Yeast communities in Sphagnum phyllosphere along the temperaturemoisture ecocline in the boreal forestswamp ecosystem and description of Candida sphagnicola sp. nov. Aleksey V. Kachalkin & Andrey M. Yurkov Antonie van Leeuwenhoek Journal of Microbiology ISSN 0003-6072 Volume 102 Number 1 Antonie van Leeuwenhoek (2012) 102:29-43 DOI 10.1007/s10482-012-9710-6 1 23 Your article is protected by copyright and all rights are held exclusively by Springer Science+Business Media B.V.. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your work, please use the accepted author’s version for posting to your own website or your institution’s repository. You may further deposit the accepted author’s version on a funder’s repository at a funder’s request, provided it is not made publicly available until 12 months after publication. 1 23 Author's personal copy Antonie van Leeuwenhoek (2012) 102:29–43 DOI 10.1007/s10482-012-9710-6 ORIGINAL PAPER Yeast communities in Sphagnum phyllosphere along the temperature-moisture ecocline in the boreal forest-swamp ecosystem and description of Candida sphagnicola sp. nov. Aleksey V. Kachalkin • Andrey M. Yurkov Received: 12 October 2011 / Accepted: 3 February 2012 / Published online: 14 February 2012 Ó Springer Science+Business Media B.V. 2012 Abstract The effects of the temperature-moisture factors on the phylloplane yeast communities inhabiting Sphagnum mosses were studied along the transition from a boreal forest to a swamp biotope at the Central Forest State Biosphere Reserve (Tver region, Russia). We tested the hypothesis that microclimatic parameters affect yeast community composition and structure even on a rather small spatial scale. Using a conventional plating technique we isolated and identified by molecular methods a total of 15 species of yeasts. Total yeast counts and species richness values did not depend on environmental factors, although yeast community composition and structure did. On average, Sphagnum in the swamp biotope supported a more evenly structured yeast community. Relative abundance of ascomycetous yeasts was significantly higher on swamp moss. Rhodotorula mucilaginosa dominated in the spruce forest and Cryptococcus magnus was more abundant in the swamp. Our study confirmed the low occurrence of tremellaceous yeasts in the Sphagnum phyllosphere. Of the few isolated ascomycetous yeast and yeast-like species, some were differentiated from hitherto known species in physiological tests and phylogenetic analyses. We describe one of them as Candida sphagnicola and designate KBP Y-3887T (=CBS 11774T = VKPM Y-3566T = MUCL 53590T) as the type strain. The new species was registered in MycoBank under MB 563443. Keywords New yeast species
Sphagnum
Peatland
Epiphytes
Molecular systematics
Candida A. V. Kachalkin and A. M. Yurkov contributed equally to the manuscript. Introduction Electronic supplementary material The online version of this article (doi:10.1007/s10482-012-9710-6) contains supplementary material, which is available to authorized users. A. V. Kachalkin (&) Faculty of Soil Science, Lomonosov Moscow State University, Leninskie Gory, 119991 Moscow, Russia e-mail: kachalkin_a@mail.ru A. M. Yurkov Geobotanik, Fakultät für Biologie und Biotechnologie, Ruhr-Universität Bochum, Universitätsstraße 150, 44801 Bochum, Germany e-mail: andrey.yurkov@rub.de Plant surfaces harbour numerous and diverse microbial communities in which yeasts are among the most frequent members (Fonseca and Inacio 2006). Leaf topography and frequently fluctuating physical properties result in a multitude of diverse habitats of different scales and properties (Andrews and Harris 2000; Morris 2001; Fonseca and Inacio 2006). Besides microclimatic conditions, plant age, leaf morphology, and amount of nutrients influence establishment of yeast population on leaves and account for the 123 Author's personal copy 30 extraordinary diversity of phylloplane communities (Fonseca and Inacio 2006; Nix-Stohr et al. 2008). Nevertheless, studies available to this date report yeasts of the genera Cryptococcus, Sporobolomyces, and Rhodotorula dominating on various plant species worldwide suggesting, thus, low specificity of phylloplane yeasts towards the plant species (Fonseca and Inacio 2006; Inacio et al. 2010). Although yeasts inhabiting the phyllosphere were studied intensively, the diversity and distribution of yeasts in the phylloplane of non-vascular plants such as Sphagnum moss received little attention so far (Fonseca and Inacio 2006). Unlike vascular plants, which release a variety of sugars, alcohols and amino acids, the Sphagnum phyllosphere is enriched with a large spectrum of complex organic acids and phenolic compounds (Rasmussen et al. 1995, Rydin et al. 2006; Montenegro et al. 2009). Consequently, yeast communities developing on Sphagnum mosses differ considerably from those found on higher plants (e.g., Kachalkin et al. 2008). In addition to the specific chemical composition of exudates, Sphagnum species shape microbial communities through increased water holding capacity, lower temperature, acidification and nitrogen immobilization in the environment (van Breemen 1995; Painter 1998; Maksimova and Chernov 2004; Kachalkin et al. 2008). These effects, in turn, significantly influence ecosystem processes such as decomposition and nutrient turnover in forest litter (e.g., Wardle et al. 2003). Thereby, Sphagnum mosses are well-recognised edificatory plant species in oligotrophic lowlands but also within the boreal lower storey. Sphagnum mosses are widespread and since a large proportion of the boreal peatlands is dominated by Sphagnum species, there might be as much as 1.5 9 106 km2 of Sphagnum cover in these habitats (Rydin et al. 2006). Thereby, Sphagnum mosses constitute a substantial part of terrestrial leaf surface area. Despite this fact, our knowledge about yeasts associated with Sphagnum species is limited to a few studies surveyed phyllosphere (e.g., Maksimova and Chernov 2004; Kachalkin et al. 2008) and those ones analysed peat layer rather than living moss parts (reviewed by Thormann and Rice 2007). Variability in yeast population sizes among plants and leaves, is sometimes correlated with leaf position in the canopy, exposure and age (Fonseca and Inacio 2006). Similarly, yeast community composition and structure might vary markedly between experimental sites reflecting local properties such as temperature and 123 Antonie van Leeuwenhoek (2012) 102:29–43 humidity (e.g., Inácio et al. 2002). The ultimate importance of the environmental factors for microbial species dispersal and development, often referring back to the idea of Beijerinck (1913) and Baas Becking (1934) that ‘‘everything is everywhere, but the environment selects’’, is now questionable as distinct biogeographical patterns have been demonstrated for bacteria (Whitaker 2006) and fungi (Taylor et al. 2006), including yeasts (Lachance et al. 2001; Liti et al. 2009). Nevertheless, the mechanisms that determine the distribution of microorganisms in their natural habitats are still poorly understood. This is partly due to the complexity of systems such as soil and the phyllosphere (see also Botha 2006; Fonseca and Inacio 2006; Meyer and Leveau 2011), which limits our ability to uncover mechanisms influencing abundance, composition and structure of microbial communities (Herrera et al. 2010). Consequently, a few model substrates, namely decaying tissues of cacti, floral nectar, tree fluxes, fruits and insects, were used to study influence of spatial (Lachance et al. 2001; Ganter et al. 2004; Libkind et al. 2007; Maksimova et al. 2009) and environmental (Herrera et al. 2010) factors on yeasts. In phylloplane, exposure, water and nutrient availability are key limiting factors (Fonseca and Inacio 2006; Whipps et al. 2008). Although there is experimental evidence that the total yeast population is limited by nutrients and water availability (Andrews and Harris 2000; Fonseca and Inacio 2006; Nix-Stohr et al. 2008), little is known about shifts in yeast community composition and structure associated with these environmental parameters. Habitat heterogeneity driven by light and moisture is difficult to assess on a particular species of vascular plant as its distribution is often restricted to a certain ecological optimum or biome. Sphagnum mosses are widespread in the boreal zone and can be simultaneously found in open wetlands and in a dark boreal forest bed. Thereby, analysis of Sphagnum phyllosphere enables the assessment of microbial community responses to microclimatic parameters since that the effects of a particular plant species on the microbial population could be considerably minimised. Finally, Sphagnum moss provides rather extreme but stable environmental conditions that render this habitat a good model for assessing the influence of ecological factors such as light exposure and precipitation ratio on microbial population in the phylloplane. Here, we report results of a study which was aimed at characterising the effects of gradual changes in Author's personal copy Antonie van Leeuwenhoek (2012) 102:29–43 vegetation cover along a temperature-moisture gradient on the yeast community in the phyllosphere of Sphagnum mosses. The goal of this investigation was to study abundance, diversity, composition and structure of the yeast communities in an ecocline (sensu Whittaker 1960), a transition zone between two biotopes, a boreal spruce forest and an oligotrophic swamp. We hypothesize that alteration of the microclimatic parameters like substrate moisture and light exposure affects the distribution of phylloplane yeasts even on a rather small spatial scale. We test the observation of Kachalkin et al. (2008) regarding the specificity of yeast inhabiting the phyllosphere of Sphagnum species. With this study, we increase our knowledge about yeasts associated with the moss layer and report isolation of a novel ascomycetous yeasts. This species is described here as Candida sphagnicola. Materials and methods Study sites and sampling The study was performed in the Central Forest State Biosphere Reserve (www.clgz.ru), Tver region, Russia (approximate coordinates: 56.48°N, 32.92°E) in June Forest biotope 31 2008. In general, this area is characterized by low environmental pollution and land management. About 47% of the territory is covered with primeval spruce forests that have never been logged or managed. Since 1985 the Central Forest State Biosphere Reserve is included into UNESCO Man and the Biosphere Program (MAB), which is intended to promote and be an example of optimal relationship between man and nature (see: www.unesco.ru/en/?module=news&action= theme&id=101). Sphagnum mosses (green parts) were sampled every 2 m along a transect line across an area associated with the gradual transition from a coniferous forest to a swamp biotope (Fig. 1). This gradient is additionally accompanied by an alteration of the primary vegetation, Sphagnum moisture, and insolation, i.e., amount of the solar radiation. The analysed forest biotope consisted of a Norway spruce–blueberry plant association (Picea abies and Vaccinium myrtillus) with some Scots pines (Pinus sylvestris) and mossy (Sphagnum girgensohnii) ground cover. The transition from forest to swamp biotope was sharp and characterised by a change in primary vegetation, specifically, more occasional and suppressed growth of spruce and birch (Betula pubescens), together with the clear dominance of Tussock cottongrass Swamp biotope 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 Distance, m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Sampling points Fig. 1 Schematic representation of the study area, sampling sites and distances between them 123 Author's personal copy 32 Antonie van Leeuwenhoek (2012) 102:29–43 (Eriophorum vaginatum) in the ground cover. The mosses Sphagnum angustifolium and Polytrichum commune constituted the main moss vegetation in the swamp and built a thick peat layer. A total of 40 sites were sampled at both biotopes and a composite sample (approx. 100 g of Sphagnum) was collected at each sampling site. Moisture content of Sphagnum moss was measured gravimetrically for each sample and was calculated as the proportion of the original (wet) Sphagnum moss weight to the dry weight. Insolation was assessed qualitatively through the estimation of the foliage projective cover. in the molecular studies by taking one to three isolates of each phenotypic group from each sample. PCRfingerprinting with microsatellite-specific oligonucleotides were used to group pure cultures. Strains showing identical electrophoretic profiles were considered as conspecific and only 1–2 representatives of them were chosen for further identification by sequencing of rDNA regions. Other details on oligonucleotide primer sequences and PCR conditions are given by Yurkov et al. (2012). Isolation of cultures Yeast cultures were identified using nucleotide sequences of the three regions from the nuclear ribosomal DNA cistron, the small subunit (18S or SSU) rDNA, the internal transcribed spacer (ITS) region and the D1/D2 domains of the large subunit (26S/28S or LSU) rDNA. Protocols describing DNA extraction, amplification, purification and sequencing are given by Glushakova et al. (2010) and Yurkov et al. (2012). For species identification the nucleotide sequences were compared with sequences deposited in the NCBI (www.ncbi.nih.gov) and CBS (www.cbs.knaw.nl) databases, respectively. Nucleotide sequences were deposited in GenBank under the accession numbers given in Table 1. For several reasons yeasts inhabiting Sphagnum moss were studied in composite samples rather than in individual leaf samples. First, estimations of the total yeast counts reported by Kachalkin et al. (2008) suggest that an individual leaf of a Sphagnum moss is too small to carry the sufficient amount of yeast cells that can be detected using cultivation techniques. Second, the extreme leaf-to-leaf variation in microbial counts was repeatedly observed in phylloplane (Inacio et al. 2010 and references therein). Thus, homogenisations and pooling of samples (plants or leaves) should even the distribution of yeast populations when multiple samples are collected (see Yurkov et al. 2011). Third, the denseness of plants growing in a peat layer allow boundless yeast cells exchange between different leaves and plants. The yeast community of Sphagnum mosses was analysed by plating on glucose-peptone-yeast extract (GPY) agar supplemented with 4 ml/l lactate (final pH 4–4.5). Four sub-samples of 1 g were taken from each composite sample, placed in a 50 ml plastic tube, suspended 1:20 (w/v) in sterile demineralised water, and vortexed for 20 min. An aliquot of 0.1 ml was used for inoculation. Each sub-sample was plated in duplicate. The plates were incubated at 10°C for 1 month to prevent fast development of moulds. Plates were examined after 7, 14, 21 and 28 days of incubation. Colonies were differentiated into macromorphological types using a dissection microscope, counted, and 1–2 representatives of every colony type per plate were brought into pure culture. Cultures were grouped according to their morphology and a few basic physiological properties. Using this preliminary grouping, we reduced the number of strains involved 123 Identification of cultures Novel yeast species A total of four strains representing the novel species Candida sphagnicola were studied (Table S1). Strains K-280 (=KBP Y-3887T) and K-708 were isolated in the course of this survey. Further attempts to obtain additional isolates of C. sphagnicola resulted in two more cultures. Strain K-962 was isolated from a sample of Sphagnum girgensohnii collected in October 2008 in the same area of the Central Forest State Biosphere Reserve and strain KBP Y-3971 was isolated from the phylloplane of Alternate-leaved Golden Saxifrage (Chrysosplenium alternifolium) collected in May 2008 also from the swamp biotope at Losiny Ostrov (Elk park) National Park (Moscow, Russia) by Glushakova AM (Glushakova and Chernov 2010). Additional information about the strains is given in the Table S1. Phenotypic characterization of all Candida sphagnicola isolates was carried out according to Yarrow (1998). Mating tests were performed on potato-dextrose (PDA, Difco), MYP, dilute GPY, and water agars. Author's personal copy Antonie van Leeuwenhoek (2012) 102:29–43 33 Table 1 Species list and relative abundance (%) of yeasts and yeast-like fungi on Sphagnum mosses in the forest and the swamp biotopes Species Rhodotorula mucilaginosa Classification Pucciniomycotina Number of strainsa Sequence example Relative abundance, %b Forest biotope Swamp biotope 37.1 21/5 HE572519 59.8* 6/2 HE572520 2.0 22.3** 6/2 HE572521 3.6 10.6 4/2 HE572522 8.2 5.3 6/2 HE572523 9.5 3.7 3/1 HE572524 3.8 7.3 2/1 HE572525 6.4* 2/1 HE572526 1.4 2.3 2/1 HE572527 0.2 3.2 1/1 HE572528 \0.1 3.2 1/1 FN667995 \0.1 3.2 2/1 HE572529 2.7* 1/1 HE572530 1.8 \0.1 1/1 HE572531 \0.1 1.6 2/2 FN868154 0.7 \0.1 Microbotryomycetes Sporidiobolales Cryptococcus magnus Agaricomycotina Tremellomycetes Filobasidiales Dothidella sp. Pezizomycotina Dothideomycetes Sporobolomyces roseus Pucciniomycotina Microbotryomycetes Sporidiobolales Cryptococcus victoriae Agaricomycotina Tremellomycetes Tremellales Aureobasidium pullulans Pezizomycotina Dothideomycetes Dothideales Candida friedrichii Saccharomycotina \0.1 Saccharomycetes Saccharomycetales Rhodotorula lysiniphila Pucciniomycotina Cystobasidiomycetes Erythrobasidiales Candida sp. KBP Y-3853 Saccharomycotina Saccharomycetes Saccharomycetales Cystofilobasidium capitatum Agaricomycotina Tremellomycetes Cystofilobasidiales Pichia membranifaciens Saccharomycotina Saccharomycetes Saccharomycetales Cryptococcus diffluens Agaricomycotina 0.2 Tremellomycetes Filobasidiales Cryptococcus wieringae Agaricomycotina Tremellomycetes Filobasidiales Candida zeylanoides Saccharomycotina Saccharomycetes Saccharomycetales Candida sphagnicola sp. nov. Saccharomycotina Saccharomycetes Saccharomycetales a Number of strains studied using a PCR-fingerprinting technique/number of sequenced strains b Relative abundance (average values) of each yeast species was calculated for each biotope and is based on colony counts obtained from a total of 272 (2 9 136) plates. Significant differences (Wilcoxon T-test) are indicated by * p B 0.05, ** p B 0.01 123 Author's personal copy 34 Antonie van Leeuwenhoek (2012) 102:29–43 Phylogenetic analysis was based on nucleotide sequences of the SSU, the ITS region and the LSU. Alignments were made using the MAFFT algorithm (Katoh et al. 2002). The final alignment (TreeBase S11989) contained 2,854 characters: 1,735 positions corresponded to the 18S rDNA, 556 positions to the ITS region, and 561 positions to the 26S rDNA. The model of DNA substitution (GTR?G?I) and parameters for maximum likelihood analysis were derived by using Modeltest, version 3.7 (Posada and Crandall 1998). Maximum likelihood (ML) analysis was performed using RAxML version 7.0.3 with 1,000 rounds of bootstrap replicates (Stamatakis et al. 2008). Genetic distances were computed using PAUP* 4b10 (Swofford 1998), whereby distance settings were the same as the ones obtained for the priors of the ML analysis. A parsimony network was constructed from aligned LSU and ITS sequences with the program TCS 1.21 (Clement et al. 2000) using a 95% connection limit and gaps treated as missing data. biotope type on total yeast counts. Normality of distribution was additionally tested for the total yeast quantity. Differences in species distribution among biotopes were assessed using the Wilcoxon signedrank test (also referred to as Wilcoxon T-test). Effects were considered to be statistically significant at the level p B 0.05. Statistical evaluations were performed with Statistica 8–9 (StatSoft Inc., USA). Statistical data analyses Yeast quantity and diversity For each sample, yeast quantity and community structure were determined. Yeast quantity was calculated as CFU (colony forming units) per gram of moss at dry weight. Frequency of occurrence was calculated as the number of samples, where a species was observed, as a proportion of the total number of samples. Relative abundance was calculated as the proportion of a particular species in the sample and is based on colony counts. Probability of dominance was calculated as the ratio of number of samples where a species showed the highest abundance over the total number of samples where this species was observed. Yeast diversity was studied using Shannon–Weaver index. Six out of 40 sampling sites were excluded from the analysis because they yielded no yeast cultures either due to low yeast quantity in a particular replicate or due to fast development of moulds, which made isolation and appropriate quantification of yeasts difficult. Quantity values were Log10-transformed for the analysis. Because yeast distribution in phylloplane is unimodal either normal or lognormal (Fonseca and Inacio 2006; Inacio et al. 2010), a one-way analysis of variance (ANOVA) using a general linear model with fixed effects (biotope type: the forest and the swamp) model was used to determine significant effect of a On average, the yeast quantity increased gradually from the boreal spruce forest to the swamp, from 3.6 to 4.0 Log10 (CFU/g, dry weight), respectively (Fig. 2). Yeast counts did not differ significantly between neighbouring sampling sites, but did differ statistically between the two extreme points: forest and swamp (ANOVA: F = 16.6, p \ 0.001). The total yeast quantity values expressed as CFU/g highly depend on the moisture level of the analysed substrate, e.g., Sphagnum moss. As a result, yeast counts calculated as colony forming units per gram of Sphagnum moss at natural humidity did not differ between biotopes (data not shown). On average, 4.5 and 4.0 colonies per plate were observed in forest and swamp Sphagnum samples, respectively. This allows a reliable differentiation of colonies and adequate isolation of representative strains for identification. In total 15 yeast species were isolated and identified during this study (Table 1). They belong to the four lineages of Fungi Agaricomycotina (5), Pucciniomycotina (3), Saccharomycotina (5) and Pezizomycotina (2). On average, two yeast species were found in a Sphagnum sample. Forest and swamp sites yielded an equal number of yeast species, namely 12. Nine of the species were shared between the biotopes. The Shannon diversity 123 Results Studied biotopes differed considerably in respect to the temperature-moisture regime. The transition from the forest to the swamp was characterized by increased insolation, 60–75 and 15–25% of the foliage projective cover, respectively. Similarly, moisture content was much higher in Sphagnum samples collected in the swamp (527–831%; mean: 719%) than in the forest (132–799%; mean: 241%). Author's personal copy Antonie van Leeuwenhoek (2012) 102:29–43 35 5.5 *** Forest Yeast quantity, Log10 CFU/g 5.0 Swamp 4.5 4.0 3.5 3.0 2.5 1 5 10 15 20 25 30 35 40 Sampling sites Fig. 2 Quantity of yeast (log10 (CFU) per gram) on Sphagnum mosses (dry weight) in forest (points 1–20) and swamp (points 21–40) biotopes. Bars are standard errors and middle points the respective means index was higher in the swamp biotope (mean: 0.98 and maximum: 2.08) than in the forest (mean: 0.62 and maximum: 1.70). significantly between the biotopes (Table 1). The abundance of other yeasts varied between the sampling sites and did not differ significantly between the biotopes (Table 1). Community structure Relative abundance of ascomyceteous yeasts clearly distinguished the communities of the forest and swamp biotopes. On average, ascomycetous yeasts constituted 10.9 and 18.4% of the total abundance in the forest and the swamp, respectively. At the species level, the relative abundances of the two most frequent species, Rhodotorula mucilaginosa (Wilcoxon T-test: Z = 2.08, p = 0.038) and Cryptococcus magnus (Wilcoxon T-test: Z = -3.04, p = 0.002), differed significantly between the studied biotopes (Table 1; Fig. 3). R. mucilaginosa dominated on Sph. girgensohnii (forest biotope) and Cr. magnus was the most abundant yeast on Sph. angustifolium (swamp). Remarkably, the share of Cr. magnus increased significantly with the decrease of the abundance of R. mucilaginosa (Fig. 3). These two species cooccurred in the yeast community only in the transition zone forest-to-swamp and in the swamp biotope (Fig. 3). Yeasts C. friedrichii (Wilcoxon T-test: Z = 2.023, p = 0.043) and Cr. diffluens (Wilcoxon T-test: Z = 1.992, p = 0,046) were repeatedly found on Sph. girgensohnii and their abundance differed Discussion Yeast communities in the phyllosphere of Sphagnum species differ from those of the vascular plants and it was suggested that differences are determined by the biochemical characteristics of the environment rather than the temperature-moisture regime in the turf (Kachalkin et al. 2008). Here, we present results of a study addressing at the effects of the temperaturemoisture regime in Sphagnum moss on yeasts. More specifically, we focused on the effects of the spatial transition from forest to swamp biotope. The cultivable yeast community was quantitatively analysed using a plating approach with subsequent identification of isolated pure cultures based on rDNA sequence data. Yeast quantity and diversity The number of cultivable yeast cells increased gradually from the boreal spruce forest to the swamp (Fig. 2). Additionally, yeast numbers reflected the 123 Author's personal copy 36 Antonie van Leeuwenhoek (2012) 102:29–43 Forest biotope Swamp biotope 1 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 19 21 22 23 24 25 26 27 28 29 31 33 34 36 37 38 39 40 Rhodotorula mucilaginosa Cryptococcus magnus Cr. victoriae Dothidella sp. Sporobolomyces roseus Aureobasidium pullulans Candida friedrichii C. membranaefaciens Cystofilobasidium capitatum R. lysiniphila Candida sp. KBP Y-3853 Cr. diffluens C. zeylanoides Cr. wieringae C. sphagnicola sp. nov. Relative abundance: 100-50 % 49-20 % 19-5 % 4-0 % Fig. 3 Species richness and relative abundance of yeasts species on Sphagnum mosses in the forest (points 1–20) and the swamp (points 21–40) biotopes. Sampling points, which yielded no yeast cultures, are not shown Yeast quantity, Log10 CFU/g moisture content of moss samples and, therefore, showed a clear separation of the two vegetation types with a sharp discontinuity between the forest and the swamp (Fig. 4). Although the total yeast quantity is often given in literature as CFU/g of plant material, we found that the total yeast population values on Sphagnum species can be biased by the moisture level of the analysed sample. We observed that moisture content of a sample varied by a factor of 6.3 (from 132 to 831%) on a rather small distance, i.e., 80 m (Fig. 4). Remarkably, yeast counts adjusted to the moss dry weight did not differ considerably between neighbouring sampling sites. Similarly, the same number (12) of yeast species were isolated from the two biotopes (Table 1). Thus, our results suggest that the general community indices quantity and diversity do not depend on the temperature-moisture regime of the Sphagnum moss. 4.4 4.2 4.0 R2 = 0.43 3.8 3.6 3.4 3.2 100 300 500 700 900 Humidity of Sphagnum moss, % Fig. 4 Quantity of yeast (log10 (CFU) per gram) in the forest (circles) and the swamp (squares) biotopes in relation to the moisture content (%) of Sphagnum mosses 123 The influence of organic nutrients for the establishment of phylloplane community has been long considered (e.g., Fonseca and Inacio 2006; Nix-Stohr et al. 2008). Recently, experiments showed the application of exogenous nutrients to increase yeast population in the phylloplane of tall fescue (Nix-Stohr et al. 2008). It was demonstrated that application of organic nitrogen stimulates yeast population, while sugar and inorganic nitrogen sources had little effect. Therefore, nitrogen rather than carbon determine carrying capacity of the phylloplane. Phenolic compounds in the Sphagnum layer bind nitrogen making it unavailable to microorganisms (Painter 1998). Neither insolation (temperature and ultraviolet radiation) nor humidity (water availability) affected yeast numbers and species richness values in our study. This implies that the size of yeast population in the phyllosphere of Sphagnum species could be limited by the carrying capacity of this environment, the maximum yeast population size that the environment can sustain through resources (e.g., nitrogen sources) and habitats availability. Although species richness values did not differ between the biotopes, the Shannon diversity index values increased by 60% for swamp-collected relative to forest-collected Sphagnum yeast communities. Because Shannon diversity index relies on the species richness values and the relative abundance of every species, higher values of Shannon diversity index in the swamp suggest greater community evenness in this biotope (see also McGill et al. 2007). In this case, swamp community is composed of species that are likely to be found in equal abundance, as based on colony counts. On average, in forest samples the most frequent species (the first rank) accounted for 59.8% of Author's personal copy Antonie van Leeuwenhoek (2012) 102:29–43 the total yeast counts, while the second and the third ranks accounted for 9.5 and 8.2%, respectively (Table 1). In contrast, swamp samples were dominated by two frequently obtained species with 37.1 and 22.3%, closely followed by the third rank with 10.6% indicating that the evenness of the yeast community is affected by the temperature-moisture gradient. Although the concept of species abundance distribution (SAD) was introduced to ecology more than 100 years ago, our knowledge of the shape of SADs of microorganisms is still poor (McGill et al. 2007). In contrast to the hyperdiverse phyllosphere communities (e.g., Unterseher et al. 2011), yeasts inhabiting Sphagnum moss form species-poor communities with uneven community structure (dominated by few of the total encountered species). This pattern was previously reported for highly specialized yeast communities in soils (Yurkov et al. 2011) and floral nectar (Pozo et al. 2011) where environmental conditions strongly affect yeast population sizes and select towards a few dominating specialist species. In Sphagnum, a number of physico-chemical properties such as nitrogen oligotrophy, water regime, and complex organic acids (van Breemen 1995) may be an underlying cause of the observed community pattern (Fig. 3). Community composition and structure Unlike total yeast counts and species richness, species composition on different Sphagnum mosses differed significantly along the temperature-moisture ecocline (Fig. 3; Table 1). The proportion of ascomycetes in the swamp was nearly twice as high as in the forest. A possible explanation for this trend could be that ascomycetous yeasts are sensitive to water stress due to their inability to produce extracellular polysaccharide capsules (EPS). The production of EPS is a common trait for phylloplane-inhabiting basidiomycetous yeasts (Fonseca and Inacio 2006 and references therein). EPS have already been shown to protect yeasts from harsh environmental conditions but also to improve species growth rates through binding and accumulation of nutrients within capsular material (Fonseca and Inacio 2006 and references therein). On the species level, the most pronounced differences between the forest and the swamp was the abundance of the four yeast species: Cr. magnus, R. mucilaginosa, C. friedrichii, and Cr. diffluens. The 37 first species occurred on Sph. angustifolium (in the swamp), whereas the other three species were typical for Sph. girgensohnii, under the forest cover. The relative abundance of other yeast species did not show any significant trend and, in our eyes, illustrates extreme spatial patchiness of the Sphagnum cover (Fig. 3). The probability of dominance of the yeast R. mucilaginosa on Sphagnum mosses in the forest and in the swamp biotope was 92.9 and 61.5%, respectively. Cr. magnus was the most abundant species in the 25% of analysed samples collected in the swamp. Lower water content of the forest moss and dry periods in the late summer could partly explain the significant increase of the R. mucilaginosa abundance in the forest biotope. It was shown before that R. mucilaginosa sustains unfavorable environmental conditions, including drying (Moore et al. 1989). Alternatively, the presence of R. mucilaginosa on Sphagnum moss might be due to the accidental fall of yeast cells from the upper vegetation layers as this yeast is a recognized phylloplane inhabitant (e.g., Fonseca and Inacio 2006). Particularly, R. mucilaginosa together with Cr. magnus were repeatedly observed among epiphytous yeasts in the boreal zone of European Russia (e.g., Yurkov et al. 2008a; Glushakova and Chernov 2010). We assume that unlike R. mucilaginosa, Cr. magnus development in the forest Sphagnum is largely limited by the dry summer period. This assumption is additionally supported by the striking differences in moss moisture content values between the forest and the swamp (Fig. 4). Interestingly, the increased proportion of Cr. magnus along with the decreased abundance of R. mucilaginosa in swamp biotope could not be explained by their differential survival on Sphagnum due to humidity differences, as both of these species seem to tolerate high water content. R. mucilaginosa has been reported regularly from a variety of aquatic habitats, like oligotrophic lakes (Libkind et al. 2003; Brandão et al. 2011), sea water (Nagahama et al. 2001; Bass et al. 2007; Coelho et al. 2010) and glaciers (Butinar et al. 2007; de Garcı́a et al. 2007). Cr. magnus together with other filobasidious yeasts were found previously on Sphagnum mosses in Russia (Kachalkin et al. 2008) and Germany (Yurkov et al. 2008b). Thus, we conclude that the differences in the taxonomic structure of yeast communities reflect changes in the environmental conditions in the Sphagnum layer other than humidity. 123 Author's personal copy 38 Tolerance to ultraviolet (UV) radiation is likely to be an important selection pressure for survival of phyllosphere colonists, both fungi and bacteria (Fonseca and Inacio 2006; Whipps et al. 2008). It is noteworthy that a large majority of yeasts found on the phylloplane produce photoprotective compounds such as carotenoid pigments (Yurkov et al. 2008c; Libkind et al. 2009), mycosporines (Brandão et al. 2011; Libkind et al. 2011) and ubiquinone (Yurkov et al. 2008c). Similar to oligotrophic lakes studied by Libkind et al. (2009), swamps are typically exposed to increased UV radiation due to low vegetation cover. However, we did not observe any correlation between the proportion of pigmented yeasts and the foliar projection cover. This is most probably due to the wide range of mechanisms protecting yeasts from UV radiation. The two most frequent species in the swamp biotope R. mucilaginosa and Cr. magnus were shown to synthesize two different groups of photoprotective compounds, i.e., carotenoids and mycosporines. The red yeast R. mucilaginosa does not produce mycosporines but the synthesis of the carotenoid pigment torularhodine (Yurkov et al. 2008c; Moliné et al. 2010) provides this yeast an outstanding UV tolerance (Moliné et al. 2010). In contrast, Cr. magnus can synthesise only low amounts of carotenoids (Yurkov et al. 2008c), but it is able to produce mycosporines (Brandão et al. 2011). Our results confirm a previous observation regarding the low abundance of tremellaceous yeasts (e.g., Cryptococcus victoriae) in the Sphagnum phylloplane (Kachalkin et al. 2008). The share of these species did not exceed 9.5% in this study. The high abundance of non-pigmented yeasts, particularly the members of the Microbotryomycetes lineage, was reported to be characteristic for epiphytous communities on mosses (Maksimova and Chernov 2004; Kachalkin et al. 2008; Kachalkin 2010). However, in the present study we did not observe these yeasts. In our opinion, this disagreement with the two earlier surveys is due to the dissimilarity between sampling regions (compare with Kachalkin et al. 2008) and, additionally, due to the seasonal variation, i.e., sampling in June (this study) and September (Maksimova and Chernov 2004), respectively. Notably, the non-pigmented Microbotryomycetes are known to be psychrotolerant and they were repeatedly detected in extreme habitats, like glaciers (Turchetti et al. 2008) and Antarctica (Connell et al. 2008). Thus, the development of the cold- 123 Antonie van Leeuwenhoek (2012) 102:29–43 adapted species in the late summer and early autumn is not surprising as the night temperatures drop down during this period. The dissimilarities in the yeast community composition in Sphagnum observed at the two different sampling seasons (Maksimova and Chernov 2004; this study) might hint to seasonal variation in species occurrence similar to the one reported for higher plants (e.g., Nix et al. 2008; Glushakova and Chernov 2010). This observation should be addressed in additional studies with more sampling sites and time points. Novel yeast species Analysis of the D1/D2 large subunit rDNA gene sequences of the surveyed yeasts suggested that three ascomycetes represented hitherto undescribed species (Table 1). The yeast Candida sp. KBP Y-3853 (=VKPM Y-3486 = MUCL 52871) belongs to the Yarrowia lipolytica clade (Saccharomycotina) and differs from the genetically most closely related species, Candida oslonensis, by 9 nucleotide substitutions. Dothidella sp. KBP Y-4207 resembled morphologically some of so-called ‘‘black yeasts’’, which are regularly observed as epiphytes and endophytes (Fonseca and Inacio 2006). Sequencing results placed this yeast in the class Dothideomycetes with the closest match (5 nucleotide substitutions) among the recognized species being Plowrightia periclymeni (FJ215702). Other conspecific strains were isolated previously in the USA (North Carolina) from tissues of Magnolia grandiflora (EF420084) and in the USA (Maine) from leaves of Vaccinium angustifolium (EU051628). The presence of this fungus in the phyllosphere of different plants in remote regions additionally illustrates a great, undiscovered diversity of fungi associated with plants. Finally, three strains of an anamorphic species were isolated from Sph. girgensohnii. Additionally, strain KBP Y-3971 acquired from the KBP culture collection (Faculty of Soil Science, Lomonosov Moscow State University) was used for physiological tests (Table 2) and mating experiments. Phylogenetic analysis showed that the four strains are conspecific (Table S1) and placed the novel species into the Candida glaebosa clade (Lachance et al. 2011), a group also comprising C. fluviatilis and C. palmioleophila. The nearest sequence match among currently recognized species was obtained with Candida fluviatilis showing Author's personal copy Antonie van Leeuwenhoek (2012) 102:29–43 39 Table 2 Physiological characteristics distinguishing Candida sphagnicola sp. nov. (this study) and closely related species (according Kurtzman and Fell (1998) and CBS database) C. sphagnicola sp. nov.* D-glucose fermentation C. fluviatilis C. palmioleophila C. glaebosa C. saitoana C. pseudoglaebosa - ? - - - W L-sorbose - - - - V ? Cellobiose ? ? - ? ? ? Lactose ? ? - ? V ? Melibiose W - ? ? ? ? Raffinose ? - ? ? ? ? Melezitose W ? ? - V ? Inulin - - - - ? - Soluble starch - ? ? - V - D-xylose W ? ? ? ? ? L-arabinose D-ribose ? - - - V V - L-rhamnose W/V - - - - - D-glucosamine ? ? ? V V ? Salicin ? ? - ? ? ? Growth at 50% glucose - - - - V ? Growth at 10% NaCl - ND - W/V - ND Growth at 37°C - ? ? - V - Abbreviations used: ?, growth; -, no growth; V variable growth, W weak growth, ND no data * Note following strains were used in assimilation tests: KBP Y-3887T, K-708, K-962 and KBP Y-3971 2 nucleotide substitutions and 1 indel in the SSU rDNA, 6 substitutions in the LSU rDNA and 8 substitutions in the ITS region from C. fluviatilis (Fig. 5). The latter species differs from C. palmioleophila by 2 nucleotide substitutions and 1 indel in the SSU rDNA, 6 sustitutions in the LSU rDNA and 8 substitutions and 3 indels in the ITS region. We analysed the average genetic distances within the Candida glaebosa clade and found that pairwise distances between species ranged between 5.4 and 10.0 nucleotides (nts) per 1,000 base pairs (Fig S1). The largest distance was observed between C. palmioleophila and the novel species found during this study. Among the other species pairs, genetic distances were uniform and ranged between 5.3 and 6.5 nts per 1,000 bp. High sequence similarities of ribosomal gene regions, not exceeding five nucleotide positions in the D1/D2 domains, were also reported for ascomycetous yeasts of the genera Debaryomyces (Dlauchy et al. 2011), Schwanniomyces (Martorell et al. 2005) and Ogataea (Nagatsuka et al. 2008; Glushakova et al. 2010). Some species of the genera Debaryomyces, Schwanniomyces and Candida show additionally highly similar sequences of the ITS region (e.g., Martorell et al. 2005; Dlauchy et al. 2011 and references therein). The parsimony network analysis (with 95% connection limit) based on LSU and ITS sequence data was found to be useful in delineation of some closely related ascomycetous yeasts (e.g., Lachance et al. 2010). But this type of analysis might fail to delineate species displaying the low level of sequence divergence. For example, Dlauchy et al. (2011) reported that all species of the genus Debaryomyces were connected into a single a network based on D1/D2 sequence data. Although authors used LSU sequence data alone, reports on the high similarity of the ITS region (e.g., Martorell et al. 2005) suggest delimitation of species of the genus Debaryomyces with the parsimony networks can be difficult. The parsimony network analysis using LSU and ITS sequences performed in this study for members of the Candida glaebosa clade resulted in 123 Author's personal copy 40 Antonie van Leeuwenhoek (2012) 102:29–43 Scheffersomyces spartinae CBS 6059 (AB054292 / EU343815 / U45764) Candida glaebosa CBS 5691 (FJ153125 / FJ153208 / U45757) 60 83 100 Candida pseudoglaebosa CBS 6715 (AB013544 / CBS database / U71072) Candida saitoana CBS940 (AB013523 / CBS database / U45762) 100 54 0.02 Candida fluviatilis CBS 6776 (AB013521 / CBS database / U45717) Candida sphagnicola sp. nov. KBP Y-3887T (HE602781 / FN868154) 78 Candida palmioleophila CBS 7418 (AB013520 / EU568917 / U45758) Candida oleophila CBS 2219 (AB013534 / AY528671 / U45793) 56 100 Candida zeylanoides CBS 619 (AB013509 / AY542871 / U45832) Candida fragi CBS 7702 (AB013573 / AY344066 / U71071) Schwanniomyces polymorphus CBS 186 (AB054275 / AF121136 / U45836) Fig. 5 Maximum likelihood analysis of an alignment of the SSU rDNA, the ITS region and the LSU (D1/D2 domains) rDNA for Candida sphagnicola sp. nov. The numbers given on branches are frequencies ([50%) with which a given branch appeared in 1,000 bootstrap replications. The scale indicates the number of expected substitutions accumulated per site. The tree is rooted with Schwanniomyces polymorphus (AB054275/ AF121136/U45836) two networks (Fig S2). The first network consisted of the novel species, C. fluviatilis and C. palmioleophila, and another one consisted of C. saitoana, C. glaebosa and C. pseudoglaebosa. The sister group to the novel species, which consists of C. saitoana, C. glaebosa and C. pseudoglaebosa, is also characterized by low sequence divergence (Fig S1) and the three species were delimited on the basis of DNA reassociation and isoenzyme patterns (Lachance et al. 2011). Currently available data led us to conclusion that high sequence similarity of ribosomal gene regions is likely to be a common attribute of species of the Candida glaebosa clade. This observation should be addressed in additional studies utilizing more strains and additional molecular markers. Despite the low divergence of rDNA sequences, we think that erection of a novel species is sufficiently supported. Based on the sequence divergence along the nearly entire SSU rDNA gene, the ITS regions and the D1/D2 domains of the LSU rDNA gene being in the range observed for other currently recognized closely related species, we provide the formal description of this species for which the name Candida sphagnicola is proposed. We do not describe the two other taxa as their proper description requires additional studies. 123 Description of Candida sphagnicola sp. nov. Kachalkin & Yurkov On Glucose Peptone Yeast extract Agar (GPYA), after 3 days at 20°C the cells are spherical or ellipsoidal (1.5–3.0 9 2.0–5.0 lm) and proliferate by multilateral budding (Fig. 6). After 7 days on GPYA at 20°C streak culture is whitish and slightly glistening with a smooth surface. After 7 days primitive pseudohyphae are produced on Potato-Dextrose Agar (PDA) at 10 and 20°C. Ascospores were not detected. Glucose is not fermented. Assimilation of carbon compounds: D-glucose, D-galactose, sucrose, maltose, cellobiose, trehalose, lactose, raffinose, D-ribose, D-glucosamine, ethanol, glycerol, ribitol, D-mannitol, salicin, DL-lactic acid, succinic acid, citric acid, 2-ketogluconic acid, arbutin, melibiose (weak), Author's personal copy Antonie van Leeuwenhoek (2012) 102:29–43 41 was reported to be difficult because of the high variability observed within several species (Lachance et al. 2011). Latin diagnosis of Candida sphagnicola sp. nov. Kachalkin & Yurkov Fig. 6 Phase contrast micrograph of Candida sphagnicola sp. nov. KBP Y-3887. Vegetative cells reproducing by budding after 3 days on GPY agar at room temperature, bar = 10 lm melezitose (weak), D-xylose (weak), L-rhamnose (weak). No growth occurs on L-sorbose, inulin, soluble starch, L-arabinose, D-arabinose, erythritol, dulcitol, D-glucuronic acid, 5-ketogluconic acid and inositol. Nitrate and nitrite are not assimilated. No growth occurs in 50% glucose–0.5% yeast extract and in YM agar supplemented with 10% NaCl. Starch-like compounds are not produced. Urease activity is negative. The maximum growth temperature is 29°C. The type strain is KBP Y-3887T, isolated from Sphagnum girgensohnii Russ. (Central Forest State Biosphere Reserve, Tver region, Russia). It has been deposited in the collection of the Yeast Division of the Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands, as strain CBS 11774T (=VKPM Y-3566T = MUCL 53590T). The specific epithet sphagnicola refers to the substrate, Sphagnum moss, from which the species has been isolated. The sequence of the 18S (SSU) rDNA gene is deposited in GenBank under the accession number HE602781. The sequence of the ITS (ITS1-5.8S-ITS2) and 26S (LSU) rDNA D1/D2 regions is deposited in GenBank under the accession number FN868154. The final alignment is deposited in the TreeBase (S11989). The novel species could be distinguished from the closely related species C. fluviatilis and C. palmioleophila by the D-glucose fermentation and assimilation of cellobiose, lactose, melibiose, raffinose, soluble starch, D-ribose, L-rhamnose, salicin and growth at 37°C. However, separation of the members of C. glaebosa clade using traditional physiological tests In agaro cum dextroso et peptono et extracto levidinis (GPY) post dies 3 ad 20°C cellulae ovoidae aut elongatae (1.5–3.0 9 2.0–5.0 lm), singulae, per gemmationem multilateraleum reproducentes. Cultura in striis in agaro GPYA post dies 7 ad 20°C, cremicolor, semi-nitida, glabra. In agaro cum extracto Solani tuberose et dextroso (PDA) post dies 7 ad 10°C et 20°C pseudomycelium primitivum interdum formatur. Ascosporae non formantur. Glucosum non fermentatur. D-glucosum, D-galactosum, sucrosum, D-maltosum, cellobiosum, trehalosum, lactosum, raffinosum, D-ribosum, D-glucosaminum, ethanolum, glycerolum, ribitolum, D-mannitolum, salicinim, D L-lacticum, acidium succinicum, acidium citricum, acidium 2-ketogluconicum, arbutinum, melibiosum (exigue), melezitosum (exigue), D-xylosum (exigue) et L-rhamnosum (exiguum) assimilantur at non L-sorbosum, inulinum, amylum solubile, L-arabinosum, D-arabinosum, erythritolum, dulcitolum, acidum D-glucuronicum, acidium 5-ketogluconicum nec inositolum. Non kalium nitricum nec natrium nitrosum assimilantur. Non crescit in medio cum 50% glucose et 0.5% extracto levidinis. Non crescit in medio cum extracto malti (YM) et 10% sodii cloridii. Materia amyloidea iodophila non formatur. Ureum non finditur. Temperatura maxima crescentiae: 29°C. Cultura typica KBP Y-3887T isolata ex folia Sphagnum girgensohnii, viva et exsiccata numero CBS 11774T (=VKPM Y-3566T = MUCL 53590T) in collectione zymotica Centraalbureau voor Schimmelcultures, Trajectum ad Rhenum, Hollandia, sustentat. Acknowledgments The authors are grateful to Chernov IYu (Moscow State University, Russia), Daniel H-M (BCCM/ MUCL, Belgium), Schäfer AM (Ruhr-Universität Bochum, Germany) and three anonymous reviewers for their valuable suggestions on the manuscript. This work was supported by the Russian Foundation for Fundamental Research (RFBR) 10-0400332-a and the German Research Foundation (DFG) YU 152-1/1. We thank Glushakova AM for providing a strain for the analysis and Maksimova IA for line drawings. 123 Author's personal copy 42 References Andrews JH, Harris RF (2000) The ecology and biogeography of microorganisms on plant surfaces. Annu Rev Phytopathol 38:145–180 Baas Becking L (1934) Geobiologie of inleiding tot de milieukunde. 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