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Aquatic Botany 93 (2010) 25–31

Contents lists available at ScienceDirect

Aquatic Botany

journa l homepage: www.e lsev ier .com/ locate /aquabot

olecular analysis of green-tide-forming macroalgae in the Yellow Sea

infeng Wanga,b, Peng Jianga,∗, Yulin Cuia,b, Nan Lib,c, Mingqing Wangd, Hanzhi Lina,b,eimin Hee, Song Qina,∗

Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Nanhai Road 7, Qingdao 266071, ChinaGraduate University of Chinese Academy of Sciences, Beijing 100049, ChinaYantai Institute of Coastal Zone Research for Sustainable Development, Chinese Academy of Sciences, Yantai 264003, ChinaCollege of Marine Life Science, Ocean University of China, Qingdao 266003, ChinaCollege of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 200090, China

r t i c l e i n f o

rticle history:eceived 5 September 2009eceived in revised form 25 February 2010ccepted 3 March 2010vailable online 9 March 2010

eywords:he Yellow Sea

a b s t r a c t

In the summer of 2008, free-floating green algae bloomed in the Yellow Sea. Samples were collectedin a wide area (119◦32′–122◦00′E, 32◦25′–36◦49′N). We calculated the sequence divergences of nuclearITS, chloroplast rbcL, and psbA data of free-floating samples collected from the Yellow Sea and Ulvaceaefrom Europe and Japan. In the ITS sequence, 19 out of the 21 Yellow Sea samples of 2008 were iden-tical to those of a sample taken at Qingdao in 2007. A low divergence (0.2%) was found in remainingtwo samples. Similar evidence was shown by pairwise distances of rbcL and psbA gene sequence data,implying the uniformity of the Yellow Sea blooms in 2007 and 2008. The ITS sequence of the Yellow

ree-floatingnteromorphaTSbcLsbA

Sea samples differed 8.1–10.8% from free-floating Enteromorpha or Ulva reported worldwide. ITS-basedmolecular phylogenetic results and rbcL sequence data grouped the free-floating alga in the YellowSea into one clade with Enteromorpha procera, Enteromorpha linza and Enteromorpha prolifera. Further-more, both morphological characteristics and ribotype network of the ITS sequences imply that theblooming algae in 2007 and 2008 were E. prolifera. The haplotypes of the Yellow Sea free-floatingE. prolifera are closely related to those from the Japanese coast but less to European and American

algae.

. Introduction

Enteromorpha or Ulva can multiply strongly from fragmentationr reproduction, forming a “green-tide” and resulting in ecologicalnd social problems (Fletcher et al., 1990; Valiela et al., 1997; Bäckt al., 2000; Blomster et al., 2002). Enteromorpha species have beenistinguished morphologically from Ulva sp. for having tubular andonostromatic blades (Link, 1820).Infrageneric classification and phylogeographic studies of

nteromorpha are difficult to perform due to the species’ exten-ive distribution (Van den Hoek et al., 1995), tiny intraspecificifferences (Bliding, 1963), a high variability in morphology andytology resulting from the change of developmental stages and

nvironmental conditions (De Silva and Burrows, 1973; Mshigenind Kajumulo, 1979; Tanner, 1986), and the free-floating natureMalta et al., 1999; Blomster et al., 2002). In recent years, molec-lar analyses have been conducted to address these difficulties.long with the sequences in the GenBank, nuclear rDNA ITS and

∗ Corresponding authors. Tel.: +86 532 82898500; fax: +86 532 82880645.E-mail addresses: jiangpeng@qdio.ac.cn (P. Jiang), sqin@qdio.ac.cn (S. Qin).

304-3770/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.aquabot.2010.03.001

© 2010 Elsevier B.V. All rights reserved.

chloroplast rbcL have been frequently cited to identify species ofUlvaceae (Malta et al., 1999; Hayden et al., 2003; Shimada et al.,2003), and are often used as genetic markers in phylogeographicstudies on Ulva and Enteromorpha (Blomster et al., 1999; Haydenand Waaland, 2002; Leskinen et al., 2004; Shimada et al., 2008).

In the summer of 2008, Enteromorpha sp. bloomed in QingdaoCity off the coast of the Yellow Sea, causing concern among scien-tists and coastal managers (Sun et al., 2008; Leliaert et al., 2009).Studies were carried out on the morphological characteristics, lifehistory, physiological parameters, and reasons for the bloomingof the green alga (Fu et al., 2008; Ding et al., 2009). Using ITSsequences, Jiang et al. (2008) demonstrated that 10 free-floatingEnteromorpha sp. samples from Qingdao coast in 2007 were onespecies. The attached Ulvaceae species in this area did not consti-tute the biogeographic cradle of the blooms. Subsequently, Leliaertet al. (2009) reported on the 2008 bloom event based on ITS andrbcL gene sequences. However, the green-tide forming samplesfrom the Qingdao coast in these two studies were obtained only

within a span of a year and from a restricted area. In the presentstudy, nuclear rDNA ITS, chloroplast rbcL, and psbA sequences offree-floating samples from a much larger area of the Yellow Sea arereported and discussed for species identification, genetic diversity,and phylogeographical structure.

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. Materials and methods

.1. Samples

Twenty-two free-floating green algae were collected from theellow Sea (119◦32′–122◦00′E, 32◦25′–36◦49′N) in the summersf 2007 and 2008 (Table 1; Fig. 1). Each sample was washed withoiled seawater three to four times and cultivated in VSE media.

.2. Morphological observation

Macroscopic observations on the fresh fronds, including theolor, texture and branching of the thalli, as well as the arrange-ent, shape, and chloroplast position of the cells, were conducted

nder a Nikon Eclipse 50i microscope (Nikon, Kawasaki, Japan).

.3. DNA extraction and PCR amplification

Genome DNA was extracted from fresh plants accordingo the method described by Doyle and Doyle (1990). PCR

able 1ree-floating Enteromorpha samples collected form the Yellow Sea.

Samples Collection sites location Date

S096 P01 (35◦53′N, 120◦10′E) 2007-07-29S266 P02 (34◦59′N, 119◦49′E) 2008-07-02S275 P03 (36◦43′N, 121◦19′E) 2008-06-23S313 P04 (34◦48′N, 119◦32′E) 2008-07-11S326 P05 (36◦46′N, 121◦40′E) 2008-07-17S349 P06 (35◦30′N, 120◦27′E) 2008-07-13S350 P07 (35◦44′N, 120◦40′E) 2008-07-13S351 P08 (35◦45′N, 120◦31′E) 2008-07-13S363 P09 (32◦44′N, 121◦16′E) 2008-07-17S364 P09 (32◦44′N, 121◦16′E) 2008-07-17S376 P10 (33◦30′N, 121◦45′E) 2008-07-25S378 P11 (34◦30′N, 121◦30′E) 2008-07-24S379 P12 (34◦30′N, 121◦45′E) 2008-07-24S380 P13 (35◦15′N, 121◦15′E) 2008-07-23S381 P14 (35◦38′N, 122◦00′E) 2008-07-23S383 P15 (34◦58′N, 120◦18′E) 2008-07-25S385 P16 (34◦00′N, 121◦15′E) 2008-07-25S386 P17 (33◦45′N, 121◦30′E) 2008-07-25S402 P18 (33◦29′N, 121◦17′E) 2008-06-26S403 P18 (33◦29′N, 121◦17′E) 2008-06-26S914 P19 (32◦25′N, 121◦27′E) 2008-06-30S924 P20 (33◦16′N, 120◦48′E) 2008-07-01

Fig. 1. Sites of sample collection in the Yellow Sea.

ny 93 (2010) 25–31

amplification of the ITS region (including ITS1, 5.8S, and ITS2)was performed using the primers and thermal cycling pro-file of Leskinen and Pamilo (1997). In the case of rbcL, theprimers and reaction profile used were those recorded inManhart (1994). The psbA genes were amplified using degenerateprimers (forward: 5′-YTHTAYCCWATHTGGGAAGC-3′ and reverse:5′-GGGAAGTTRTGNGCRTTRCG-3′) and thermal cycling profile(94 ◦C for 10 min, followed by 30 cycles of 1 min at 94 ◦C, 1 min at57 ◦C, 50 s at 72 ◦C, and finally 10 min at 72 ◦C). The design of thesenew degenerate psbA primers was according to the alignment of 22different aquatic psbA sequences in the Refseq database of GenBank.The primers were tested using DNA from Spirulina sp., Chlamy-domonas reinhardtii, Laminaria japonica, and two other randomlycollected surface water samples from the seashore with DNA fromEscherichia coli as the negative contrast (Li et al., unpublished data).

PCR products were checked and purified on 1% TAE agarosegels and sequenced by Shanghai Sangon Corp. (Sangon, Shanghai,China).

2.4. Sequence data and phylogenetics analysis

Sequences were aligned and edited using ClustalX (Larkinet al., 2007) and BioEdit (Hall, 1999), respectively. Nucleotidefrequencies, substitution rates, and pairwise distances were calcu-lated using Mega4.0 with the Kimura 2-parameter model (Tamuraet al., 2004). For comparative analysis, 47 ITS and 19 rbcL sequencesof Enteromorpha or Ulva, including free-floating species reportedpreviously, were downloaded from the GenBank (Table 2

). With Ulvaria obscura var. blyttii from the order Ulvales serv-ing as outgroup, phylogenetic trees were built using maximumparsimony (MP) and neighbor joining (NJ) methods with Mega4.0(Tamura et al., 2007). Robustness of the MP and NJ trees was testedby bootstrapping with 1000 replicates of the data.

The ITS sequences of Enteromorpha prolifera with clear biogeo-graphic records were also downloaded from the GenBank (Table 2).Based on 20 different ITS haplotypes of E. prolifera containingsequences of 22 samples from this study and 29 samples from pre-vious studies (Tables 1–2), a statistical parsimony network tree wasconstructed to illuminate the phylogeographical structure, distri-bution, and genetic variation of the Enteromorpha species using TCS1.21 (Clement et al., 2000).

3. Results

3.1. Morphology of the green alga

Free-floating green algae of the Yellow Sea sampled in 2007 and2008 have the same morphology. The fresh fronds were light green,about 12 cm long, monostromatic, filamental, with tubular thallus,and a gas inflation in some segments of the tube (Fig. 2a and b).They were thickly branched, with tiny and multiple branchlets onthe bough (Fig. 2b–d). Cells were irregularly arranged and filledwith chromatophores visible on the surface (Fig. 2d–f).

3.2. Pairwise distances of ITS, rbcL, and psbA sequences

All ITS sequences of the free-floating green algae were thesame length (585 bp). Alignment displayed no indels, but onlyone to two substitutions among the sequences. The nucleotidefrequencies were 0.202 (A), 0.18 (T/U), 0.331 (C), and 0.287 (G). Thetransition/transversion ratio k1 = 1000 (purines) and k2 = 976.793

(pyrimidines). The overall transition/transversion bias wasR = 596.721, where R = [A × G × k1 + T × C × k2]/[(A + G) × (T + C)].Nineteen out of 21 free-floating samples from 2008 were identicalto the samples from 2007. With a variance of only 1–2 bp, the diver-gence value was at 0.2% when these 19 samples were compared

J. Wang et al. / Aquatic Botany 93 (2010) 25–31 27

Fig. 2. Morphological observation of the free-floating green algae from the Yellow Sea. (a) Monostromatic filamental and tubular thalli (arrow shows gas inflation of thetube), scale bar = 1 cm. (b) Thallus of Enteromorpha sample, scale bar = 1 cm. (c) Thick branches of the thallus, scale bar = 1 mm. (d) Tiny branchlets and irregularly arrangedcells, scale bar = 100 �m. (e) Cells in surface view, scale bar = 60 �m. (f) Chloroplasts and pyrenoids in the cells (arrow shows pyrenoid). Scale bar = 40 �m.

Table 2Reference data from published ITS and rbcL sequences of Enteromorpha and Ulva.

Species Source and/or reference Accession no.

ITS rbcL

E. compressa Portaferry, Strangford Lough, Northern Ireland (Blomster et al., 1998) AF035350 AY255859E. intestinalis Woolwich, Thames, London, England (Blomster et al., 1998) AF035342 AY255860E. intestinalis floatinga Olkiluodonvesi, Finland (Blomster et al., 2002) AF499452

Sheep Bay, Valdez-Cordova County, AK, USA (Hayden and Waaland, 2004) AY422552E. linza Yoshino River, Tokushima Prefecture, Japan (Shimada et al., 2003) AB097649 AB097620

Ythan Estuary, Aberdeenshire, Scotland (Tan et al., 1999) AJ000203Humboldt Bay, CA, USA (Hayden et al., 2003) AY260557 AY255861

E. muscoidesb Los Torunos, Puerto Réal, Cádiz, Spain (Blomster et al., 1999) AF127170 AY255862E. muscoides floating The Backstrand, Co. Waterford, Ireland (Blomster et al., 1999) AF127167E. prolifera Ythan Estuary, Aberdeenshire, Scotland (Tan et al., 1999) AJ234304 AY255864

Ythan Estuary, Aberdeenshire, Scotland (Tan et al., 1999) AJ234305Red Hill Marina, Salton Sea, Imperial County, CA, USA (Hayden and Waaland, 2004) AY422510 AY422554Nobusha River, Rumoi, Hokkaido, 100 m upstream, Japan (Shimada et al., 2008) AB298308Nobusha River, Rumoi, Hokkaido, 50 m upstream, Japan (Shimada et al., 2008) AB298308Furubira River, Yoichi, Hokkaido, 10 m upstream, Japan (Shimada et al., 2008) AB298308Koyoshi River, Yurihonjo, Akita, 10 m upstream, Japan (Shimada et al., 2008) AB298308Nadachi River, Nadachi, Niigata, 10 m upstream, Japan (Shimada et al., 2008) AB298308Fishing Port of Kurobe, Toyama, Japan (Shimada et al., 2008) AB298308Daishouji River, Kaga, Ishikawa, 100 m upstream, Japan (Shimada et al., 2008) AB298308Yura River, Maizuru, Kyoto, 1 km upstream, Japan (Shimada et al., 2008) AB298308Honjo, Matsue, Shimane, salted lake, Japan (Shimada et al., 2008) AB298308Honjo, Matsue, Shimane, salted lake, Japan (Shimada et al., 2008) AB298309Irie, Matsue, Shimane, salted lake, Japan (Shimada et al., 2008) AB298310Saikyou River, Nishinoomote, Kagoshima, 90 m upstream, Japan (Shimada et al., 2008) AB298311Mumomi River, Fukuoka, Fukuoka, 2.7 km upstream, Japan (Shimada et al., 2008) AB298312Shimanto River, Shimanto, Kochi, 500 m upstream, Japan (Shimada et al., 2008) AB298313Natori River, Natori, Miyagi, 20 m upstream, Japan (Shimada et al., 2008) AB298314Juhoh River, Hitachi, Ibaraki, 10 m upstream, Japan (Shimada et al., 2008) AB298314

28 J. Wang et al. / Aquatic Botany 93 (2010) 25–31

Table 2 (Continued )

Species Source and/or reference Accession no.

ITS rbcL

Yanyuu River, Ohshima, Kagoshima, 70 m upstream, Japan (Shimada et al., 2008) AB298314Ura River, Ohshima, Kagoshima, 90 m upstream, Japan (Shimada et al., 2008) AB298314Nakakiku River, Ohshima, Kagoshima, 200 m upstream, Japan (Shimada et al., 2008) AB298314Sanze River, Tsuruoka, Yamagata, 10 m upstream, Japan (Shimada et al., 2008) AB298315Sekiguchi River, Yamada, Iwate, 10 m upstream, Japan (Shimada et al., 2008) AB298316Toyo River, Sakata, Yamagata (4), 20 m upstream, Japan (Shimada et al., 2008) AB298317Shio River, Motobu, Okinawa, 80 m upstream, Japan (Shimada et al., 2008) AB298318Shimanto River, Shimanto, Kochi, 10 m upstream, Japan (Shimada et al., 2008) AB298319Shimanto River, Shimanto, Kochi, 7 km upstream, Japan (Shimada et al., 2008) AB298320Shio River, Motobu, Okinawa, 80 m upstream, Japan (Shimada et al., 2008) AB298321Red Hill Marina, Salton Sea, Imperial County, CA, USA (Hayden and Waaland, 2004) AY422554

E. procerac Unknown (Hayden et al., 2003) AY260558 AY255863U. armoricana floating Miyajima, Hiroshima Prefecture, Japan (Shimada et al., 2003) AB097661 AB097631U. fasciata Kihei, Maui, USA (Hayden et al., 2003) AY260561 AY255867U. fasciata floating Miyajima, Hiroshima Prefecture, Japan (Shimada et al., 2003) AB097664 AB097634U. lactuca Ballyhenry Island, Strangford Lough, Northern Ireland (Tan et al., 1999) AJ234310 AF499669

Nemuro, Hokkaido, Japan (Shimada et al., 2003) AB097651 AB097622U. ohnoi floating Tosa, Kochi Prefecture, Japan (Hiraoka et al., 2003) AB116031 AB116037U. pertusa floating Hakata, Fukuoka Prefecture, Japan (Shimada et al., 2003) AB097654 AB097625U. rigida Cádiz, Spain (Hayden et al., 2003) AY260565

Pelluco Beach, SE of Puerto Montt, Chile (Hayden and Waaland, 2004) AY422564U. scandinavica Langstone Harbour, Portsmouth, England (Tan et al., 1999) AJ234317 AY255870Ulvaria obscura var. blyttii Padilla Bay, WA, USA (Hayden et al., 2003) AY260571 AF499673

a Floating, reported free-floating species.b As E. clathrata.c As E. linza.

Fig. 3. MP tree of ITS sequences of free-floating Enteromorpha and Ulva. U., Ulva; E., Enteromorpha; floating, reported free-floating species. Ulvaria obscura var. blyttii servedas outgroup taxa. The robustness of the tree was tested by bootstrapping with 1000 replicates of the data

J. Wang et al. / Aquatic Botany 93 (2010) 25–31 29

F ., Entea repli

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3

wsossSsfslp(flg

ig. 4. MP tree of rbcL sequences of free-floating Enteromorpha and Ulva. U., Ulva; Es outgroup taxa. The robustness of the tree was tested by bootstrapping with 1000

ith the two others. The results suggest very close identity of theamples taken in both years. On the other hand, the ITS sequencesf the Yellow Sea samples differed 8.1–10.8% from free-floatingnteromorpha or Ulva reported worldwide, suggesting a differencemong them. An analysis of the rbcL sequences showed that 9 outf 10 samples from the Yellow Sea shared 948 bp nucleotides; onlyne rbcL sequence (S376) had a difference of 2 bp (0.2% pairwise).even out of 10 psbA partial sequences (702 bp) were identical;he pairwise distances were 0.1–1.3% from those of the other threeamples (S313, S351, and S386).

.3. Phylogenetic analysis based on ITS and rbcL sequences

The topological trees of both ITS and rbcL of Enteromorphaere polyphyletic. Since the ITS sequences of 19 free-floating

amples of 2008 and those of Qingdao in 2007 were identical,nly one of these samples (S364), along with the two divergentamples (S402 and S403), was used in the phylogenetic analy-is. Similarly, the rbcL sequences of one of the identical samples,386, and that of one different sample, S376, were used to con-truct the phylogenetic tree. In MP phylogenetic trees inferredrom both ITS and rbcL sequences (Figs. 3 and 4), the free-floatingamples of the Yellow Sea were grouped into a clade with E. pro-

ifera, Enteromorpha linza, and Enteromorpha procera. The samehylogenetic topology was found when using the NJ methodnot shown), whereas the previously reported samples of free-oating Enteromorpha and Ulva in Europe and Japan fell into otherroups.

romorpha; floating, reported free-floating species. Ulvaria obscura var. blyttii servedcates of the data

3.4. Ribotypes network analysis of E. prolifera

Statistical parsimony network for ITS sequences of E. proliferadivided 17 ribotypes into four groups: Group A represented threeribotypes of the Yellow Sea samples (01–03), and connected withGroup B (04–07). Ribotypes 04 and 05 contained ten specimensfrom the coast of the Sea of Japan. Ribotype 06 included five spec-imens from Ohshima, Kagoshima, and the Pacific coast of Japan.Group B was divided into two different lineages; Group C con-sisted of two ribotypes (08–09); while Group D was comprised ofeight ribotypes (10–17) (Fig. 5). The number of nucleotide substi-tutions or indels was larger in three ITS sequences (i.e., AJ234304,AJ234305, and AY422510). Since there is no line connecting thesethree with the other 17 sequences, they are not shown in the net-work.

4. Discussion

4.1. Characterization of the green alga bloom in the Yellow Sea

Large-scale blooms of free-floating Enteromorpha and Ulvahave been frequently reported worldwide (Fletcher et al., 1990;Hernandez et al., 1997; Taylor, 1999; Bäck et al., 2000; Hiraoka

et al., 2003; Largo et al., 2004). In the present study, evidenceshowed that the free-floating green algae bloom arose from a sin-gle species of Enteromorpha which is broadly distributed in theYellow Sea. Meanwhile, a significant difference in ITS sequenceswas found between the free-floating green algae of the Yellow Sea

30 J. Wang et al. / Aquatic Botany 93 (2010) 25–31

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ig. 5. Ribotype network and geographical distribution of haplotype of E. proliferaibotypes corresponds to one base substitution. (b) Geographical distribution of dif

nd those of free-floating species of Enteromorpha or Ulva recordedlsewhere in the world. Molecular phylogenetic analysis groupedhe free-floating Enteromorpha species into one clade with E. pro-era, E. linza, and E. prolifera. This is similar to the results of thebcL analysis conducted by Leliaert et al. (2009), who indicatedhat free-floating algae in the Yellow Sea belonged to an LPP grouplinza–procera–prolifera species complex, Shimada et al., 2008). E.rocera is currently regarded as a synonym of E. linza; it has annteromorpha-like tubular base and an Ulva-like distromatic blade.n contrast, the free-floating Enteromorpha of the Yellow Sea had a

onostromatic filamental, tubular, densely branched thalli featur-ng E. prolifera. Furthermore, ribotype network analysis suggestedhat the free-floating Enteromorpha sample in 2007 and 2008 was. prolifera.

.2. Phylogeography of E. prolifera

E. prolifera from Scotland (AJ234304 and AJ234305) and the

SA (AY422510) were excluded from the ribotype network of ITS

equence because of their large sequence divergence from otheribotypes. However, the three ribotypes were shown within the E.rocera, E. linza, and E. prolifera group by Tan et al. (1999), Haydent al. (2003), and Hayden and Waaland (2004), suggesting that the

uences. (a) Statistical parsimony network tree. Each line between two connectinghaplotype groups.

divergences resulted not from biogeographic diversity, but possi-bly from inaccurate gene amplification or sequencing. Conversely,ribotypes of the Yellow Sea sample (Group A) have a close relation-ship with those of 04, 05, and 06 of Japan (Group B). Ribotypes 04and 05 originated from Fukuoka (Kyushu) or Shimane and extendedinto that of the Sea of Japan coast (Group C). The result is similarto that of Shimada et al. (2008), who speculated that E. proliferaexpanded into the Sea of Japan through Kyushu. The same routewas also hypothesized by Brodie et al. (1991) for Chondrus nipponi-cus and by Masuda et al. (1992) for Laurencia nipponica. Ribotype 06occurred at Ohshima and Kagoshima in Japan and expanded mainlyinto the Pacific coast of Japan (Group D). The network (Fig. 5) illu-minates the phylogeny and evolution of the E. prolifera rather thanthe green-tide source or direction.

Acknowledgements

This study is supported by the National Science & Technology

Pillar Program of China (No. 2008BAC49B01), the Innovative KeyProject of the Chinese Academy of Sciences (No. KZCX2-YW-209),and the Science and Technology Project of Qingdao City (No. 06-2-2-12-JCH). We thank the crews of open-cruise R/V Beidou of theYellow Sea Fisheries Research Institute, Chinese Academy of Fishery

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ciences and R/V Dongfanghong II of Ocean University of China. Were also grateful to Prof. Pu Xu, Associate Prof. Songdong Shen, Asso-iate Prof. Ming Zhu, and Qinqin Lu, Quanfu Wang, Depin Jia, Conguo, and Zhengyi Liu for their assistance in sampling, and Prof.aniel Kister for improving the English of the manuscript.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.aquabot.2010.03.001.

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