Ming A Yang Y Liu S Wang H Li Y Li H Nong Y Cai D Jia H Tao Y et al Effects of nearnatural forest management on soil greenhouse gas flux in Pinus massoniana (Lamb) and Cunninghamialanceolata (Lamb) Hook plantations Forests 2018 9 229 [CrossRef]

21 Wen L Lei P Xiang W Yan W Liu S Soil microbial biomass carbon and nitrogen in pure and mixedstands of Pinus massoniana and Cinnamomum camphora differing in stand age For Ecol Manag 2014 328150ndash158 [CrossRef]

22 Van Der Heijden MGA Bardgett RD Van Straalen NM The unseen majority Soil microbes as drivers ofplant diversity and productivity in terrestrial ecosystems Ecol Lett 2008 11 296ndash310 [CrossRef] [PubMed]

23 Singh JS Gupta VK Soil microbial biomass A key soil driver in management of ecosystem functioningSci Total Environ 2018 634 497ndash500 [CrossRef] [PubMed]

24 Chen C Chen HYH Chen X Huang Z Meta-analysis shows positive effects of plant diversity onmicrobial biomass and respiration Nat Commun 2019 10 1332 [CrossRef] [PubMed]

25 Boer W Folman LB Summerbell RC Boddy L Living in a fungal world Impact of fungi on soil bacterialniche development FEMS Microbiol Rev 2005 29 795ndash811 [CrossRef] [PubMed]

26 Haumlttenschwiler S Tiunov A Scheu S Biodiversity and litter decomposition in terrestrial ecosystemsAnnu Rev Ecol Evol 2005 36 191ndash218 [CrossRef]

27 Fornara DA Tilman D Hobbie SE Linkages between plant functional composition fine root processesand potential soil N mineralization rates J Ecol 2009 97 48ndash56 [CrossRef]

28 Kerdraon D Drewer J Castro B Wallwork A Hall J Sayer E Litter traits of native and non-nativetropical trees influence soil carbon dynamics in timber plantations in panama Forests 2019 10 209 [CrossRef]

29 Yang Y Liu S Chen L Wang H Lu L Short-term effects of manipulated throughfall reduction on thequantity and quality of litterfall in a Pinus massoniana plantation Acta Ecol Sin 2018 38 4470ndash4478

30 Zhou X Wen Y Goodale UM Zuo H Zhu H Li X You Y Yan L Su Y Huang X Optimal rotationlength for carbon sequestration in Eucalyptus plantations in subtropical China New For 2017 48 609ndash627[CrossRef]

31 Bao SD Soil Agro-Chemistrical Analysis China Agricultural Press Beijing China 2000 pp 1ndash49532 Evans EH Day JA Fisher A Price WJ Smith CMM Tyson JF Atomic spectrometry update

Advances in atomic emission absorption and fluorescence spectrometry and related techniques J AnalAtom Spectrom 2004 19 775 [CrossRef]

33 Reis B Zagatto E Jacintho A Merging zones in flow injection analysis Part 4 Simultaneousspectrophotometric determination of total nitrogen and phosphorus in plant material Anal Chim Acta 1980119 305ndash311 [CrossRef]

34 Liu W Wu J Fan H Li Y Yuan Y Liao Y Huang R Hu L Fang H Guo H Carbon pools in an agesequence of Eucalyptus plantation forests Ecol Environ Sci 2013 22 12ndash17

35 Liu L Gundersen P Zhang T Mo J Effects of phosphorus addition on soil microbial biomass andcommunity composition in three forest types in tropical China Soil Biol Biochem 2012 44 31ndash38 [CrossRef]

36 You Y Wang J Huang X Tang Z Liu S Sun OJ Relating microbial community structure to functioningin forest soil organic carbon transformation and turnover Ecol Evol 2014 4 633ndash647 [CrossRef]

37 Dohrmann R Kaufhold S Three new quick CEC methods for determining the amounts of exchangeablecalcium cations in calcareous clays Clay Clay Miner 2009 57 338ndash352 [CrossRef]

Forests 2019 10 879 19 of 20

38 Pan G Chen A Comparison of methods for determination of cation exchange properties of different soilsform southern China Trop Subtrop Soil Sci 1992 1 38ndash44

39 Vance ED Brookes PC Jenkinson DS An extraction method for measuring soil microbial biomass C SoilBiol Biochem 1987 19 703ndash707 [CrossRef]

40 Brookes PC Landman A Pruden G Jenkinson DS Chloroform fumigation and the release of soilnitrogen A rapid direct extraction method to measure microbial biomass nitrogen in soil Soil Biol Biochem1985 17 837ndash842 [CrossRef]

41 Bossio DA Scow KM Gunapala N Graham KJ Determinants of soil microbial communities Effects ofagricultural management season and soil type on phospholipid fatty acid profiles Microb Ecol 1998 361ndash12 [CrossRef]

42 Wang X Wang X Zhang W Shao Y Zou X Liu T Zhou L Wan S Rao X Li Z et al Invariantcommunity structure of soil bacteria in subtropical coniferous and broadleaved forests Sci Rep 2016 619071 [CrossRef]

43 Bossio DA Fleck JA Scow KM Fujii R Alteration of soil microbial communities and water quality inrestored wetlands Soil Biol Biochem 2006 38 1223ndash1233 [CrossRef]

44 Kourtev PS Ehrenfeld JG Haggblom M Exotic plant species alter the microbial community structureand function in the soil Ecology 2002 83 3152 [CrossRef]

45 Lv T Carvalho PN Zhang L Zhang Y Button M Arias CA Weber KP Brix H Functionality ofmicrobial communities in constructed wetlands used for pesticide remediation Influence of system designand sampling strategy Water Res 2017 110 241ndash251 [CrossRef] [PubMed]

46 Dumontet S Cavoski I Ricciuti P Mondelli D Jarrar M Pasquale V Crecchio C Metabolic andgenetic patterns of soil microbial communities in response to different amendments under organic farmingsystem Geoderma 2017 296 79ndash85 [CrossRef]

47 Liu B Li Y Zhang X Wang J Gao M Effects of chlortetracycline on soil microbial communitiesComparisons of enzyme activities to the functional diversity via Biolog EcoPlatestrade Eur J Soil Biol 2015 6869ndash76 [CrossRef]

48 Saiya-Cork KR Sinsabaugh RL Zak DR The effects of long term nitrogen deposition on extracellularenzyme activity in an Acer saccharum forest soil Soil Biol Biochem 2002 34 1309ndash1315 [CrossRef]

49 Steinauer K Tilman D Wragg PD Cesarz S Cowles JM Pritsch K Reich PB Weisser WWEisenhauer N Plant diversity effects on soil microbial functions and enzymes are stronger than warming ina grassland experiment Ecology 2015 96 99ndash112 [CrossRef] [PubMed]

50 Wardle DA Bonner KI Nicholson KS Biodiversity and plant litter Experimental evidence which doesnot support the view that enhanced species richness improves ecosystem function Oikos 1997 79 247ndash258[CrossRef]

51 Fanin N Kardol P Farrell M Nilsson M Gundale MJ Wardle DA The ratio of Gram-positive toGram-negative bacterial PLFA markers as an indicator of carbon availability in organic soils Soil Biol Biochem2019 128 111ndash114 [CrossRef]

52 Chen L Zeng X Tam NF Lu W Luo Z Du X Wang J Comparing carbon sequestration and standstructure of monoculture and mixed man-grove plantations of Sonneratia caseolaris and S apetala in SouthernChina For Ecol Manag 2012 284 222ndash229 [CrossRef]

53 Rouhi-Moghaddam E Hosseini SM Ebrahimi E Tabari M Rahmani A Comparison of growthnutrition and soil properties of pure stands of Quercus castaneifolia and mixed with Zelkova carpinifolia in theHyrcanian forests of Iran For Ecol Manag 2008 255 1149ndash1160 [CrossRef]

54 Binkley D Campoe OC Gspaltl M Forrester DI Light absorption and use efficiency in forests Whypatterns differ for trees and stands For Ecol Manag 2013 288 5ndash13 [CrossRef]

55 Duan W Ren H Fu S Wang J Zhang J Yang L Huang C Community comparison and determinantanalysis of understory vegetation in six plan-tations in South China Restor Ecol 2010 18 206ndash214[CrossRef]

56 Zhang K Dang H Tan S Wang Z Zhang Q Vegetation community and soil characteristics of abandonedagricultural land and pine plantation in the Qinling Mountains China For Ecol Manag 2010 259 2036ndash2047[CrossRef]

Forests 2019 10 879 20 of 20

57 Hobbie SE Ogdahl M Chorover J Chadwick OA Oleksyn J Zytkowiak R Reich PB Tree SpeciesEffects on Soil Organic Matter Dynamics The Role of Soil Cation Composition Ecosystems 2007 10 999ndash1018[CrossRef]

58 Bosatta E Aringgren GI Theoretical analysis of microbial biomass dynamics in soils Soil Biol Biochem 199426 143ndash148 [CrossRef]

59 Wolters V Joergensen RG Microbial carbon turnover in beech forest soils at different stages of acidificationSoil Biol Biochem 1991 23 897ndash902 [CrossRef]

60 Anderson TH Domsch KH Ratios of microbial biomass carbon to total organic carbon in arable soilsSoil Biol Biochem 1989 21 471ndash479 [CrossRef]

61 Insam H Domsch KH Relationship between soil organic carbon and microbial biomass on chronosequencesof reclamation sites Microb Ecol 1988 15 177ndash188 [CrossRef]

62 Anderson T Domsch KH Carbon assimilation and microbial activity in soil Z Pflanz Bodenkd 1986 149457ndash468 [CrossRef]

63 Cook BD Allan DL Dissolved organic carbon in old field soils Total amounts as a measure of availablere-sources for soil mineralization Soil Biol Biochem 1992 24 585ndash594 [CrossRef]

64 Lucas-Borja ME Candel D Jindo K Moreno JL Andreacutes M Bastida F Soil microbial communitystructure and activity in monospecific and mixed forest stands under Mediterranean humid conditionsPlant Soil 2012 354 359ndash370 [CrossRef]

65 Bauhus J Pareacute D Co Teacute L Effects of tree species stand age and soil type on soil microbial biomass and itsactivity in a southern boreal forest Soil Biol Biochem 1998 30 1077ndash1089 [CrossRef]

66 Feng WT Zou XM Schaefer D Above- and belowground carbon inputs affect seasonal variations of soilmicrobial bio-mass in a subtropical monsoon forest of southwest China Soil Biol Biochem 2009 41 978ndash983[CrossRef]

67 Yu X Liu X Zhao Z Liu J Zhang S Effect of monospecific and mixed sea-buckthorn (Hippophaerhamnoides) plantations on the structure and activity of soil microbial communities PLoS ONE 2015 10e0117505 [CrossRef]

68 Balser TC Firestone MK Linking Microbial Community Composition and Soil Processes in a CaliforniaAnnual Grassland and Mixed-Conifer Forest Biogeochemistry 2005 73 395ndash415 [CrossRef]

69 Jian S Li J Chen J Wang G Mayes MA Dzantor KE Hui D Luo Y Soil extracellular enzymeactivities soil carbon and nitrogen storage under nitrogen fertilization A meta-analysis Soil Biol Biochem2016 101 32ndash43 [CrossRef]

70 Wittmann C Kaumlhkoumlnen MA Ilvesniemi H Kurola J Salkinoja-Salonen MS Areal activities andstratification of hydrolytic enzymes involved in the biochemical cycles of carbon nitrogen sulphur andphosphorus in podsolized boreal forest soils Soil Biol Biochem 2004 36 425ndash433 [CrossRef]

71 Garland JL Analysis and interpretation of community-level physiological profiles in microbial ecologyFEMS Microbiol Ecol 1997 24 289ndash300 [CrossRef]

72 Lehman RM Colwell FS Ringelberg DB White DC Combined microbial community-level analysesfor quality assurance of terrestrial subsurface cores J Microbiol Meth 1995 22 263ndash281 [CrossRef]

73 Zak JC Willig MR Moorhead DL Wildman HG Functional diversity of microbial communities Aquantitative approach Soil Biol Biochem 1994 26 1101ndash1108 [CrossRef]

copy 2019 by the authors Licensee MDPI Basel Switzerland This article is an open accessarticle distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (httpcreativecommonsorglicensesby40)