MICROBIOLOGY ECOLOGY. Diverse communities ofarbuscular mycorrhizal fungi inhabit sites with very high altitude intibet Plateau.

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1 RESEARCH ARTICLE Diverse communities ofarbuscular mycorrhizal fungi inhabit sites with very high altitude intibet Plateau Yongjun Liu 1,2, Junxia He 1, Guoxi Shi 1, Lizhe An 1, Maarja Öpik 3 & Huyuan Feng 1 1 Key Laboratory of Arid and Grassland Ecology of Ministry of Education, School of Life Sciences, Lanzhou University, Lanzhou, China; 2 College of Life Science and Engineering, Northwest University for Nationalities, Lanzhou, China; and 3 Department of Botany, Institute of Ecology and Earth Sciences, University of Tartu, Tartu, Estonia MICROBIOLOGY ECOLOGY Correspondence: Huyuan Feng, Key Laboratory of Arid and Grassland Ecology of Ministry of Education, School of Life Sciences, Lanzhou University, Room 322, Lanzhou , China. Tel: ; fax: ; fenghy@lzu.edu.cn Received 24 January 2011; revised 6 June 2011; accepted 19 June Final version published online 18 July DOI: /j x Editor: Philippe Lemanceau Keywords arbuscular mycorrhiza; Dracocephalum heterophyllum; pioneer species; colddominated ecosystem; high altitude; Tibet Plateau. Introduction The vast majority of terrestrial plants form mutualistic associations with arbuscular mycorrhizal fungi (AMF), which belong to the phylum Glomeromycota with about 200 described species (Smith & Read, 2008). AMF can increase nutrient uptake as well as improve the stress tolerance of their hosts (Smith et al., 2003; Aroca et al., 2007). AMF also exert strong effects on the plant communities (van der Heijden et al., 1998; Vogelsang et al., 2006). In particular, the diversity of AMF and plants can be inter-related (Johnson et al., 2004; Landis et al., 2004). A high number of AMF taxa has been found in tropical and boreal forests (Husband et al., 2002; Öpik et al., 2008, 2009), whereas the AMF richness is often low in disturbed and degraded habitats (Öpik et al., 2006; Tian et al., 2009). Even where richness remains high, environmental factors such as ph have been shown to determine community composition even at relatively small spatial scales (Dumbrell et al., 2010). At a much smaller, within sample site scale, studies indicate Abstract Diversity of arbuscular mycorrhizal fungi (AMF) is well studied in many ecosystems, but little is known about AMF in cold-dominated regions with very high altitude. Here, we examined AMF communities associated with two plant species in the Tibet Plateau. Roots and rhizosphere soils of Dracocephalum heterophyllum (pioneer species) and Astragalus polycladus (late-successional species) were sampled at five sites with altitude from 4500 to 4800 m a.s.l. A total of 21 AMF phylotypes were identified from roots and spores following cloning and sequencing of 18S rrna gene, including eight new phylotypes and one new family-like clade. More AMF phylotypes colonized root samples of D. heterophyllum ( ) than of A. polycladus ( ). Vegetation coverage was the most important factor influencing AMF community composition in roots. Globally infrequent phylotype Glo-B2 in Glomus group B was the most dominant in roots, followed by globally frequent phylotype Glo-A2 related to Glomus fasciculatum/intraradices group. Our findings suggest that a diverse AMF flora is present in the Tibet Plateau, comprising both potentially habitat-selective and generalist fungi. that AMF have different levels of host preference or specificity (e.g. Husband et al., 2002; Vandenkoornhuyse et al., 2003), suggesting that the host plant is also an important factor in assembling the AMF community. Thus, it is likely that the diversity and distribution of AMF are codetermined by the biotic and abiotic factors, and that the balance between the two depends on spatial scale and local factors. Diversity of AMF has been well studied in grasslands, forests, and some other ecosystems of mainly the temperate climatic zone (Öpik et al., 2010). Little is known about AMF diversity in regions with very high altitude (above 3500 m a.s.l.; but see Gai et al., 2009 and some studies in montane sites with altitude below 2000 m a.s.l.: for example Sýkorová et al., 2007; Wu et al., 2007), where the habitats are often cold-dominated. Kytöviita & Ruotsalainen (2007) suggested that AMF could not improve nutrient capture of host plants in low temperature habitats, but this does not imply that AMF in such habitats cannot persist as plant symbionts. On the contrary, a high diversity of AMF spores

2 356 Y. Liu et al. and phylotypes has been reported in sites with altitude ranging from 3500 to 5200 m a.s.l. (Gai et al., 2009) and in a low-arctic meadow (Pietikäinen et al., 2007), respectively. Nonetheless, it is widely accepted that the major plant fungal associations in high-altitude and polar regions are ectomycorrhiza and dark septate endophytes rather than AM (Gardes & Dahlberg, 1996; Newsham et al., 2009; Gao & Yang, 2010); this perhaps suggests that most plants in these regions are not good hosts for AMF, and that AMF should have a different life-history strategy in these regions. For example, these fungi would select suitable host plants (Gardes & Dahlberg, 1996) and the spores might be dormant for a long time (Pietikäinen et al., 2007). Even if diverse AMF communities could be present in the regions with very high altitude, which AMF species can persist and which host plants they colonize is still unclear. The evolution of AMF traits may be highly dependent on abiotic factors such as soil temperature and hypoxia (Helgason & Fitter, 2009). Specific AMF have been found in extreme habitats such as geothermal and gypsum soils (Appoloni et al., 2008; Alguacil et al., 2009). Therefore, we predict that AMF species and their traits in regions with very high altitude may be different from those in ecosystems with more moderate abiotic conditions. To better understand the biodiversity of AMF and its determinants in the regions with very high altitude, we chose to study AMF associated with two plant species with different life-history strategies in the Tibet Plateau of China (mean altitude over 4500 m a.s.l.). We used molecular techniques to describe AMF communities to address the following questions: (1) what is the AMF diversity in the roots of a pioneer and a late-successional plant species in the central Tibet Plateau? (2) Which environmental factors determine the richness and community composition of AMF associated with these two plant species? (3) Are the AMF taxa in this region different from those in other ecosystems? Materials and methods Study sites and sampling This study was conducted in the central Tibet Plateau, where the average altitude is above 4500 m a.s.l. and the mean annual temperature ranges between 3 and 7 1C. Five study sites were chosen along the Qinghai-Tibet highway from Xidatan to Amdo, in a line running approximately north-east to south-west (Table 1, Supporting information, Fig. S1). In each site, a pioneer plant species Dracocephalum heterophyllum (Lamiaceae) was dominant in the region nearby the highway (disturbed once due to the construction of highway 30 years ago, o 50 m from highway), whereas the natural plant communities (late-successional stage; 4 60 m from highway) were dominated by Cyperaceae, Caryophyllaceae or leguminous plants such as Astragalus polycladus (Table 1). Because the plants of Cyperaceae and Caryophyllaceae are traditionally regarded as little AM-dependent (Wang & Qiu, 2006; Smith & Read, 2008), we chose a leguminous species, A. polycladus, as the Table 1. Sampling sites and their vegetation details Early-successional plant community Late-successional plant community Study site Latitude, longitude Xidatan (X) N E Kunlunshan (K) Wudaoliang (W) Tongtianhe (T) Amdo (A) N E N E N E N E Altitude (m a.s.l.) Vegetation coverage Species richness Dominant plant species (coverage) 4524 c. 30% 2 D. heterophyllum (c. 25%), Aster alpinus (c.5%) 4746 c. 70% 4 D. heterophyllum (c.55%), Avena fatua (c. 5%), Poa sp. (c. 5%) 4568 c. 30% 2 D. heterophyllum (c. 20%), Saussurea wellbyl (c.10%) Vegetation coverage Species richness Dominant plant species (coverage) c. 45% 10 Androsace tapete (c.15%), Astragalus polycladus (c.10%), Arenaria kansuensis (c.8%) w c. 65% 9 Kobresia pygmaea (c.40%) w, Astragalus polycladus (c.10%) c. 15% 1 Astragalus polycladus (c.15%) 4603 c. 25% 1 D. heterophyllum (c. 25%) c. 65% 10 K. pygmaea (c.30%) w, Stipa aliena (c.10%), Astragalus polycladus (c.8%) 4773 c. 75% 6 D. heterophyllum (c.50%), Potentilla bifurca (c.7%), Elymus nutans (c.5%) c. 75% 11 K. pygmaea (c.35%) w, Potentilla parvifolia (c.8%), Arenaria kansuensis (c.5%) w, Astragalus polycladus (c.4%) Site codes are shown in parentheses. w Potentially non-am species of Caryophyllaceae or Cyperaceae (Smith & Read, 2008).

3 Diverse AM fungi in Tibet Plateau 357 representative of late-successional plant species. Before sampling, the vegetation coverage, plant species coverage and species richness were estimated on three plots of 1 1m at each plant community using Sutherland s method (Sutherland, 1996). Vegetation coverage and plant species richness varied in different sites, with low vegetation coverage in sites of Xidatan and Wudaoliang due to serious permafrost degradation and rodent damage. More details of the sampling sites are presented in Table 1. Sampling was conducted in the beginning of August In each site, three individuals of D. heterophyllum and A. polycladus were randomly sampled (30 samples in total: 2 plant species 3 plant individuals 5 sampling sites). Plants together with their rhizosphere soil were carefully excavated and placed in plastic bags. Roots were separated from each plant individual, washed with tap water and cut into approximately 1-cm-long fragments. Due to the limited quantity of roots, we randomly picked about 10 root fragments from samples of the same plant species and pooled these in order to verify the AM status of the study plant species following the method of McGonigle et al. (1990). The remaining roots (30 samples) were stored at 20 1C until DNA extraction. Soils (30 samples) were airdried and used for AMF spore extraction and soil chemical analyses. Spore extraction and soil chemical analysis AMF spores were extracted from 30 soil samples of 100 g by wet sieving (a pair of sieves: 750 and 38-mm mesh) and sucrose centrifugation (Brundrett et al., 1994), and the spore densities were counted. Spores from soil samples of the same plant species in each site were pooled (resulting in 10 pooled spore samples) and then grouped according to spore morphology and color using a dissecting microscope. Spore number of each morphotype in each pooled sample was counted, and the spore morphotypes were further identified using the molecular method as described below. Soil total nitrogen, organic carbon and available (Olsen) phosphorus contents were analyzed using the methods described previously (Liu et al., 2009). Soil ph was measured in 1 M KCl (10 g soil in a 50 ml solution). DNA extraction and amplification For each root sample, about 30 root fragments (c. l-cm lengths) were randomly picked and washed four times with sterilized distilled water. Root DNA was extracted using Plant DNA Extraction Kit (Tiangen Biotech, China) following the manufacturer s instructions, and then diluted 1 : 10 with ddh 2 O to be used as PCR template. For spore samples, up to three clean and healthy-looking spores of each morphotype per pooled sample were transferred into microcentrifuge tubes and vortexed at maximum speed. Spores were further rinsed four times with sterilized distilled water. Single spores were transferred into tubes with 10 ml ddh 2 O, and crushed with forceps under the dissecting microscope. A total of 120 single spores were incubated at 65 1C for 30 min and the liquid used as DNA template in PCR. All root- and spore-derived DNA samples were subjected to nested PCR. The first PCR reaction was performed with the universal fungal primers GeoA2 and Geo11 to amplify a c. 1.8 kb fragment of the 18S rrna gene (Schwarzott & Schüßler, 2001). PCR reactions were carried out in a final volume of 25 ml with2ml template and 0.5 mm of each primer using the Pfu PCR mastermix system (Tiangen Biotech) with the following cycling conditions: 94 1C for 120 s; 30 (94 1C for 30 s; 59 1C for 60 s and 72 1C for 150 s); and 72 1C for 600 s. Successful products of the first amplification were diluted 1 : 100 and 2 ml of this dilution was used as a template in the second PCR with universal eukaryotic primer NS31 (Simon et al., 1992) and AMFspecific primer AML2 (Lee et al., 2008). Most first PCR products of spores could not be detected on agarose gel, and thus 1 ml of the first PCR product was used as template for the second PCR. The NS31-AML2 primer combination was used because amplification using AML1-AML2 (Lee et al., 2008) was unsuccessful in our lab. PCR reactions were run with the same conditions as described above using the following cycling conditions: 94 1C for 120 s; 30 (94 1C for 30 s; 58 1C for 60 s and 72 1C for 80 s); and 72 1C for 600 s. All the PCR reactions were run on a GeneAmp s PCR system 2700 (Applied Biosystems). PCR products were examined on a 1.5% (w/v) agarose gel with ethidium bromide staining. Cloning, restriction fragment length polymorphism (RFLP) typing and sequence analysis All root-derived PCR products were used for constructing clone libraries (30 in total). PCR products of expected length (c. 560 bp) were purified using the Gel and PCR clear up System (Promega). After an A-tailing procedure, the DNA products were ligated into pgem-t vector (Promega) and cloned into Escherichia coli DH5a (Tiangen Biotech) according to the manufacturer s instructions. For each clone library, 50 randomly selected white clones were immersed in 30 ml ddh 2 O and subjected to three cycles of freezing and thawing for preparation of plasmid templates. Inserts were reamplified with primers NS31/AML2 with the same PCR conditions as above and double-digested with the restriction enzymes HinfI and Hin1II (MBI, Lithuania). Digested products were examined on a 2.5% (w/v) agarose gel. One representative of each root-derived RFLP type was sequenced with primer T7 by Major Biotech Company (Shanghai, China).

4 358 Y. Liu et al. The spore-derived PCR products were directly subjected to RFLP typing as described above. One representative of each spore-derived RFLP type was cloned as described above. One confirmed positive clone was sequenced from each spore-derived clone library with primer T7 as above. Sequences were edited using the CONTIGEXPRESS module of VECTOR NTI Suite 6.0 (InforMax Inc., MD), and submitted to BLAST search against public nucleotide sequence databases (Altschul et al., 1997). All AMF sequences have been submitted to GenBank database under the accession numbers GU GU and HQ HQ The representative sequences obtained in this study, the most closely related sequences from GenBank and the representative sequences of major families of Glomeromycota were used in the phylogenetic analysis. Sequences were aligned using CLUSTALX (Thompson et al., 1997) and manually edited using GENEDOC (Nicholas et al., 1997). Bayesian (GTR1G1I model) and neighbor-joining (Kimura 2-parameter model with 1000 bootstrap replications) phylogenetic analyses were performed using MRBAYES 3.1 (Ronquist & Huelsenbeck, 2003) and MEGA 4.0 (Tamura et al., 2007), respectively. Bayesian analysis was performed with two independent runs of Markov chain Monte Carlo with 10 6 generations (sampling frequency = 10), and a conservative burn-in of generations (25%) was chosen. Statistical data analysis Due to limited identification success of AMF spores, the molecular spore identification data are presented, but the community composition of AMF spores was not analyzed further. Root-colonizing AMF community composition was analyzed on the basis of number of clones of each phylotype in a root sample. The effects of host plant and sampling site (as independent factors) on AMF richness per root sample and spore density per soil sample were analyzed using twoway ANOVA. Differences in AMF richness, spore density and soil variables between samples were tested using Fisher s least significant difference at the 5% level after one-way ANOVA. All above statistical analyses were performed using the SPSS 13.0 (SPSS Inc., IL). Sampling effort curves of rootcolonizing AMF richness (Mao Tau) were computed using ESTIMATES 8.0 (Colwell, 2006). Before analysis of AMF community composition, raw data of clone counts were square root transformed in order to down-weigh the importance of abundant phylotypes. The dissimilarities of AMF communities among samples were computed by nonmetric multidimensional scaling (NMDS) with Bray Curtis distance using the function isomds in the R PACKAGE VEGAN version (R Development Core Team, 2010). To explore the relationships between environmental variables and AMF community composition, the environmental variables (including data of soil properties, altitude, latitude, longitude, plant species richness and vegetation coverage; all data except the latitude and longitude were square root transformed) were fitted as vectors onto the NMDS plot using the function envfit in the R PACKAGE VEGAN version (R Development Core Team, 2010). We used the online database MaarjAM ( botany.ut.ee; Öpik et al., 2010; status of October 20, 2010) to compare the AMF phylotypes obtained in this study with the AMF detected in other ecosystems. Because the MaarjAM database comprises AMF 18S rrna gene sequences derived from 105 published papers of different ecosystems, we could determine the detection frequencies of our phylotypes in different ecosystems. We first used our sequences to BLAST against the MaarjAM database and grouped our sequences into the corresponding molecular virtual taxa with the sequence identity Z98%, and then searched how many studies had detected the corresponding molecular virtual taxa. Results Spore density and molecular identification of spores AMF spore density varied in soil samples, with the highest and lowest spore density being present in rhizosphere soils of D. heterophyllum ( spores per 100 g soil; mean SE) and A. polycladus ( ) in Xidatan (Table 2). Spore density was different between sampling sites (F = 11.45, P o 0.001), but not between host plant species (F = 3.14, P = 0.09); site and plant species had a significant interaction (F = 36.24, P o 0.001). Amplification of spore-derived DNA was unsuccessful for most, but not all spore morphotypes (Table 2, Table S1). In total, 56 out of 120 single spores that were subjected to molecular analysis (46.7%) were successfully amplified and subjected to RFLP typing (Table 2). A total of 17 RFLP types were detected, and one representative of each spore-derived RFLP type was sequenced. Most spores of the same morphotype shared the same RFLP pattern (data not shown), so that the remaining spores were classified by RFLP typing. A BLAST search showed that eight sequences belonged to Glomeromycota (Fig. 1), and nine sequences were related to Ascomycota, possibly representing sequences of parasitizing ascomycetes in spores (Hijri et al., 2002). Molecular identification of AMF in roots Before molecular analysis, the AM status of the plant species studied was confirmed by root staining and microscopy, showing high levels of root AM colonization: 53.5% and 58.9% of root length was colonized by AMF for

5 Diverse AM fungi in Tibet Plateau 359 Table 2. Spore density and success rate of molecular identification of single spores in rhizosphere soils of each plant species per site Sampling sites Plant species Spore density (spores per 100 g soil) No. of morphotypes in each pooled sample No. of analyzed single spores No. of successfully amplified single spores No. of AMF phylotypes X DH a AP f K DH c AP d W DH ef AP b T DH f AP de A DH de AP de If the second PCR reaction yielded a c. 560-bp product, it was considered as a successful amplification regardless of whether the PCR production was an AMF or a non-amf sequence. Spore densities are shown with mean SE (n = 3). Significant differences of spore densities between samples were tested using Fisher s least significant difference at the 5% level and indicated by different letters. Please note that spore morphotypes were analyzed following pooling of three replicate samples per site (in total of 10 samples; see Materials and methods). Site codes are as in Table 1. DH, Dracocephalum heterophyllum; AP, Astragalus polycladus. D. heterophyllum and A. polycladus, respectively. Both arbuscules (12.8%) and vesicles (10.6%) were observed in the roots of D. heterophyllum; only vesicles (15.7%), but no arbuscules were observed in the roots of A. polycladus. All 30 root samples yielded PCR products of expected length in the first (c. 1.8 kb) and second PCR reactions (c. 560 bp). A total of 1500 clones were screened, and 1464 clones containing insert of correct size were submitted to RFLP typing. One representative clone of each root-derived RFLP type of each sample group (same plant species in same site) was sequenced, yielding a total of 117 sequences. The remaining clones were classified by RFLP typing. BLAST search showed that 75 sequences had high homology to members of Glomeromycota, 17 sequences were related to Ascomycota, one sequence to Basidiomycota, nine sequences to Metazoa and Cercozoa, and 13 sequences to plants. Two sequences appeared to be chimeric. Altogether, 822 AMF clones/sequences (56%) were retained in the further analyses. Phylogenetic analysis Both Bayesian and neighbor-joining phylogenetic analyses of 44 representative AMF sequences from roots and eight spore-derived sequences revealed 21 sequence groups with sequence identity Z98% (Fig. 1), hereafter referred to as phylotypes. Most of the phylotypes had high credibility values and bootstrap support ( 4 80%). Seven phylotypes were related to sequences of described species, six to uncultured AMF and eight phylotypes (Glo-A1, Glo-A6, Glo-A8, Aca-1, Clade-1, Clade-2, Clade-3 and Amb-2) were undescribed previously ( 97% identity with published sequences; Fig. 1). Twenty phylotypes belonged to six families and three orders of Glomeromycota (Fig. 1): nine Glomeraceae A (Glomus group A), two Glomeraceae B (Glomus group B), five Diversisporaceae, one Acaulosporaceae, one Pacisporaceae and two Ambisporaceae phylotypes. The remaining phylotype Clade-1 within order Diversisporales could not be placed in the known families and should be considered as a new family-like clade (Fig. 1). Clade-2 and Clade-3 were highly divergent from the genus Diversispora, but were placed with high credibility values in the family Diversisporaceae (Fig. 1), possibly representing two genera; Clade-2 and Glomus fulvum (AM418543) belong to the recently described genus Redeckera (Schüßler & Walker, 2010); Clade-3 appears to be a new genus. Phylotypes of Glomerales were most abundant in roots, representing approximately 84% of the clones, followed by Diversisporales (13%) and Archaeosporales (3%). Two phylotypes, Glo-B2 (33% of all AMF clones) and Glo-A2 (30%), were most abundant in both plant species and all sampling sites. Specific phylotypes (occurred in one host-site combination) such as Glo-A3, Glo-A4 and Glo-A5 were often rare (Table S2). A total of seven phylotypes were detected from spores, but only two of them (Glo-B1 and Div-2) were also detected in roots (Table 3). Most frequent phylotypes among spores were not found in roots: Pac-1 was detected at three sites mostly from rhizospheres of A. polycladus (Xidatan, Kunlunshan, Amdo) and Div-3 in three sites of D. heterophyllum (Kunlunshan, Wudaoliang, Amdo), respectively (Table S1).

6 360 Y. Liu et al. Fig. 1. Bayesian phylogenetic tree inferred from representative AMF 18S rrna gene sequences identified in this study and reference sequences from GenBank. Numbers above and below the branches are credibility values of Bayesian analysis and the bootstrap values of the neighbor-joining analysis, respectively (values 4 70% are shown). Sequences obtained from roots are shown in bold and coded as follows: the first letter indicates the sampling site (see the site codes in Table 1), the subsequent two letters indicate the host plant (DH: Dracocephalum heterophyllum; AP: Astragalus polycladus). Sequences obtained from single spores are shown in bold and coded with spore-rflp. The GenBank accession numbers of all new sequences are shown in parentheses. Sequence groups (Glo-A1, etc.) identify distinct clusters of sequences with similarity Z98%.

7 Diverse AM fungi in Tibet Plateau 361 Search against the MaarjAM database Thirteen out of 21 AMF phylotypes detected in the present study could be grouped into the corresponding molecular virtual taxa of the MaarjAM database (Table 3). Eight phylotypes showed low similarity (93 97%) to the sequences of this database and those in GenBank ( 97%). The most dominant phylotype in the present study (Glo-B2) has been detected by seven studies in the MaarjAM database (as VT00056, no related species is known, Table 3), but it was often rare in these studies (e.g. Öpik et al., 2009). Phylotype Glo-A2, second most dominant in this study, has been earlier detected in 37 studies in three climatic zones (as VT00113 related to Glomus fasciculatum group), followed by Glo-A9 (VT00065 related to Glomus geosporum group, 15 studies) and Div-2 (VT00062, 14 studies) that were infrequent in our study. Composition and diversity of AMF communities in roots Sampling effort curves clearly indicate that a large proportion of the total AMF diversity colonizing roots was captured for both plant species (Fig. 2). Fifteen AMF phylotypes were detected in the roots of D. heterophyllum, whereas only five phylotypes were detected in the roots of A. polycladus (Fig. 2, Table S2). Both host plant (F = , P o 0.001) and sampling site (F = 36.07, P o 0.001) showed significant Fig. 2. Sampling effort curves (Mao Tau) for AMF phylotypes detected in roots of Dracocephalum heterophyllum and Astragalus polycladus. effects on AMF richness, with significant site and host plant interaction (F = 28.07, P o 0.001). The mean number of AMF phylotypes per root sample of D. heterophyllum ( ; mean SE) was higher than that of A. polycladus ( ). Higher AMF richness was detected in D. heterophyllum roots at Amdo (8 0.58) and Kunlunshan (7 0) as compared with the other three sites (Fig. 3). The AMF richness in A. polycladus roots followed a different pattern: it was highest in Amdo and Tongtianhe (3 0), medium in Xidatan ( ) and lowest in Wudaoliang Table 3. Number of publications in the MaarjAM database ( Öpik et al., 2010) that have earlier reported the AMF phylotypes of this study Clones in roots/from spores Molecular virtual taxon Related species Temperate zone Subtropical zone Tropical zone Subarctic zone Glo-A1 18/- Glo-A2 250/- VT00113 Glomus fasciculatum Glo-A3 5/- VT00064 Glomus constrictum Glo-A4 3/- VT00198 Glomus hoi Glo-A5 3/- VT Glo-A6 8/- Glo-A7 83/- VT Glo-A8 -/1 Glo-A9 -/1 VT00065 Glomus geosporum Glo-B1 49/1 VT00193 Glomus claroideum, G. etunicatum Glo-B2 272/- VT Pac-1 -/7 VT00284 Pacispora scintillans Aca-1 7/- Clade-1 47/- Clade-2 18/- Clade-3 11/- Div-1 8/- VT00060 Diversispora celata, Glomus eburneum Div-2 16/2 VT Div-3 -/4 VT00263 Diversispora spurca Amb-1 27/- VT00283 Ambispora fennica Amb-2 -/1 Codes of molecular virtual taxa (VT) in the MaarjAM database ( Öpik et al., 2010). Total

8 362 Y. Liu et al. 1.0 X K W T A NMDS axis Fig. 3. The richness of AMF phylotypes detected in root samples in each site. Mean richness SE (n = 3) are shown. Abbreviations of sample sites are as in Table 1. Bars with different letters are significantly different at P and Kunlunshan (1 0) (Fig. 3). Taken as a whole, the total number of detected AMF phylotypes in roots was highest in Amdo (11 phylotypes), and lowest in Wudaoliang and Tongtianhe (both four, Table S2). NMDS ordination of AMF communities showed a low stress level (0.09), indicating good representation of phylotype composition. NMDS ordination yielded sample groupings according to host plant (Fig. 4). There was also a trend for samples from each site to cluster together regardless of host plant, except for Kunlunshan and Amdo samples from D. heterophyllum grouping together, and A. polycladus samples grouping with samples from the same host from other sites (Fig. 4). Ten environmental factors fitted as vectors onto the NMDS plot showed that eight factors were significantly correlated with the AMF community (Fig. 4); of these, vegetation coverage was most strongly related to the AMF community composition (r 2 = 0.539, P o 0.001). Environmental factors Soil properties varied among samples (Table S3). In particular, soil available P content, ranging from 0.59 to 2.2 mg kg 1 (Table S3), was affected by sampling site (F = , P o 0.001), but not by host plant species (F = 2.95, P = 0.101), with significant interaction between them (F = 19.40, P o 0.001). Soil available P (r = 0.37, P = 0.05; Pearson s correlation) and C/N ratio (r = 0.44, P = 0.02) were negatively correlated with the vegetation coverage. None of soil variables were correlated with altitude (data not shown). Vegetation coverage showed a positive relationship with plant richness (r = 0.77, P o 0.001) and altitude (r = 0.63, P o 0.001), suggesting that the vegetation coverage was higher in high-altitude sites than in lowaltitude sites of this study NMDS axis-1 Fig. 4. Joint plot of NMDS ordination of AMF communities colonizing roots of different host plant species and the vectors of significant (P 0.05) environmental variables across sites. The length of the arrow is proportional to the strength of correlation between environmental variable and community dissimilarities. Filled symbols: Dracocephalum heterophyllum; open symbols: Astragalus polycladus. Abbreviations of sample sites are as in Table 1. Discussion Diverse and novel AMF are present in the Tibet Plateau Our results show that a relatively high AMF diversity was present in the high-altitude alpine environment of the central Tibet Plateau. A total of 21 AMF phylotypes (from roots and spores) detected in five study sites of this study is close to the number of 23 morphospecies detected in the southern Tibet Plateau (Gai et al., 2009), where the climate is less extreme than at the sites of this study. Although comparison of the morphospecies detected by Gai et al. (2009) and the phylotypes detected in this study is difficult, a clear difference is the lack of Scutellospora in our study, but found by Gai and colleagues. This suggests that there is variation in AMF community composition among locations in the central and southern Tibet Plateau, also seen among sampling sites of this study, indicating even higher numbers of AMF species in this region. Four to 13 AMF phylotypes were detected per study site, which is comparable to those known in temperate natural ecosystems (Öpik et al., 2006, 2010). Our results are also consistent with a study showing that at the level of individual plants, the mean number of AMF taxa detected from plant roots in the low-arctic meadow habitat was not lower than that in temperate ecosystems (Pietikäinen et al., 2007). Eight out of 21 AMF phylotypes detected in this study are not well affiliated to known AMF species or phylotypes.

9 Diverse AM fungi in Tibet Plateau 363 Among these, Clade-1 and Clade-3 are sufficiently divergent that they may represent a new family and a new genus within Diversisporales (Fig. 1). A high proportion of new AMF taxa detected in this study confirms the view that there may be many AMF taxa awaiting discovery (Helgason et al., 2002; Fitter, 2005), in particular in less-studied geographic regions and biomes (Öpik et al., 2010). The most dominant phylotype (Glo-B2) in this study has been infrequently detected in other ecosystems (Table 3), suggesting that this AMF perhaps prefers habitats like those in the present study. On the other hand, the second most abundant phylotype was a globally widespread taxon related to G. fasciculatum/ intraradices group (Glo-A2; Table 3; Öpik et al., 2006, 2010). These findings suggest that the AMF communities in colddominated environments in the Tibet Plateau include some unique taxa and have also some taxa in common with more moderate habitats. The unique taxa in this system may include AMF specialized to this extreme habitat with possibly specific functional properties. For example, Ambispora fennica (Amb-1; Fig. 1), detected in two sites in this study, may prefer cold habitats because this species was first isolated from subarctic region in Finland ( N; Walker et al., 2007) and has been rarely detected in plant roots in other climatic regions (Table 3). AMF richness patterns Our study system revealed a unique pattern: more AMF phylotypes colonized roots of early-successional D. heterophyllum in sites disturbed 30 years ago than roots of A. polycladus in undisturbed vegetation (16 and five phylotypes, respectively). The same trend was apparent at the level of individual root samples, whereby the phylotype number was higher for D. heterophyllum ( per sample) than for A. polycladus ( ). It is generally held that disturbance affects AMF diversity negatively (e.g. Öpik et al., 2006; Tian et al., 2009). Unfortunately, our study system does not allow disentangling the effects of disturbance/ successional stage and host plant, because D. heterophyllum occurs almost exclusively in formerly disturbed vegetation, while A. polycladus occurs only in late-successional vegetation. Therefore, we cannot make firm conclusions about the disturbance effect on the AMF richness. We can still say that there were abundant arbuscules in the roots of D. heterophyllum, but not A. polycladus, suggesting active symbiosis in the first case, but not the latter. Spore density and richness, however, did not differ between vegetation of different disturbance histories, suggesting that there is no effect of differing vegetation composition or disturbance history. At the same time, only two of the AMF phylotypes were detected both from spores and (rarely) in roots, which might mean that different factors influence sporulating and root-colonizing fractions of AMF communities, resulting in different richness estimates. Clearly, more data are needed, using both molecular and morphological methods about ecosystems of different successional stages in this region, and only then can we fully understand the ecological importance and traits of AMF in this unique ecosystem. In fact, natural communities in central Tibet Plateau are dominated by Kobresia sedges that are known to host diverse communities of ectomycorrhizal fungi in Himalayan meadows (Gao & Yang, 2010), but can also exhibit AMF colonization (Gai et al., 2006). Sedges can show quite high AM colonization levels and positive growth response to AMF in extreme habitats such as ultramafic soils (Lagrange et al., 2011). Whether ectomycorrhizal fungi are dominant over AMF in late-successional plant species in these habitats, and AMF predominate in plants of early succession, remains to be clarified in future studies. We found that vegetation coverage was an important factor influencing the AMF community composition. The observed higher AMF richness in sites with higher vegetation coverage suggests that decrease in vegetation coverage could decrease AMF diversity (Tian et al., 2009). Also, other soil factors such as available P and organic C were related with AMF community composition (Fig. 4), in concordance with previous studies, showing that abiotic factors may influence the patterns of AMF communities (Lekberg et al., 2007; Dumbrell et al., 2010). Nonetheless, it is difficult to separate environmental selection from effect of host plants and plant communities on AMF (Schechter & Bruns, 2008), because these organisms are always codetermined by biotic and abiotic factors (Bever et al., 2001). In conclusion, the present study is the first investigation of the molecular diversity of AMF in the Tibet Plateau of China. A high AMF diversity with many new taxa detected in this region suggests that there are unique AMF in cold ecosystems with very high altitude as well as generalist fungi common in many types of ecosystems. AMF communities colonizing roots varied in different plant species, with more AMF phylotypes inhabiting the roots of pioneer plant species than late-successional plant, suggesting that the pioneer plant may be a better host for AMF in the Tibet Plateau. Because the vegetation area in the Tibet Plateau has decreased sharply in recent decades due to anthropogenic disturbances and climate change (Harris, 2010), evaluation of the AMF diversity patterns as well as the ecological traits of AMF in this region is necessary if protection and restoration of degraded regions is to be effective. Acknowledgements We are grateful to Prof. Tuo Chen and Dr Gaosen Zhang from Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, and Prof. Shiweng Li from Lanzhou Jiaotong Unviersity for

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Number of spores of AMF phylotypes detected from rhizospheres of two plant species at five study sites. Table S2. Number of clones of AMF phylotypes detected in the roots of two plant species at five study sites. Table S3. The characteristics of rhizosphere soils of each plant species in each site. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.