Does a fungal species drive ectomycorrhizal root traits in Alnus spp
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Does a fungal species drive ectomycorrhizal root traits in Alnus spp.? Ivika Ostonen, Leho Tedersoo, Triin Suvi, and Krista Lo ˜ hmus Abstract: Ectomycorrhizal (EcM) fungi contribute significantly to the shaping of short-root morphology, playing an im- portant role in balancing the costs and benefits of root growth and nutrient uptake and exchange in boreal forests. We aimed to assess the effect of various EcM fungal taxa on root traits at seven sites dominated by grey alder, Alnus incana (L.) Moench, and black alder, Alnus glutinosa (L.) Gaertn. Mean root size, specific root length, specific root area, root tis- sue density, and root-tip frequency of EcM short roots were measured in EcM anatomotypes in relation to the effects of host species, soil moisture level, and nutrient status. Redundancy analysis revealed that anatomotype, alder species, site, and soil parameters (N, P, K, Ca, and Mg concentrations, pH, organic-matter content) accounted for 42.3% (p < 0.001) of the total variation in EcM root morphology. Variation decreased in the following order: anatomotypes (27.9%) > soil pa- rameters and sites (19.9%) > alder species (5.1%). EcM fungus species had the primary influence on EcM short-root size. EcM roots of the dominant anatomotype, Alnicola spp., had the highest specific root length and specific root area in both alder species. Short-root morphology depends most strongly on the fungal taxa involved, which indicates that the type of mycobiont has an important influence on the functional properties of fine roots. Re ´sume ´: Les champignons ectomycorhiziens (EcM) contribuent de manie `re significative a ` de ´terminer la morphologie des racines courtes, jouant ainsi un ro ˆle important dans le maintien de l’e ´quilibre entre les cou ˆts et les be ´ne ´fices associe ´s a ` la croissance racinaire et a ` l’absorption ainsi qu’aux e ´changes dans les fore ˆts bore ´ales. Nous visions a `e ´valuer l’effet des tax- ons des champignons EcM sur les caracte ´ristiques des racines dans sept stations domine ´es par Alnus incana (L.) Moench et Alnus glutinosa (L.) Gaertn. La dimension moyenne des racines courtes mycorhize ´es, la longueur spe ´cifique des racines (LSR) et la surface spe ´cifique des racines (SSR) ont e ´te ´ mesure ´es chez les types anatomiques d’EcM en relation avec les effets de l’espe `ce ho ˆte, de l’humidite ´ du sol et du statut des nutriments. L’analyse de redondance a re ´ve ´le ´ que le type anat- omique, l’espe `ce d’aulne, la station et les parame `tres du sol (N, P, K, Ca, Mg, pH et matie `re organique) expliquent 42,3 % (p < 0,001) de la variation totale de la morphologie des racines mycorhize ´es. La variation de ´crite diminuait dans l’ordre suivant : types anatomiques (27,9 %) > parame `tres du sol et stations (19,9 %) > espe `ces d’aulne (5,1 %). Le champignon EcM influenc ¸ait surtout la dimension des racines courtes mycorhize ´es. Les racines mycorhize ´es par le type anatomique dominant, Alnicola spp., avaient les valeurs de LSR et de SSR les plus e ´leve ´es chez les deux espe `ces d’aulne. La morpho- logie des racines courtes de ´pend avant tout des taxons fongiques implique ´s, ce qui indique que le type de mycobiontes a une grande influence sur les proprie ´te ´s fonctionnelles des racines fines. [Traduit par la Re ´daction] Introduction Ectomycorrhizal (EcM) associations play a key role in mineral nutrition of Pinaceae, Fagaceae, Betulaceae, and Salicaceae in boreal and temperate forest ecosystems (Brun- drett 2002). EcM short roots with primary structure are re- sponsible for water and nutrient uptake and it is well known that the formation of EcM roots involves alterations in the morphology of the plant root and fungal mycelium. Morpho- logical and anatomical features of ectomycorrhizas are more or less characteristic of the fungal species forming the ana- tomotype (Agerer 1987–1998). Considerable variation in a range of physiological functions, such as enzyme activities (Courty et al. 2005), nutrient uptake (Abuzinadah and Read 1989; Perez-Moreno and Read 2000; Landeweert et al. 2001; Chalot et al. 2002), and heavy-metal tolerance (Jentschke and Godbold 2000), has been shown among EcM species. There could be great differences in EcM effec- tiveness in facilitating tree growth (Burgess et al. 1993). EcM diversity and community structure are related to the growth rate of the host (Korkama et al. 2006). EcM fungi can explore the surrounding soil by means of extramatrical mycelia, which are either concentrated close to the mycor- rhizal mantle or form far-reaching rhizomorphs of different exploration types, and each EcM fungus (or exploration type) may utilize a distinct foraging strategy (Agerer 2001). Owing to improvements in species-identification method- ology (Horton and Bruns 2001), the number of studies fo- cusing on EcM fungi and EcM community structure in different sites and hosts has increased. Much less is known about the effects of different anatomotypes (and (or) EcM fungus species) on EcM root morphology and about EcM Received 16 January 2009. Accepted 9 June 2009. Published on the NRC Research Press Web site at cjfr.nrc.ca on 26 September 2009. I. Ostonen, 1 T. Suvi, and K. Lo ˜hmus. Institute of Ecology and Earth Sciences, University of Tartu, 46 Vanemuise Street, 51014 Tartu, Estonia. L. Tedersoo. Institute of Ecology and Earth Sciences, University of Tartu, 46 Vanemuise Street, 51014 Tartu, Estonia; Natural History Museum, Tartu University, 46 Vanemuise Street, 51014 Tartu, Estonia. 1 Corresponding author (e-mail: [email protected]). 1787 Can. J. For. Res. 39: 1787–1796 (2009) doi:10.1139/X09-093 Published by NRC Research Press
Transcript of Does a fungal species drive ectomycorrhizal root traits in Alnus spp
Does a fungal species drive ectomycorrhizal roottraits in Alnus spp.?
Ivika Ostonen, Leho Tedersoo, Triin Suvi, and Krista Lohmus
Abstract: Ectomycorrhizal (EcM) fungi contribute significantly to the shaping of short-root morphology, playing an im-portant role in balancing the costs and benefits of root growth and nutrient uptake and exchange in boreal forests. Weaimed to assess the effect of various EcM fungal taxa on root traits at seven sites dominated by grey alder, Alnus incana(L.) Moench, and black alder, Alnus glutinosa (L.) Gaertn. Mean root size, specific root length, specific root area, root tis-sue density, and root-tip frequency of EcM short roots were measured in EcM anatomotypes in relation to the effects ofhost species, soil moisture level, and nutrient status. Redundancy analysis revealed that anatomotype, alder species, site,and soil parameters (N, P, K, Ca, and Mg concentrations, pH, organic-matter content) accounted for 42.3% (p < 0.001) ofthe total variation in EcM root morphology. Variation decreased in the following order: anatomotypes (27.9%) > soil pa-rameters and sites (19.9%) > alder species (5.1%). EcM fungus species had the primary influence on EcM short-root size.EcM roots of the dominant anatomotype, Alnicola spp., had the highest specific root length and specific root area in bothalder species. Short-root morphology depends most strongly on the fungal taxa involved, which indicates that the type ofmycobiont has an important influence on the functional properties of fine roots.
Resume : Les champignons ectomycorhiziens (EcM) contribuent de maniere significative a determiner la morphologie desracines courtes, jouant ainsi un role important dans le maintien de l’equilibre entre les couts et les benefices associes a lacroissance racinaire et a l’absorption ainsi qu’aux echanges dans les forets boreales. Nous visions a evaluer l’effet des tax-ons des champignons EcM sur les caracteristiques des racines dans sept stations dominees par Alnus incana (L.) Moenchet Alnus glutinosa (L.) Gaertn. La dimension moyenne des racines courtes mycorhizees, la longueur specifique des racines(LSR) et la surface specifique des racines (SSR) ont ete mesurees chez les types anatomiques d’EcM en relation avec leseffets de l’espece hote, de l’humidite du sol et du statut des nutriments. L’analyse de redondance a revele que le type anat-omique, l’espece d’aulne, la station et les parametres du sol (N, P, K, Ca, Mg, pH et matiere organique) expliquent 42,3 %(p < 0,001) de la variation totale de la morphologie des racines mycorhizees. La variation decrite diminuait dans l’ordresuivant : types anatomiques (27,9 %) > parametres du sol et stations (19,9 %) > especes d’aulne (5,1 %). Le champignonEcM influencait surtout la dimension des racines courtes mycorhizees. Les racines mycorhizees par le type anatomiquedominant, Alnicola spp., avaient les valeurs de LSR et de SSR les plus elevees chez les deux especes d’aulne. La morpho-logie des racines courtes depend avant tout des taxons fongiques impliques, ce qui indique que le type de mycobiontes aune grande influence sur les proprietes fonctionnelles des racines fines.
[Traduit par la Redaction]
IntroductionEctomycorrhizal (EcM) associations play a key role in
mineral nutrition of Pinaceae, Fagaceae, Betulaceae, andSalicaceae in boreal and temperate forest ecosystems (Brun-drett 2002). EcM short roots with primary structure are re-sponsible for water and nutrient uptake and it is well knownthat the formation of EcM roots involves alterations in themorphology of the plant root and fungal mycelium. Morpho-logical and anatomical features of ectomycorrhizas are more
or less characteristic of the fungal species forming the ana-tomotype (Agerer 1987–1998). Considerable variation in arange of physiological functions, such as enzyme activities(Courty et al. 2005), nutrient uptake (Abuzinadah and Read1989; Perez-Moreno and Read 2000; Landeweert et al.2001; Chalot et al. 2002), and heavy-metal tolerance(Jentschke and Godbold 2000), has been shown amongEcM species. There could be great differences in EcM effec-tiveness in facilitating tree growth (Burgess et al. 1993).EcM diversity and community structure are related to thegrowth rate of the host (Korkama et al. 2006). EcM fungican explore the surrounding soil by means of extramatricalmycelia, which are either concentrated close to the mycor-rhizal mantle or form far-reaching rhizomorphs of differentexploration types, and each EcM fungus (or explorationtype) may utilize a distinct foraging strategy (Agerer 2001).
Owing to improvements in species-identification method-ology (Horton and Bruns 2001), the number of studies fo-cusing on EcM fungi and EcM community structure indifferent sites and hosts has increased. Much less is knownabout the effects of different anatomotypes (and (or) EcMfungus species) on EcM root morphology and about EcM
Received 16 January 2009. Accepted 9 June 2009. Published onthe NRC Research Press Web site at cjfr.nrc.ca on 26 September2009.
I. Ostonen,1 T. Suvi, and K. Lohmus. Institute of Ecology andEarth Sciences, University of Tartu, 46 Vanemuise Street, 51014Tartu, Estonia.L. Tedersoo. Institute of Ecology and Earth Sciences,University of Tartu, 46 Vanemuise Street, 51014 Tartu, Estonia;Natural History Museum, Tartu University, 46 VanemuiseStreet, 51014 Tartu, Estonia.
1Corresponding author (e-mail: [email protected]).
1787
Can. J. For. Res. 39: 1787–1796 (2009) doi:10.1139/X09-093 Published by NRC Research Press
community structure in relation to ecosystem function, par-ticularly mineral nutrition (Godbold 2005).
However, community structure of EcM fungi is an impor-tant biotic factor, with a significant effect on EcM root mor-phology, together with abiotic factors (e.g., physical andchemical characteristics of soil) at the site, and genetic vari-ability within host trees. Different functional parameters ofshort EcM roots, e.g., specific root area (SRA), specific rootlength (SRL), and root tissue density (RTD), have been usedas cost–benefit indices at the level of both the individualroot and the entire root system, assuming that resource ac-quisition is proportional to root length or surface area, andthat root cost (construction and maintenance) is proportionalto mass (Fitter 1991; Pregitzer et al. 2002; Withington et al.2006; Ostonen et al. 2007a, 2007b). However, EcM short-root parameters can be separated into independent varia-bles — mean length (L), diameter (D), and RTD — and de-pendent or derived variables calculated from the formervariables SRA and SRL, and root-tip frequency per unit drymass (RTFW) and root-tip frequency per unit length (RTFL)(Ostonen et al. 2006). The larger the SRA and the smallerthe RTD, the more effective is the strategy of allocating as-similates to the build-up of EcM roots (Ostonen et al. 1999;Wahl and Ryser 2000). At the level of the entire root sys-tem, optimality theory predicts that a tree optimizes rootfunction by maximizing its nutrient and water uptake withminimum resource investment in the root system (Leuschneret al. 2004).
The impact of fungal symbionts on EcM root characteris-tics is reported to be significant in Norway spruce; differen-ces in the volume proportions of mantle and cortex in theroot, as well as in SRA and RTD, have been revealed (Os-tonen et al. 1999; Ostonen and Lohmus 2003). In the rhizo-sphere, Lohmus et al. (2006) showed a positive correlationbetween SRA of EcM roots and the functional diversity andactivity of microbial communities at the soil–root interfaceand in the bulk soil. At the level of the forest ecosystem, apositive correlation between SRA of EcM roots and the as-similation efficiency of leaves (kg�kg–1�year–1) in alders hasbeen reported (Lohmus et al. 2006).
Alder species are indigenous to a large part of Europe.They occur in a wide variety of habitats ranging from dryto wet, and in soils ranging from from calcareous to acidic.However, while black alder (Alnus glutinosa (L.) Gaertn.) ismore commonly the dominant tree species in forests in wet-lands, both black alder and grey alder (Alnus incana (L.)Moench.) forests are typical of the riparian zone, and greyalder also tolerates relatively dry soils.
Grey alder and black alder were selected as model taxa inour study because EcM colonization of alders respondsstrongly to changes in soil conditions (Baar et al. 2000;Dilly et al. 2000), and alders host a limited set of EcMfungi, which allows comparisons to be made among and be-tween taxa at several geographical scales (Pritsch et al.1997a, 1997b; Becerra et al. 2005).
Our background hypothesis was that the different anato-motypes of EcM roots resulting from colonization of thesame host by different fungi have significant differences intheir morphological parameters. However, the pattern of theEcM community depends on both forest site type and soilnutrient availability (Toljander et al. 2006; Lilleskov et al.
2008), and differences in the proportions of the dominantanatomotypes may eventually be exhibited in the mineralnutrition of trees at different sites. If this is the case, thenrelating EcM community structure and colonization to themorphological parameters of EcM roots in boreal forests, toaccount for mineral-nutrition efficiency and (or) strategy,would increase our understanding of the function of EcMcommunity structure in the mineral nutrition of boreal-forestecosystems.
We studied the impact of EcM community structure onEcM root morphology in grey and black alder stands grow-ing in Estonian forest sites where soil conditions and pre-vious land use varied. Our main aim was to elucidate howthe morphological parameters of EcM roots depend on EcMfungus species, site and soil conditions, and host tree spe-cies.
Material and methodsThe study was performed in three grey alder and four
black alder stands (Table 1) situated throughout Estonia.The sites were selected to cover different soil conditions, toensure a wide spectrum of EcM fungus species colonizingalder roots. Previous land use was taken into consideration.Two 29-year-old black alder plantations (Songa and Sirgala)originated from the same set of mother trees (Vares et al.2004). The Jarvselja site is situated in a primeval mixed for-est in southeast Estonia that has remained unmanagedfor >200 years. Wet plots were established in a forest areain a minerotrophic black alder swamp and dry plots in anadjoining Vaccinium myrtillus – spruce forest where blackalder forms a subdominant tree layer (Tedersoo et al. 2008).The Inka black alder and Porijoe grey alder study areas(Mander et al. 2008) are in the flood plain of small riversthat host a natural, unmanaged alder stand. The undisturbedgrey alder forest at Saka lies on a talus slope of the NorthEstonian (Ordovician) Klint, where the soils on Cambrianclays are influenced by carbonate-rich ground water fromlimestone terraces (Rooma and Paal 2001). The Leidisoogrey alder forest is situated on the bank of a young peatbog in western Estonia, where the soils are influenced bycontrasting hydrological conditions and calcareous bedrock;pHKCl variation within the site is almost 3 units (Table 1).
At all sites, three plots were established in a relatively dryarea and three in a wet area with substantial seasonal water-logging. Soil moisture status was estimated qualitatively.
Root sampling and processingEcM roots were sampled in alder stands in September
2005 at Porijogi, in September 2006 at Songa, and in Sep-tember 2007 at the other five sites. In each plot, three rootsamples (15 cm � 15 cm to 10 cm depth) were collectedusing a flat spade. The root fraction was cleaned of adheringsoil and clay by careful rinsing in tap water. Roots wereplaced in large Petri dishes filled with water and identifiedto tree species according to the presence and shape of EcMand nodules. The EcM roots of alders were separated intoanatomotypes (Agerer 1991) using a light microscopeequipped with Normarski differential interference contrastat 1000� magnification, scored for relative abundance, andsubsequently identified using sequence analysis of nuclear
1788 Can. J. For. Res. Vol. 39, 2009
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rDNA internal transcribed spacer (ITS) and large subunit re-gions. Sequencing was performed with the primer ITS5 (5’-ggaagtaaaagtcgtaacaagg-3’). At polymorphic sites, reversesequencing with the primer ITS4 (5’-tcctccgcttattgatatgc-3’)or LF340 (5’-tacttgtkcgctatcgg-3’) was also run to improvesequence quality. Large subunit regions were amplified inmost species using the primers ctb6 (5’-gcatatcaataagcg-gagg-3’) and (or) LR5 (5’-tcctgagggaaacttcg-3’). Sequenceswere assembled, trimmed, checked, and edited in SE-QUENCHER 4.5 software (GeneCodes Corp., Ann Arbor,Michigan) and 97% ITS region identity was used as the bar-coding threshold. A more detailed description of processingmolecular typing and DNA barcoding of Alnus-associatedectomycorrhizal fungi on root tips at the seven studied sitesis presented in Tedersoo et al. (2009).
A random EcM root subsample was taken from each ana-tomotype sample. The sets of 5–113 subsamples per anato-motype totalled 50–1150 tips throughout all stands. Weacquired more subsamples from dominant anatomotypes.The mean values of the morphological parameters in eachstand also reflected the proportions of the different anatomo-types. EcM roots were mostly only first order; however,some anatomotypes included ramified EcM roots of the firstand second orders. The root tips were washed with tap waterto remove soil particles and then cleaned with a small softbrush to remove all remaining soil particles. Root tips werecounted under a microscope. EcM root diameter, length, andprojection area were measured with WinRHIZOTM Pro2003b (Regent Instruments Inc., Quebec, Quebec). The air-dry roots were dried at 70 8C for 2 h to constant mass andthen weighed.
Morphological parameters such as mean D (mm), L (mm),and (W (mg), root-tip volume (V, mm3), SRA (m2�kg–1), SRL(g�m–1), RTD (kg�m–3), RTFW (number�(mg dry mass)–1)and RTFL (number�cm–1) were measured or calculated(Ostonen et al. 2007a) separately for each anatomotype.
Soil samplingApproximately 50 g of rhizosphere soil was carefully sep-
arated from roots, pooled by plot, and placed in plastic bagsaccording to the rooting depth of each sample in each plot.Soil samples were dried at 70 8C and mineral nutrients wereanalyzed. Total N was determined by the Kjeldahl methodwith a Tecator ASN 3313. P was extracted by 15 ammo-nium lactate and measured by flow-injection analysis. Avail-able K was determined from the same solution by the flamephotometric method (AOAC956.01). Exchangeable Ca andMg in the soil were measured from ammonium acetate ex-tract (pH 7.0). Organic-matter content was determined onthe basis of LOI for 2 h at 360 8C. Soil pH was measuredin 1 mol/L 20 potassium chloride solution.
Statistical methodsThe normality of short-root variables for different tree
species in a site and for five dominant anatomotypes waschecked by means of Lilliefors and Shapiro–Wilk tests. Thevariables were normally distributed except for RTFW andRTFL, which were log-transformed. Rare anatomotypes,representing one to four samples per stand, were used onlyfor analyzing differences between stands and tree speciesand were excluded from the analysis between anatomotypes.T
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Ostonen et al. 1789
Published by NRC Research Press
Multiple comparison of means was applied using Tukey’stest for unequal sample sizes and 95% confidence intervals.The software STATISTICA 7.0 was used; the significancelevel a = 0.05 was accepted in all cases.
The effects of site, tree species, anatomotype, and soil nu-trient variables on morphological parameters of EcM rootswere studied using three-way mixed ANOVA for each rootparameter separately, with plot as a random factor nestedwithin site, and site nested within host species. Soil pH andnutrient concentrations were used as covariates. The fivemost frequent anatomotypes — Alnicola spp., Tomentellaaff. sublilacina, Tomentella aff. ellisii, Lactarius cyathulifor-mis, Genea–Humaria lineage sp. — were included in theanalysis. Morphological parameters of EcM roots and soilnutrient concentrations (mg�kg–1) were log-transformed andsoil nutrient concentrations (%) were arcsine-square-root-transformed prior to the analyses to meet the assumptionsof parametric tests.
Principal component analysis and redundancy analysis(RDA) (CANOCO program; ter Braak and Smilauer 2002)were used to detect and visualize relationships between rootcharacteristics and anatomotypes, and between site and soilconditions and tree species. The significance of RDA resultswas tested with a permutation test (p < 0.01).
Results
Sources of variation in EcM root morphologyThe results of principal component analysis for all meas-
ured and calculated short-root characteristics showed thattwo axes accounted for 81% of the total variation in short-root traits. According to the RDA, all factors — anatomo-type, alder species, site, and soil parameters (N, P, K, Ca,Mg concentrations, pH, organic-matter content) — ac-counted for 42.3% (p < 0.001) of the total variation inshort-root morphology (Fig. 1). The described variation inshort-root morphology decreased in the following order:anatomotypes (27.9%) > soil parameters and sites(19.9%) > alder species (5.1%). Even when rare anatomo-types (<5% at any site) were removed, the dominant typesstill explained 26.9% of total variation in short-root mor-phology (Fig. 1).
AnatomotypesThe number of different anatomotypes and molecular spe-
cies of EcM fungi differed between alder stands, amountingto 40 species in total. The smallest number of different EcMfungal species was found in Sirgala and the highest numberin Porijoe, 11 and 18 molecular species, respectively. A de-tailed description of the community composition of Alnus-associated EcM fungi on root tips at the seven studied sitesis presented in Tedersoo et al. (2009). In all studied alderforests, the anatomotypes Alnicola spp. and T. aff. sublila-cina dominated, ranging from 65% to 85% of all detectedanatomotypes (Table 2). The dominant anatomotypes pos-sessed similar short-distance exploration types with regardto their extrametrical mycelia (Agerer 2001). Alnicola spp.comprised several closely related molecular species thatcould not be distinguished by anatomotyping. However,there was no clear trend in EcM root morphology amongspecies within the anatomotype group Alnicola spp. In the
RDA ordination, Tomentella species tended to diverge ac-cording to root morphological parameters (Fig. 1). Anato-motype was the main factor determining the diameter,length, and mass of EcM short roots in alder species, as re-vealed by three-way ANOVA (Table 3). The mean root di-ameter in L. cyathuliformis was significantly larger thanin Alnicola spp. and T. aff. sublilacina (Table 4). Genea–Humaria lineage sp. and Paxillus filamentosus differedsignificantly from other anatomotypes in being shorter ingrey and black alder stands, respectively (Fig. 1, Table 4).
According to the results of three-way mixed ANOVA, allstudied main factors — anatomotype, host tree species, site,and their interactions — had a significant effect on SRA andSRL (Table 3). Of the main effects, only host tree speciesand site significantly influenced RTD of EcM short roots.However, host species � anatomotype and site � anatomo-type interactions also had a significant impact on RTD ofEcM short roots (Table 3). SRA and SRL were the highestand RTD was the lowest for EcM short roots infected by Al-nicola spp. (Fig. 1, Table 4) for both alder species. In T. aff.sublilacina, mean RTD was up to 1.3–1.5� higher for bothalder species than for other dominant anatomotypes(Table 4). Mean RTFW was significantly higher in Alnicolaspp. than in T. aff. sublilacina for both alder species(Table 4).
Tree speciesShort roots of black alder were longer and heavier than
those of grey alder (t test, p < 0.05). EcM short roots colon-ized by Alnicola spp. and T. aff. sublilacina were signifi-cantly longer in black alder stands (Table 4). However,according to the results of three-way mixed ANOVA, the ef-fect of alder species on the length of EcM short roots wasinsignificant (Table 3). The functional characteristics SRAand SRL were significantly higher in grey alder EcM shortroots than in those of black alder (Table 5). Alder specieshad a significant effect on SRA, SRL, and RTD of the dom-inant anatomotype: SRA and SRL were higher in Alnicolaspp. and RTD was lower in A. incana (Table 4).
Site and soil conditionsEcM root parameters did not differ significantly between
waterlogged and dry plots, either for all anatomotypes com-bined or for different anatomotypes separately.
According to RDA, site and soil characteristics togetheraccounted for 19.9% of total variability in morphologicalparameters of EcM roots. Soil characteristics alone ac-counted for 15.7%, most of which (12.7%) was due to N(%), P (mg�kg–1), K (mg�kg–1), and organic-matter content.
Site significantly affected the functional characteristicsSRA, SRL, and RTD (Table 3). RTD was affected moststrongly by site, and soil Mg concentration (Table 3) corre-lated positively with RTD (r = 0. 36, p < 0.0001). However,for two dominant anatomotypes the correlation betweenRTD and soil Mg concentration was significantly positiveonly for Alnicola spp. (r = 0.43, p < 0.0001); no statisticallysignificant correlation for RTD of T. sublilacina-colonizedEcM roots was found. According to three-way mixed AN-OVA, D and L of EcM short-roots were significantly af-fected by percent soil N; in the case of L, soil Pconcentration and LOI were even more important (Table 3).
1790 Can. J. For. Res. Vol. 39, 2009
Published by NRC Research Press
Values for the same host species also differed signifi-cantly among sites (Table 5). In grey alder stands, mean Dwas lower at Saka than at Leidisoo and Porijoe. The oldernatural black alder stands at Inka and Jarvselja had signifi-cantly longer, thinner roots than the younger planted standsat Sirgala and Songa (Table 5). However, grey and black al-der stands were grouped differently by the functional param-eters SRA and RTD than by D and L (Table 5).
Discussion
The mycorrhizal structures of EcM roots of Alnus spp.have been extensively analyzed from the mycological per-spective (Brunner and Horak 1990; Brunner et al. 1992;Pritsch et al. 1997a, 1997b; Becerra et al. 2005; Tedersoo
et al. 2009). Data on the effect of EcM fungus species andcommunity structure on short-root morphology and eventu-ally on the mineral nutrition of trees are still scarce becauseidentification techniques have only recently become avail-able and because of difficulties in culturing most EcM fungi.In this study, we show that EcM anatomotype is the mostimportant factor determining short-root traits in the two Al-nus species, which confirms the postulated differences in theecological functions of EcM fungal taxa below ground. Inparticular, the present results demonstrate that the particularEcM fungus strongly affects the length, mass, and diameterof root tips. However, D values for EcM short roots variedless than L values in all cases, because variation in D valueson elongation of root cells; differences in L values involve
Fig. 1. Ordination diagram of redundancy analysis indicating the relative importance of ectomycorrhizal (EcM) short-root traits (arrows) inrelation to EcM anatomotypes (~), sites (*, Alnus glutinosa; *, Alnus incana), and tree species (&, A. glutinosa; &, A. incana). Abbre-viations: D, EcM short-root diameter (mm); W, EcM short-root-tip dry mass (mg); V, EcM short-root-tip volume (mm3); L, EcM short-root-tip length (mm); RTD, root tissue density (kg�m–3); SRA, specific root area (m2�kg–1); SRL, specific root length (m�g–1); RTFW, EcMshort-root-tip frequency per unit dry mass (number�mg–1); RTFL, EcM short-root-tip frequency per unit length (number�cm–1).
Table 2. Abundance (%) of the most common EcM anatomotypes used for determining root traits in Alnus incana andA. glutinosa stands in seven sites.
Alnus incana Alnus glutinosa
Leidisoo Saka Porijoe Inka Songa Jarvselja SirgalaAlnicola spp. 48 44 55 55 41 46 34Tomentella aff. sublilacina 29 22 25 21 24 39 32Tomentella aff. ellisii 5 16 <1 3 25 0 2Lactarius cyathuliformis s.l. 5 2 4 8 6 <1 0Genea–Humaria lineage sp. 2 5 7 <1 0 0 0Tomentella sp. 1 7 0 0 2 <1 5 0Tomentella aff. stuposa 1 <1 <1 4 0 2 11Paxillus filamentosus 0 3 <1 0 0 0 13Geopyxis cf. carbonaria 0 4 0 <1 <1 0 7
Ostonen et al. 1791
Published by NRC Research Press
both elongation of existing cells and formation of new cellswithin the apical meristem of a short root.
Two anatomotypes, Alnicola spp. and T. aff. sublilacina,dominated in both grey and black alder forests in Estonia.A further three anatomotypes in grey alder and four in blackalder were more abundant than all other detected anatomo-types (Tedersoo et al. 2009).
We showed in our study that the functional parametersSRA and SRL incorporated the effects of all studied factors— EcM fungal partner, impact of tree species, and site andsoil conditions: SRA and SRL differed significantly betweenanatomotypes, sites, and tree species. Of short-root morpho-logical characteristics, L, D, and RTD independently charac-terize the size and inner structure of a root. At the sametime, SRA and SRL, which are inversely dependent on Dand RTD (Ostonen et al. 1999), are more intimately relatedto root function. However, SRA and SRL of short roots ofT. aff. sublilacina and L. cyathuliformis s.l. were similar, de-spite significant differences between D and RTD for theseanatomotypes. The mean D value and mean RTD may re-spond differently to different environmental conditions, andhave opposite effects on SRL and SRA (Ryser 1998, 2006;Ostonen et al. 2007b). We found D to be strongly dependenton anatomotype and RTD on site. The strong dependency ofRTD on site conditions suggests that it is a predictive func-tional indicator of mineral nutrition.
The inflow of N has been shown to be more efficient inP. filamentosus (Van der Heijden and Kuyper 2003). In ourstudy, RTD was lowest for EcM roots colonized by P. fila-mentosus., suggesting more efficient mineral nutrient ex-change than in roots colonized by dominant anatomotypes.A low RTD of roots characterized fast-growing grass species(Wahl and Ryser 2000), and we expect that a short root witha lower RTD can be considered more efficient becausewater and nutrient inflow would be facilitated. Based onSRA, SRL, and RTD, nutrient and water uptake should bemost efficient in Porijoe, as RTD of EcM roots is low andSRA is high. A positive correlation between RTD and theproportion of cell walls in the stele has been reported forgrasses (Wahl and Ryser 2000). The relationship betweenRTD variation in EcM short roots and the particular EcMfungal taxon needs further investigation.
The anatomotypes Alnicola spp. and T. aff. sublilacinawere long and relatively thin at all seven sites, whereas Pe-zizales spp. formed short, stout root tips. Van der Heijdenand Kuyper (2003) distinguished two ecological strategiesfor EcM short roots in Salix repens: a root-manipulationstrategy based on elongation of roots, and root replacement.EcM fungi with a root-manipulation strategy strongly in-creased root length and their N economy was more effectivethan that of species with a root-replacement strategy. Our re-sults suggest that T. aff. sublilacina and, particularly, Alni-cola spp. follow the root-manipulation strategy, like thesister genus Hebeloma on Salix spp. (Van der Heijden andKuyper 2003). Further, Paxillus spp. seem to use a root-re-placement strategy. Root tips of Paxillus spp. were shorter,thicker, and more numerous per unit root length than thoseof Alnicola spp. EcM roots of Paxillus spp. had, on average,1.5 times the number of root tips per unit root length thanthose of Alnicola spp.
Because of the diversity of the mycelial structures of EcMTab
le3.
Fst
atis
tics
and
pva
lues
for
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cts
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mov
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esto
prov
ide
mor
est
atis
tical
pow
er(D
,ec
tom
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ort-
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diam
eter
;W
,ec
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ort-
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ass;
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ific
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itle
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gnif
ican
t).
1792 Can. J. For. Res. Vol. 39, 2009
Published by NRC Research Press
fungi, different exploration types could be distinguished,which are able to colonize the soil and organic substratesdifferently (Agerer 2001). In our study, two dominant anato-motypes — Alnicola spp. and T. aff. sublilacina — were ofsimilar short-distance exploration types; however, the genusPaxillus, with its differentiated rhizomorphs, is characterizedas a long-distance exploration type by Agerer (2001). BothP. filamentosus and T. aff. stuposa were exceptionally abun-dant in the middle-aged A. glutinosa stand at Sirgala, whichis in an early-successional stage and has been planted on re-claimed opencast oil shale mining spoil. RDA revealed sim-ilarities between the Sirgala site and the studied grey alderforests, based on both short-root morphology and EcM fun-gal community structure. One explanation of the similaritiesin short-root morphology of the black alder plantation andgrey alder stands at Sirgala could be that the habitat in Sir-gala is drier than in the other black alder forests. On theother hand, the differences in short-root morphology andEcM fungal community structure could be related to lowerN and P concentrations in developing soil. The external my-celium of Paxillus involutus contributed significantly to Nand P nutrition of N- and P-deficient Norway spruce seed-lings (Brandes et al. 1998). It has been proved that EcMfungi may differentially affect root length, relative N- andP-uptake efficiency (per unit root length) in Salix spp. Forexample, Hebeloma sp., with long EcM root tips, was 2–3times less efficient in N uptake than Paxillus sp., withshorter EcM root tips (Van der Heijden and Kuyper 2003).
Our results indicate that both EcM fungi and the concen-trations of macronutrients affected the size of root tips. Forinstance, the length of short root tips depended significantlyon N, P, and K concentrations and organic-matter content inthe soil, and diameter depended on N and Ca concentrationsin the soil. The functional root traits RTD and SRA weresignificantly affected by the Mg concentration in the soil.Fertilizers, including N, P, K, Mg, and Ca, have been shownto affect fine-root morphology and biomass of hyphae in aPinus pinaster stand (Bakker et al. 2009), and an increasein fine-root dry matter in Norway spruce seedlings in re-sponse to addition of Mg, N, P, and K was recorded (Zhangand George 2009). Further, the effects of macronutrients onshort-root size could be mediated by EcM fungi. Speciesdensity (i.e., species richness per sample) and EcM coloniza-tion in our alder stands were significantly affected by soil Kand Ca concentrations and organic-matter content (Tedersooet al. 2009). Hence, our results confirm the existence of in-terrelations between short-root traits and EcM fungi. Nu-trient fluxes within fungal hyphae are interdependent, i.e., astrong coupling of N, K, and Mg fluxes with long-distance Ptranslocation in the mycorrhizal mycelium occurs (Jentschkeet al. 2001)
The factors that favour the dominance of different anato-motypes are still unclear. However, it was demonstrated thatEcM roots colonized by the most dominant anatomotype,Alnicola spp., had the highest SRA and SRL among all Al-nus stands, which suggests a better cost to benefit ratio forEcM short roots in Alnicola spp.
The A. glutinosa trees at Sirgala and Songa originate fromthe same set of mother trees, and the significantly higherRTFL than in the other two stands could be one piece ofevidence supporting the view that genetic variation within aT
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Not
e:V
alue
sfo
llow
edby
adi
ffer
ent
lette
rdi
ffer
sign
ific
antly
(Tuk
ey’s
test
,p
<0.
05)
with
inA
lnus
spec
ies.
Val
ues
inbo
ldfa
cety
pedi
ffer
sign
ific
antly
betw
een
the
two
Aln
ussp
ecie
s(D
,ec
tom
ycor
rhiz
al(E
cM)
shor
t-ro
otdi
amet
er;
W,
EcM
shor
t-ro
ot-t
ipdr
ym
ass;
L,
EcM
shor
t-ro
ot-t
iple
ngth
;R
TD
,ro
ottis
sue
dens
ity;
SRA
,sp
ecif
icro
otar
ea;
SRL
,sp
ecif
icro
otle
ngth
;R
TFW
,E
cMsh
ort-
root
-tip
freq
uenc
ype
run
itdr
ym
ass;
RT
FL,
EcM
shor
t-ro
ot-t
ipfr
eque
ncy
per
unit
leng
th).
Ostonen et al. 1793
Published by NRC Research Press
tree species may have an important effect on root traits also.Our results are consistent with Korkama et al. (2006), whodemonstrated a significant difference in root-tip frequencyper fine-root (<2 mm) length between slow- and fast-grow-ing clones of Norway spruce.
The results of our work show that to characterize the ef-fects of different environmental factors in a forest ecosys-tem, a complex of EcM root traits should be analyzed.Reported changes in root morphology along ecological gra-dients (Godbold et al. 2003; Leuschner et al. 2004) raise thequestion of whether differences in environmental conditionscause changes in EcM community structure. Communitystructure and relative frequency/abundance of individualfungal species may change substantially in relation to topog-raphy and nutrient gradients (Lilleskov et al. 2002, 2008;Tedersoo et al. 2003, 2008; Toljander et al. 2006). No re-ports of consistent changes in EcM exploration types couldbe found in the literature, probably because single domi-nants may mask the overall shifts. Hence, a knowledge ofEcM fungal community structure and the correlation ofEcM fungus species with morphological and physiologicalpatterns in short roots is important for understanding the rel-ative roles of the functional adaptation of plants (root anat-omy) and fungi (mantle thickness, abundance of externalmycelium, species) to soil-quality and vegetation gradients.
Our data suggest that the particular species of EcM fungidrive EcM short-root traits, primarily independent variablessuch as diameter and length, and eventually functional pa-rameters such as SRA or SRL of EcM roots in alders. Theshifts in EcM communities may play an important role inmorphological adaptation and mineral nutrition of plants.To elucidate the role played by EcM fungi in determiningshort-root functional properties and adaptation to differentsoil conditions, further investigation is needed.
AcknowledgementsWe thank M. Linder, S. Meel, and T. Jairus for help in
the laboratory. This study was funded by Estonian ScienceFoundation grants 6472, 6011, 6606, 7434, 7452, and JD92and is part of Research Project No. 0180127s08, financiallysupported by the Ministry of Education and Research of theRepublic of Estonia.
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Table 5. Characteristics of ectomycorrhizal short roots of Alnus incana and A. glutinosa stands.
D (mm) L (mm) RTD (kg�m–3) SRA (m2�kg–1) SRL (m�g–1) RTFL (no.�cm–1)
Site Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE
Alnus incanaLeidisoo 0.35b 0.01 4.3 0.3 138b 4 85a 4 79a 4 2.5 0.1Saka 0.31a 0.01 4.2 0.2 135b 4 102b 4 111b 4 2.9 0.3Porijoe 0.37b 0.01 3.8 0.3 112a 4 102b 4 89a 4 3.4 0.5
Alnus glutinosaInka 0.37ab 0.01 5.7c 0.2 122a 3 95b 2 88b 3 2.0a 0.1Songa 0.38c 0.01 4.5ab 0.2 137b 3 80a 2 69a 3 2.7b 0.1Jarvselja 0.34a 0.01 5.2bc 0.2 146b 3 81a 2 77ab 3 2.1a 0.1Sirgala 0.37 0.01 4.1a 0.2 123a 3 90b 2 78ab 3 2.6b 0.1
Note: Values followed by a different letter differ significantly (Tukey’s test, p < 0.05) within Alnus species. (D, ectomycorrhizal (EcM) short-root dia-meter; W, EcM short-root-tip dry mass; L, EcM short-root-tip length; RTD, root tissue density; SRA, specific root area; SRL, specific root length; RTFW,EcM short-roottip frequency per unit dry mass; RTFL, EcM short-roottip frequency per unit length.
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