Diversity and community assembly of moss-associated fungi in ice-free coastal outcrops of continental Antarctica

Fungal Ecology 24 (2016) 94e101

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Diversity and community assembly of moss-associated fungi in ice-free coastal outcrops of continental Antarctica Dai Hirose a, Satoru Hobara b, Shunsuke Matsuoka c, Kengo Kato d, Yukiko Tanabe d, Masaki Uchida d, Sakae Kudoh d, Takashi Osono e, * a

College of Pharmacy, Nihon University, Funabashi, Chiba, 274-8555, Japan Department of Environmental Symbiotic Sciences, Rakuno Gakuen University, Ebetsu, Hokkaido, 069-8501, Japan Center for Ecological Research, Kyoto University, Otsu, Shiga, 520-2113, Japan d National Institute of Polar Research, Tokyo, 173-8515, Japan e Department of Environmental Systems Science, Faculty of Science and Engineering, Doshisha University, Kyoto, 610-0394, Japan b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 June 2016 Received in revised form 22 September 2016 Accepted 29 September 2016 Available online 29 October 2016

To date, the relative roles of niche-related (e.g., environmental filtering and biological interactions) and non-niche-related (e.g., dispersal limitation) processes in the assembly of fungal communities have rarely been explored in ice-free coastal outcrops of continental Antarctica. Here, using variation partitioning, we show that the community composition of fungi associated with moss colonies sparsely distributed in ice-free coastal outcrops of East Antarctica is affected by a suite of environmental conditions more significantly than by geographic distance, indicating the primary importance of niche-related factors. This was mainly attributable to the widespread occurrence of the predominant, cosmopolitan fungal species, Phoma herbarum. The variance explained by spatial factors increased when the analysis was applied only to the dataset of the Lützow-Holm Bay area (by excluding the data of the Amundsen Bay area, which is 500 km from the Lützow-Holm Bay area), indicating the local importance of non-nicherelated processes. © 2016 Elsevier Ltd and British Mycological Society. All rights reserved.

Corresponding Editor: Kevin K. Newsham Keywords: Antarctica Biogeography Bryophyte Community ecology Dispersal limitation Fungal diversity

1. Introduction Understanding how fungal communities assemble is a fundamental question in fungal ecology. Deterministic theories suggest that niche-based local processes, such as trait-mediated environmental filtering and biotic interactions, largely determine patterns of species diversity and composition (Chase, 2007; Caruso et al., 2011; Ranjard et al., 2013). In contrast, stochastic theories emphasize non-niche processes and the importance of chance colonization, random extinction, and ecological drift (Hubbell, 2001; Sloan et al., 2006; Bell, 2010). Recently, evaluation of the relative roles of deterministic and stochastic spatial processes of community assembly has advanced our understanding and provided new perspectives about the diversity and assembly of fungal communities at local, regional, continental, and global scales (Caruso et al., 2011;

* Corresponding author. E-mail address: [email protected] (T. Osono). http://dx.doi.org/10.1016/j.funeco.2016.09.005 1754-5048/© 2016 Elsevier Ltd and British Mycological Society. All rights reserved.

Hazard et al., 2013; Ranjard et al., 2013; Rodrigues et al., 2013; ~lme et al., 2014). However, little is known Kivlin et al., 2014; Po about the community assembly in ice-free coastal outcrops of continental Antarctica, which comprise only about 2% of the continent, and are both 'cold and arid' and 'distant and isolated', features that impose strong environmental selection pressures and dispersal limitations on the establishment of fungi. Despite the harsh and remote environment, previous studies reported the occurrence of non-lichenized fungi in soils (Bridge and Newsham, 2009; Arenz et al., 2014) and in exotic plant substrates from Antarctica (Hirose et al., 2013). This situation appears to be in accord with the notion that many species of Antarctic fungi are cosmopolitan and have great dispersal potential (Arenz et al., 2014), and with the well-known hypothesis of Baas Becking that 'Everything is everywhere, but the environment selects' (Wit and Bouvier, 2006). A general hypothesis that can be drawn from these notions is that niche-based processes, such as trait-mediated environmental filtering associated with water availability, nitrogen level, and salinity, which characterize the environment of Antarctic terrestrial

D. Hirose et al. / Fungal Ecology 24 (2016) 94e101

systems (Convey et al., 2014), play more crucial roles in the community assembly of Antarctic fungi than do non-niche, spatial processes. However, only a few previous studies have been carried out in the Antarctic Peninsula (and none in continental Antarctica) to test this hypothesis by investigating the community composition of soil fungi at multiple locations along latitudinal and environmental gradients, demonstrating the effects of environmental factors and/or latitude on the fungal communities (Yergeau et al., 2006; Dennis et al., 2012; Newsham et al., 2016). It is still unknown whether geographic patterns of the distribution of fungal communities in continental Antarctica are more strongly related to environmental conditions than to geographic location, or vice versa. The purpose of the present study was to discriminate the relative roles of niche-related (environmental) and non-niche-related (spatial) processes in the spatial variation of fungal communities associated with moss colonies interspersed in coastal outcrops at 41 locations in six regions of continental Antarctica, located within 500 km of each other. Mosses are one of the dominant components of the terrestrial vegetation of Antarctica (Kanda and Inoue, 1994; Ochyra et al., 2008; Cannone et al., 2013), and harbor an array of fungi (Davey and Currah, 2006; Lindo and Gonzalez, 2010; Osono and Trofymow, 2012). Fungi have often been isolated from mosses from locations in Antarctica (McRae and Seppelt, 1999; Tosi et al., 2002; Stevens et al., 2007; Zhang et al., 2013; Yu et al., 2014). Here we assessed the a-diversity of moss-associated fungi and the relative importance of environmental and spatial factors affecting fungal b-diversity by applying variation partitioning (Peres-Neto et al., 2006) to test the hypothesis that niche-based processes, rather than non-niche processes, play dominant roles in the community assembly of fungi in continental Antarctica.

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and dried for 30 min. The stems were then placed on the surface of corn meal agar medium (Nissui Pharmaceutical Co., Ltd, Japan) in 100 mg/l chloramphenicol plates, and then the plates were incubated at 15
C in the dark. The incubated plates were observed microscopically nine times at 1-week intervals. Any fungal hyphae or spores appearing on the plate were isolated, transferred to a plate containing a 1:1 mixture of corn meal agar medium and malt extract agar medium supplemented with yeast extract (Nissui Pharmaceutical Co., Ltd, Japan), incubated, and identified by observing their micromorphological characteristics. 2.3. Molecular analysis of fungi

Samples were collected at 41 locations in six ice-free regions (Fig. 1): five regions in the Lützow-Holm Bay area (East Ongul Island, Langhovde, Skallen, Skarvsnes, and Breidvågnipa) and one in the Mt. Riiser-Larsen region in the Amundsen Bay area, approximately 500 km distant from the Lützow-Holm Bay area, in East Antarctica (66
450 to 69
400 S, 39
260 to 50
410 E, -17 to 267 m a.s.l.). In the study regions, moss colonies are generally restricted in spatial extent and distributed sparsely and distantly separated from each other. Samples were collected during the 51st Japanese Antarctic Research Expedition (JARE-51) from December 2009 to February 2010. At each location, five moss blocks (each 2  2 cm, 2 cm deep from the surface) were collected, making a total of 205 moss blocks. One moss stem (2 cm in length) was chosen from within the moss block with tweezers that had been sterilized with 70% ethanol, preserved in a sterilized vial (volume 1.5 mm3), and stored at 20
C. The remaining moss samples were weighed to measure their fresh weight, preserved in paper bags, and dried at room temperature. They were taken back to the laboratory in Japan and used for the isolation of fungi, identification of moss species, and chemical analyses.

Fungal isolates were transferred to fresh malt extract agar medium overlaid with a cellophane membrane, and genomic DNA was extracted from the mycelia following the modified CTAB method (Matsuda and Hijii, 1999). Molecular analysis was performed according to Tateno et al. (2015). Polymerase chain reactions (PCR) were performed for genomic DNA extracted from mycelia using Quick Taq HS DyeMix (Toyobo, Osaka, Japan). Each PCR reaction contained a 50 ml mixture [21 ml distilled water, 25 ml master mix, 3 ml of c. 0.5 ng/ml template DNA, and 0.5 ml of each primer (final concentration, 0.25 mM)]. To PCR amplify the region including the rDNA ITS and 28S rDNA D1-D2 domain, the primer pair ITS1-F (Gardes and Bruns, 1993) and LR3 (Vilgalys and Hester, 1990) were used. Each DNA fragment was amplified using a PCR thermal cycler (DNA Engine; Bio-Rad, Hercules, CA, USA) using the following thermal cycling schedule: a first cycle of 5 min at 94
C, followed by 35 cycles of 30 s at 94
C, 30 s at 50
C for annealing, and 1 min at 72
C, and a final cycle of 10 min at 72
C. The reaction mixture was then cooled at 4
C for 5 min. PCR products were purified with a QIAquick PCR Purification Kit (QIAGEN, Germany) according to the manufacturer's instructions. Purified PCR products were sequenced by FASMAC Co., Ltd. (Kanagawa, Japan). Sequencing reactions were performed using a GeneAmp PCR System 9700 (Applied Biosystems, USA) using a BigDye Terminator V3.1 Kit (Applied Biosystems), following the protocols supplied by the manufacturer. The fluorescent-labeled fragments were purified from the unincorporated terminators using an ethanol precipitation protocol. The samples were resuspended in formamide and subjected to electrophoresis in an ABI 3730xl sequencer (Applied Biosystems). The rDNA ITS sequences of the fungal isolates were compared with available sequences in the GenBank database using BLASTn searches (Altschul et al., 1997). We defined molecular operational taxonomic units (MOTUs) within the rDNA ITS sequences dataset by clustering the sequences with the BLASTclust program provided online by the Max Planck Institute (http://toolkit.tuebingen.mpg. de/blastclust#) based on 97% sequence similarity and 90% coverage criteria. We made a single count for the occurrence of a MOTU in each moss sample even when more than one isolate was detected. A MOTU was regarded as a major one when it was isolated from more than 3% (i.e. 6) of 205 moss samples tested. The sequences of MOTUs determined in this study were deposited in the DNA Data Bank of Japan (DDBJ) (LC085184eLC085206).

2.2. Isolation of fungi

2.4. Identification and chemical analyses of moss

Fungi were isolated from moss stems with a slightly modified washing method based on that of Osono et al. (2006). Moss stems were washed five times with 10 ml of sterilized 0.005% Aerosol-OT (di-2-ethylhexyl sodium sulfosuccinate) solution and then rinsed with sterilized water three times in a sterile test tube using a vertical type automatic mixer (S-100; Taitec Co., Ltd, Japan). The rinsed stems were transferred to a sterile filter paper in a 9 cm Petri dish

Samples were oven-dried to a constant mass at 40
C to determine oven-dry mass. Moisture content was calculated gravimetrically according to the equations: moisture content (%) ¼ (fresh mass - oven-dry mass)/oven-dry mass  100. Subsamples of moss tissues were observed microscopically to identify moss species. Other subsamples were ground in a laboratory mill to pass through a 0.5mm screen, and used for chemical analyses. Total carbon and

2. Materials and methods 2.1. Study area

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D. Hirose et al. / Fungal Ecology 24 (2016) 94e101

Fig. 1. Locations of the ice-free coastal outcrops from which the moss samples were collected.

nitrogen contents were measured by automatic gas chromatography (NC analyzer SUMIGRAPH NC-900, Sumitomo Chemical Co., Ltd, Osaka, Japan). The electrical conductivity (EC) was measured using an EC meter (ES-51, HORIBA, Ltd., Kyoto, Japan). A 0.1 g moss sample was mixed with 30 ml Milli-Q water in a plastic container. After shaking for 1 h, EC was determined by submerging the probe of the meter into the mixture. 2.5. Statistical analysis Rarefaction analysis was performed to examine the effect of the

number of moss samples on the number of MOTUs isolated (Colwell et al., 2012) and to compute three diversity statistics [incidence coverage-based estimator of species richness (ICE), Chao 1 richness estimator, and first-order Jackknife richness estimator (Jack1)]. This analysis was performed with EstimateS 9.1.0 (http:// www. http://viceroy.eeb.uconn.edu/EstimateS/). We used variation partitioning based on the distance-based redundancy analysis (db-RDA) to quantify the contribution of the environmental and spatial variables to the community structure of microfungi. The relative weight of each fraction (pure fractions, shared fractions, and unknown fractions) was estimated following

D. Hirose et al. / Fungal Ecology 24 (2016) 94e101

the methodology described by Peres-Neto et al. (2006). Presence/ absence data of fungal MOTUs for each moss sample were converted into a dissimilarity matrix using Simpson's index. We differentiated total dissimilarities into turnover and nestedness components, and this differentiation indicated that the turnover was responsible for most of the dissimilarity. We then constructed environmental and spatial models. First, we constructed an environmental model by applying the forward selection procedure (999 permutations with an alpha criterion ¼ 0.05) of Blanchet et al. (2008), using six environmental variables (elevation, moss species, water content, total carbon and nitrogen content, and electric conductivity). Then, we constructed a model using spatial variables extracted based on the principal components of neighbor matrices (PCNM, Borcard et al., 2004). The PCNM analysis produced a set of orthogonal variables derived from the geographical coordinates of the sampling locations (i.e., the position of sampling grids). We used the PCNM vectors that best accounted for autocorrelation and then conducted forward selection (999 permutations with an alpha criterion of 0.05) to select spatial variables that significantly influenced community dissimilarities. Based on these two models, we performed variation partitioning by calculating adjusted R2 values for each fraction (Peres-Neto et al., 2006). The analyses were performed using the ‘capscale’ command in the R package vegan. Logistic regressions were used to evaluate the effects of environmental factors on the presence/absence of fungal MOTUs, using JMP 6.0 for Macintosh.

3. Results 3.1. Fungal communities and a-diversity A total of 289 isolates were obtained from 185 (90%) of the 205 moss samples from 41 sites, equating to zero to five isolates (1.4 isolates on average) per sample. These fungal isolates were classified into 23 MOTUs (Table 1). The most frequent MOTU was Phoma herbarum, accounting for 70% (143) of the 205 samples, followed by

97

Alternaria sp. (20%, 41/205), Phaeosphaeria sp.2 (13%, 27/205), and Tetracladium sp. (5%, 11/205). The moss samples from six RiiserLarsen sites in the Amundsen Bay area, which are located approximately 500 km from the Lützow-Holm Bay area, harbored P. herbarum at a higher frequency, accounting for 68% of all the isolates, than the moss samples in the Lützow-Holm Bay area (49%) (Table 2). These four MOTUs, along with Leotiomycetes sp.1, Cladosporium sp., Dothideomycetes sp., Ascomycota sp., and Cladophialophora minutissima were regarded as major MOTUs. Rarefaction analyses indicated that this estimate of 23 MOTUs was an underestimation of the MOTU richness of fungi, with richness estimators (ICE, Chao2, and Jack1) predicting MOTU richness of 33e45 (Fig. 2). This underestimation was attributed to the high proportion of singleton MOTUs (i.e., 10 out of 23).

3.2. Variation partitioning When the data of all fungi from all sites (in the Lützow-Holm Bay area and Amundsen Bay area) were analyzed (Fig. 3a), the percentages of variation in the composition of fungal communities explained by the pure environmental (niche-related) and pure spatial (non-niche-related) fractions were 13.7% and 1.6%, respectively. The environmental factors selected included moss species and water content, total nitrogen content, and electric conductivity of moss tissues. The shared fraction between environmental and spatial variables explained 2.4% in the variation of fungal communities. The high frequency of the dominant fungal MOTU (P. herbarum) in the remote Amundsen Bay area might have masked possible effects of spatial fractions. When we analyzed the data of all fungi from the sites of the Lützow-Holm Bay area, and excluded the data from the Amundsen Bay area, the percentages explained by the spatial and shared fractions increased to 4.7% and 9.0%, respectively (Fig. 3b). This result indicated the local geographic patterns of fungal occurrence in the Lützow-Holm Bay area. In contrast, the percentage explained by the environmental fraction decreased to

Table 1 List of 23 molecular taxonomic units (MOTUs) isolated from 205 moss samples with their accession numbers, number of sample isolated, and BLAST search results. QC, query coverage; SS, sequence similarity. See text for major fungi. MOTU

Major fungi Phoma herbarum Alternaria sp. Phaeosphaeria sp.2 Tetracladium sp. Leotiomycetes sp.1 Cladosporium sp. Dothideomycetes sp. Ascomycota sp. Cladophialophora minutissima Other fungi Leotiomycetes sp.6 Pseudogymnoascus pannorum Cadophora malorum Leotiomycetes sp.2 Leotiomycetes sp.3 Leotiomycetes sp.7 Leotiomycetes sp.5 Pleosporales sp.1 Phaeosphaeria sp.3 Cosmospora sp. Phaeosphaeria sp.1 Leotiomycetes sp.4 Helotiales sp. Pleosporales sp.2

Accession number

Number of sample isolated

BLAST Best identified BLAST match taxa

Accession number

QC (%)

SS (%)

LC085205 LC085184 LC085194 LC085206 LC085198 LC085190 LC085195 LC085191 LC085188

143 41 27 11 10 9 8 8 7

Phoma herbarum Alternaria chlamydosporigena Phaeosphaeria typharum Tetracladium sp. Thelebolus microsporus Cladosporium gossypiicola Dothideomycetes sp. Ascomycota sp. Cladophialophora minutissima

AY337712 KC584231 KF251192 JX171194 KM822751 AF393702 AB752248 HQ607949 EF016382

100 95 99 99 100 99 99 86 72

99 99 98 97 99 99 98 90 99

LC085203 LC085196 LC085187 LC085199 LC085200 LC085197 LC085202 LC085186 LC085204 LC085189 LC085193 LC085201 LC085185 LC085192

5 5 3 2 1 1 1 1 1 1 1 1 1 1

Helotiales sp. Pseudogymnoascus pannorum Cadophora malorum Leuconeurospora sp. Leotiomycetes sp. Leohumicola minima Helotiales sp. Leptosphaeria doliolum Phaeosphaeria sp. Cosmospora viridescens Phaeosphaeria juncophila Helotiales sp. Varicosporium elodeae Leptosphaeria microscopica

JX852359 KP411572 JQ796752 JQ857040 JQ759534 NR121307 JX852359 JF740205 JX171188 KJ676171 AF439488 JX852359 JN995640 FN386274

95 99 100 99 100 53 95 94 100 99 92 95 96 97

95 100 99 99 96 90 96 96 100 99 98 93 99 99

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D. Hirose et al. / Fungal Ecology 24 (2016) 94e101

Table 2 Frequencies of occurrence (%) of major fungal MOTUs at six ice-free coastal outcrops. Frequency of occurrence was the proportion of moss samples from which a fungal MOTU occurred with respect to the total number of moss samples examined at each coastal outcrop (given in parentheses). A total of 205 moss samples was examined.

Phoma herbarum Alternaria sp. Phaeosphaeria sp.2 Tetracladium sp. Leotiomycetes sp.1 Cladosporium sp. Dothideomycetes sp. Ascomycota sp. Cladophialophora minutissima

Number of samples

Ongul islands (10)

Langhovde (75)

Breidvågnipa (5)

Skarvsnes (70)

Skallen (15)

Riiser-Larsen (30)

143 41 27 11 10 9 8 8 7

70.0 0.0 40.0 0.0 0.0 0.0 0.0 0.0 10.0

77.3 40.0 14.7 2.7 9.3 8.0 5.3 2.7 2.7

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 20.0

68.6 10.0 14.3 1.4 4.3 0.0 1.4 4.3 2.9

20.0 26.7 13.3 53.3 0.0 0.0 0.0 20.0 0.0

90.0 0.0 0.0 0.0 0.0 10.0 10.0 0.0 3.3

10.0% (Fig. 3b). The environmental factors selected included elevation, moss species, and total nitrogen content and electric conductivity of moss tissues. Using the data of the nine major fungal OTUs and excluding the data of minor ones resulted in a slight increase in the percentage of variation explained by the pure environmental, shared, and pure spatial fractions (Fig. 3c and d). The overall patterns of the relative importance of environmental and spatial fractions for the major fungi were similar to those obtained in the analysis of the data of all fungi from all sites (Fig. 3a) or from the Lützow-Holm Bay area alone (Fig. 3b).

with the B. pseudotriquetrum/B. archangelicum complex than with the other moss species, and was positively related with the water content of moss tissues. Tetracladium sp. was significantly more strongly associated with Hennediella heimii, was negatively related to the elevation, and was positively related with the electric conductivity of moss tissues. Cladosporium sp. was positively related with the total nitrogen content and electric conductivity of moss tissues. The Ascomycota sp. was negatively related with elevation and total nitrogen content of moss tissues. C. minutissima was negatively related with elevation and was significantly more strongly associated with C. purpureus than with the other moss species.

3.3. Effect of environmental factors 3.4. Geographic patterns Fungal MOTUs responded differently according to the environmental conditions, as shown by logistic regression analyses to evaluate the effects of environmental factors on the occurrence of MOTUs (Table 3). P. herbarum was significantly more strongly associated with the Bryum pseudotriquetrum/Bryum archangelicum complex and Ceratodon purpureus than with the other moss species, and was positively related to the total nitrogen content of moss tissues. Alternaria sp. was significantly more strongly associated

50

4. Discussion

40 Number of MOTUs

To obtain further insights into the local geographic patterns of fungal occurrence, the frequency of occurrence of major fungal MOTUs was calculated for each of the six ice-free coastal outcrops (Table 2). P. herbarum occurred at five out of the six regions with relatively frequent occurrence, whereas Alternaria sp., Phaeosphaeria sp.2, Tetracladium sp., and Ascomycota sp. occurred more frequently at Langhovde, Ongul Island, and/or Skallen, than at the other regions.

30 20 10 0 0

50 100 150 Number of samples

200

Fig. 2. Rarefaction curve showing the effect of number of moss samples on the number of fungal MOTUs. Thick line, observed species richness; thin line, estimated richness obtained using ICE; gray line, estimated richness obtained using Chao2; broken line, estimated richness obtained using Jack1.

Our results explicitly show that the community assembly of moss-associated fungi is regulated by a suite of environmental factors more significantly than by geographic distance, indicating the primary importance of niche processes (Fig. 3). This was primarily attributable to the widespread occurrence of the dominant fungus, P. herbarum, which made the spatial pattern less significant. This fungus was especially dominant at the Mt. Riiser-Larsen region in the Amundsen Bay area (Table 2), approximately 500 km from the other coastal outcrops in the Lützow-Holm Bay area (Fig. 1). In fact, P. herbarum has a worldwide distribution, not only in Antarctica (McRae and Seppelt, 1999; Tosi et al., 2002), but also in temperate regions of the Southern and Northern Hemispheres, the Tropics, and the Arctic (Ananda and Sridhar, 2002; Domsch et al., 2007; Leung et al., 2011). This fungus is highly dispersed and possesses adaptive attributes toward environmental stresses in Antarctica, such as low temperature, desiccation, and salinity, as well as ultraviolet insolation (Selbmann et al., 2002, 2005; Hughes et al., 2003). Environmental factors (i.e., niche-related processes) affected the occurrence of moss-associated fungi in continental Antarctica more strongly than spatial factors (Fig. 3). Indeed, each of the major fungal MOTUs responded differently to such environmental factors as elevation, moss species, and water content, total nitrogen content, and electric conductivity of moss tissues (Table 3). Various degrees of host specificity have been found for fungi associated

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the Lützow-Holm Bay area, the percentages explained by the spatial and shared fractions increased, whereas that explained by the environmental fraction decreased (Fig. 3), indicating the importance of local geographical patterns within the Lützow-Holm Bay area. Indeed, the spatial patterns of occurrence of some fungal MOTUs were limited to particular ice-free coastal outcrops in the Lützow-Holm Bay area (Table 2), suggesting the role of non-niche processes, such as dispersal limitation (i.e., not every fungus was necessarily everywhere). These fungi have possibly immigrated, by chance and/or only recently, to particular parts of continental Antarctica and have not yet fully expanded to nearby ice-free regions. Arenz et al. (2014) noted that fungal vectoring could occur over long distances due to atmospheric circulation and animal immigrations. The frequent occurrence of Phaeosphaeria sp.2 on the Ongul Islands (Table 2) suggested the possibility of human introduction associated with the Japanese research expeditions since 1957, although possible pathways and vectors of such immigration are unknown. It is speculated that these fungal MOTUs may rarely produce effective dispersal agents in their Antarctic habitats, such as sexual and asexual spores, due to harsh environmental conditions (Arenz et al., 2014). Unfortunately, the lack of taxonomic information for these fungi hinders further discussion of their reproductive ecology and biogeography. The number of fungal MOTUs in the present study (i.e., 23) was within the range previously reported from continental Antarctica. For example, previous studies reported that the richness of fungi associated with Antarctic moss tissues ranged from 10 to 28 species (McRae and Seppelt, 1999; Tosi et al., 2002; Yu et al., 2014). Hirose et al. (2013) reported 18 MOTUs for 43 isolates obtained from 41 dead shoots of Salix spp. transplanted and in ground contact near Syowa Station, East Antarctica. The estimated MOTUs richness outnumbered that observed here, due to the high proportion (61% of the 23 MOTUs) of minor MOTUs, including singletons. Of the other minor fungi, Pseudogymnoascus pannorum was previously isolated from B. pseudotriquetrum, C. purpureus, and some other moss species in Victoria Island (Tosi et al., 2002) and also from two High Arctic moss species (Osono et al., 2012). This fungus is known to be keratinophilic (Hayes, 2012), but there was no obvious evidence in the present study regarding an association of the occurrence of P. pannorum with such substrates as feathers or animal corpses.

Fig. 3. Relative effects of environmental and spatial factors on the community assembly of fungi associated with moss in the ice-free coastal outcrops of continental Antarctica as shown by multivariate variance partitioning. Blank, pure environment; gray, shared component with environment and space; black, pure space. Four datasets were analyzed: (a) data of all MOTUs from all sites, (b) data of all MOTUs from sites of the Lützow-Holm Bay area, (c) data of major MOTUs from all sites, (d) data of major MOTUs from sites of the Lützow-Holm Bay area. See the main text for major MOTUs. Environmental factors that had significant effect included: moss species, water content, total nitrogen content, and electric conductivity in (a) and (c), and elevation, moss species, total nitrogen content, and electric conductivity in (b) and (d).

€ bbeler, 1997; Kauserud et al., 2008), consistent with mosses (Do with the finding of the present study that the occurrence of four fungal MOTUs was significantly affected by moss species (Table 3). Previous studies obtained rather inconsistent results about the effects of environmental conditions on fungi. For example, McRae and Seppelt (1999) found no relationship between the water content of mosses and the fungi recovered from them in the Windmill Islands. Arenz and Blanchette (2011) demonstrated that fungal abundance (expressed as colony forming units) was positively correlated with total carbon content in Antarctic soils, and suggested that the presence of primary producers strongly influenced the fungal diversity. In the present study, we found no significant effect of total carbon content, indicating that organic carbon was not limiting the colonization of fungi in moss tissues. Interestingly, Tetracladium sp. and Cladosporium sp. exhibited a positive response to electric conductivity (i.e., salinity), implying adaptations to and advantages under osmotic stress (Ruisi et al., 2007). When variation partitioning was applied only to the dataset of

5. Conclusion To the knowledge of the authors, this is the first study to demonstrate the relative importance of environmental and spatial factors affecting the community assemblage of fungi in continental Antarctica. Certain niche-based processes were of relatively high importance, such as trait-mediated environmental filtering

Table 3 Effects of environmental factors on the occurrence of major fungal MOTUs. Logistic regressions were applied to the occurrence data of individual fungal MOTUs. Values indicate c2.***P < 0.001,**P < 0.01,*P < 0.05, ns non significant. (þ) and () indicate positive and negative relationship, respectively. Bp, Cp, and Hh indicate that the fungal MOTU had positive association with Bryum pseudotriquetrum/B. archangelicum complex, Ceratodon purpureus, and Hennediella heimii, respectively. Elevation Phoma herbarum Alternaria sp. Phaeosphaeria sp.2 Tetracladium sp. Leotiomycetes sp.1 Cladosporium sp. Dothideomycetes sp. Ascomycota sp. Cladophialophora minutissima

0.05 2.55 1.16 9.29 1.85 1.71 0.87 10.66 4.49

Moss species ns ns ns ** ns ns ns ** *

()

() (þ)

21.71 13.10 2.82 18.55 3.03 5.51 6.60 8.92 20.13

*** * ns ** ns ns ns ns **

Water content (Bp,Cp) (Bp) (Hh)

(Cp)

3.46 4.80 1.63 0.54 0.48 0.43 0.05 2.72 0.51

ns * ns ns ns ns ns ns ns

Total nitrogen content (þ)

5.72 0.32 0.62 1.63 0.03 4.91 0.02 5.11 2.05

* ns ns ns ns * ns * ns

Electric conductivity (þ)

(þ) ()

0.00 0.03 0.36 4.60 0.19 3.92 0.01 0.13 2.05

ns ns ns * ns * ns ns ns

(þ) (þ)

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associated with water availability, nitrogen level, and salinity, which characterize the environment of Antarctic terrestrial systems (Convey et al., 2014) and affect biotic interactions with moss species. The importance of non-niche, stochastic processes was also notable in the Lützow-Holm Bay area, possibly associated with occasional immigration and/or dispersal limitation. The functional role of these moss-associated fungi in decomposition and nutrient recycling will be investigated in another publication by the authors (Hirose et al., 2013). Future studies will be needed to expand the geographic range of the study area to maritime and sub-Antarctic regions, nearby continents in the southern hemisphere, and other parts of the globe, to obtain further insights into the scale of spatial patterns and the environmental constraints on fungal community assembly, the flow of propagules and genotype and evolution of fungal species in Antarctica, and the effects of human introduction on Antarctic fungal communities. Acknowledgments We thank Dr. Y. Motoyoshi and members of JARE-51 for their assistance during the expedition; Dr. H. Kanda and Dr. A.S. Mori for useful discussions; and Dr. Elizabeth Nakajima for critical reading of the manuscript. This study was partially supported by the National Institute of Polar Research through General Collaboration Projects no.26e28 to T.O. and by a JSPS KAKENHI Grant (No. 70370096 to M.U. and No. 15K07480 to T.O.). Two anonymous reviewers supplied helpful comments on the manuscript. References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J.H., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389e3402. Ananda, K., Sridhar, K.R., 2002. Diversity of endophytic fungi in the roots of mangrove species on the west coast of India. Can. J. Microbiol. 48, 871e878. Arenz, B.E., Blanchette, R.A., 2011. Distribution and abundance of soil fungi in Antarctica at sites on the Peninsula, ross sea region and McMurdo dry valley. Soil Biol. Biochem. 43, 308e315. Arenz, B.E., Blanchette, R.A., Farrell, R.L., 2014. Fungal diversity in Antarctic soils. In: Cowan, D.A. (Ed.), Antarctic Terrestrial Microbiology. Springer-Verlag, Berlin Heiderberg, pp. 35e53. Bell, T., 2010. Experimental tests of the bacterial distance-decay relationship. ISME J. 4, 1357e1365. Blanchet, F.G., Legendre, P., Borcard, D., 2008. Forward selection of explanatory variables. Ecology 89, 2623e2632. Borcard, D., Legendre, P., Avois-Jacquet, C., Tuomisto, H., 2004. Dissecting the spatial structure of ecological data at multiple scales. Ecology 85, 1826e1832. Bridge, P.D., Newsham, K.K., 2009. Soil fungal community composition at Mars Oasis, a southern maritime Antarctic site, assessed by PCR amplification and cloning. Fungal Ecol. 2, 66e74. Cannone, N., Convey, P., Guglielmin, M., 2013. Diversity trends of bryophytes in continental Antarctica. Polar Biol. 36, 259e271. Caruso, T., Chan, Y., Lacap, D.C., Lau, M.C.Y., McKay, C.P., Pointing, S.B., 2011. Stochastic and deterministic processes interact in the assembly of desert microbial communities on a global scale. ISME J. 5, 1406e1413. Chase, J.M., 2007. Drought mediates the importance of stochastic community assembly. Proc. Natl. Acad. Sci. U. S. A. 104, 17430e17434. Colwell, R.K., Chao, A., Gotelli, N.J., Lin, S.Y., Mao, C.X., Chazdon, R.L., Longino, J.T., 2012. Models and estimators linking individual-based and sample-based rarefaction, extrapolation, and comparison of assemblages. J. Plant Ecol. 5, 3e21. Convey, P., Chown, S.L., Clarke, A., Barnes, D.K.A., Bokhorst, S., Cummings, V., Ducklow, H.W., Frati, F., Allan Green, T.G., Gordon, S., Griffiths, H.J., HowardWilliams, C., Huiskes, A.H.L., Laybourn-Parry, J., Lyons, W.B., McMinn, A., Morley, S.A., Peck, L.S., Quesada, A., Robinson, S.A., Schiaparelli, S., Wall, D.H., 2014. The spatial structure of Antarctic biodiversity. Ecol. Monogr. 84, 203e244. Davey, M.L., Currah, R.S., 2006. Interactions between mosses (Bryophyta) and fungi. Can. J. Bot. 84, 1509e1519. Dennis, P.G., Rushton, S.P., Newsham, K.K., Lauducina, V.A., Ord, V.J., Daniell, T.J., O'Donnell, A.G., Hopkins, D.W., 2012. Soil fungal community composition does not alter along a latitudinal gradient through the maritime and sub-Antarctic. Fungal Ecol. 5, 403e408. € bbeler, P., 1997. Biodiversity of bryophilous ascomycetes. Biodivers. Conserv. 6, Do 721e738. Domsch, K.H., Gams, W., Anderson, T.H., 2007. Compendium of Soil Fungi, second ed. IHW-Verlag, Eching.

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