Taxonomic Richness of Yeasts in Japan within Subtropical and Cool Temperate Areas

<div><h3>Background</h3><p>An understanding of the role of yeasts in the environment has been uncertain because estimates of population size and diversity have often been based on species identifications that were determined from a limited number of phenotypic characteristics. DNA-based species identification has now become widely used, allowing an accurate assessment of species in different habitats. However, there are still problems in classification because some genera are polyphyletic. Consequently, the identification of yeasts and measurement of their diversity at the genus level remains difficult, as does assignment of genera to higher taxonomic ranks.</p> <h3>Methodology/Principal Findings</h3><p>A total of 1021 yeast strains was isolated from soil samples and plant materials collected from Japan’s subtropical Iriomote Island and the cool temperate Rishiri Island. Based on sequence analyses of the D1/D2 domain of the LSU rRNA gene, these 1021 strains were tentatively classified into 183 species, with apparent new species accounting for approximately half of the total species isolated (60 and 46, Iriomote and Rishiri, respectively). The yeast species composition was statistically different between the two sites with only 15 species in common. Rarefaction curves of respective sources/areas gave distinctive patterns when the threshold of sequence identity became broader, indicating that the yeast diversity was distinct at the different taxonomic levels compared.</p> <h3>Conclusions/Significance</h3><p>Our isolation study of yeasts in Japan has enabled us to expand the inventory of species diversity because a large number of new species was observed in the sampling areas. Further, we propose use of a particular diversity threshold as an “indicator” to recognize species, genera and higher taxonomic ranks.</p> </div

A neighbor-joining tree of isolates based on the D1/2 region unique sequences.

<p>The evolutionary distance (refer to the bar) was calculated according to Kimura <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050784#pone.0050784-Kimura1" target="_blank">[26]</a>. Bootstrap values <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050784#pone.0050784-Felsenstein1" target="_blank">[27]</a> were summarized by black dots (indicating at least 90% support) or by black rings (indicating at least 60% support). Blue solid ring, new species candidate isolated from a plant of Iriomote Island; blue open ring, described species isolated from a plant of Iriomote; blue solid square, new species candidate isolated from soil of Iriomote; blue open square, described species isolated from soil of Iriomote; blue solid ring, new species candidate isolated from a plant of Rishiri Island; red open ring, described species isolated from a plant of Rishiri; red solid square, new species candidate isolated from soil of Rishiri; red open square, described species isolated from soil of Rishiri. Numerals in brackets after ring or square indicate the number of isolates. Yellow color on OTUs indicates this species was isolated from both islands.</p

Selection of Orthologous Genes for Construction of a Highly Resolved Phylogenetic Tree and Clarification of the Phylogeny of Trichosporonales Species

<div><p>The order Trichosporonales (Tremellomycotina, Basidiomycota) includes various species that have clinical, agricultural and biotechnological value. Thus, understanding why and how evolutionary diversification occurred within this order is extremely important. This study clarified the phylogenetic relationships among Tricosporonales species. To select genes suitable for phylogenetic analysis, we determined the draft genomes of 17 Trichosporonales species and extracted 30 protein-coding DNA sequences (CDSs) from genomic data. The CDS regions of <i>Trichosporon asahii </i>and <i>T</i>. <i>faecale </i>were identified by referring to mRNA sequence data since the intron positions of the respective genes differed from those of <i>Cryptococcus neoformans</i> (outgroup) and are not conserved within this order. A multiple alignment of the respective gene was first constructed using the CDSs of <i>T</i>. <i>asahii</i>, <i>T</i>. <i>faecale</i> and <i>C</i>. <i>neoformans</i>, and those of other species were added and aligned based on codons. The phylogenetic trees were constructed based on each gene and a concatenated alignment. Resolution of the maximum-likelihood trees estimated from the concatenated dataset based on both nucleotide (72,531) and amino acid (24,173) sequences were greater than in previous reports. In addition, we found that several genes, such as phosphatidylinositol 3-kinase <i>TOR1 </i>and glutamate synthase (NADH), had good resolution in this group (even when used alone). Our study proposes a set of genes suitable for constructing a phylogenetic tree with high resolution to examine evolutionary diversification in Trichosporonales. These can also be used for epidemiological and biogeographical studies, and may also serve as the basis for a comprehensive reclassification of pleomorphic fungi.</p></div

Phylogenetic trees of <i>Trichosporon</i> species and related taxa based on (a) D1/D2 LSU rRNA gene sequences, (b) nucleotide sequences of 30 concatenated genes, and (c) amino acid sequences of 30 concatenated genes.

<p>The tree in (a) was constructed using the maximum likelihood method based on the Tamura-Nei model [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131217#pone.0131217.ref044" target="_blank">44</a>]. The names of clades are based on those in Fig. 161.1 in <i>The Yeasts</i>, <i>A Taxonomic Study</i>, 5^th ed. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131217#pone.0131217.ref002" target="_blank">2</a>]. Boldface indicates that the species was used for the 30-gene concatenation analysis. The trees in (b) and (c) were constructed using the maximum likelihood method based on the Tamura-Nei model [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131217#pone.0131217.ref044" target="_blank">44</a>] for (b) and a JTT matrix-based model [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131217#pone.0131217.ref045" target="_blank">45</a>] for (c). Numerals on each node represent the percentages from 100 replicating bootstrap samplings (frequencies of less than 60% are not shown) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131217#pone.0131217.ref046" target="_blank">46</a>]. Letters in brackets after the bootstrap values in (b) and (c) indicate the node name (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131217#pone.0131217.t003" target="_blank">Table 3</a>). Positions used: (a) 604 nucleotides, (b) 72,531 nucleotides, (c) 24,173 amino acids. Highest log likelihood: (a) -3894.3736, (b) -611997.4313, (c) -216445.1306. <i>B</i>., <i>Bullera</i>; <i>C</i>., <i>Cryptococcus</i>; <i>Cr</i>., <i>Cryptotrichosporon</i>; <i>T</i>., <i>Trichosporon</i>; <i>Te</i>., <i>Tetragoniomyces</i>; <i>V</i>., <i>Vanrija</i>.</p

Summary of yeast strains on plants and in soil samples from Rishiri Island and Iriomote Island.

*<p>number of unique species.</p><p>15 species were commonly isolated from both sampling areas, as given in the text.</p

Multiple alignments of symbiotic protist cellulase and catalytic domains of the members of the glycoside hydrolase family 7.

<p>Rs, <i>Reticulitermes speratus symbiotic protists</i>; Hs, <i>Hodotermopsis sjostedti symbiotic protists</i>; Nk, <i>Neotermes koshunensis symbiotic protists</i>; Md, <i>Mastotermes darwiniensis symbiotic protists</i>; Cp, <i>Cryptocercus punctulatus symbiotic protists</i>; Numbered clones (for example SM2038A27) are clone names of cellulase genes identified in this study from the symbiotic protists of termites; CBH, cellobiohydrolase; EG, endoglucanase; TrCel7A, a cellobiohydrolase component, <i>Trichoderma reesei</i> Cel7A [Uni Prot. P00725]; PgCBH-homo, <i>Pseudotrichonympha grassii</i> PgCBH-homo1 [Q95YH1]; TrCel7B, an endo-β-1,4-glucanase (EG) component, <i>T. reesei</i> Cel7B [P07981]; FoCel7B, an EG component, <i>Fusarium oxysporum</i> Cel7B [P46237]; HiCel7B, an EG component, <i>Humicola insolens</i> Cel7B [P56680]. The alignments were performed using CLUSTAL_W and subsequent manual refinement based on the three-dimensional structures of reference sequences. Arabic numerals denote the number of residues from each N terminal end. Solid and open circles under the column indicate the sites of putative proton donors and general acids/bases, respectively. Shaded columns represent conserved positions within the sequences. White letters with black shading denote cysteine residues composing the disulfide bond of <i>T. reesei</i> Cel7A. The asterisks represent the putative protistan GHF7 CBH homologue cysteine residue sites corresponding to the cysteine residue sites of <i>T. reesei</i> Cel7A. The underlined sequences in TrCel7A indicate the loop-forming regions covering the catalytic tunnel <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008636#pone.0008636-Slomovic1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008636#pone.0008636-Wang1" target="_blank">[23]</a>.</p

Phylogenetic tree of GHF11. Numbered clones (for example SM2038A64) are cellulase genes identified in this study from the symbiotic protists of termites.

<p>Letters in paretheses after each clone denote host termite species (Rs  =  <i>Reticulitermes speratus</i>, Nk  =  <i>Neotermes koshunensis</i>, Hs  =  <i>Hodotermopsis sjostedti</i>, Md  =  <i>Mastotermes darwiniensis</i>, Cp  =  <i>Cryptocercus punctulatus</i>). Accession numbers of reference sequences are denoted after species names.</p

Strains used in this study.

<p>*¹ data from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131217#pone.0131217.ref002" target="_blank">2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131217#pone.0131217.ref005" target="_blank">5</a>] and <a href="http://jcm.brc.riken.jp/en/catalogue_e" target="_blank">http://jcm.brc.riken.jp/en/catalogue_e</a>.</p><p>Strains used in this study.</p

Comparison of rarefaction curves among plant and soil samples of Iriomote and Rishiri Islands.

<p>Rarefaction values were obtained using the MOTHUR program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050784#pone.0050784-Schloss1" target="_blank">[25]</a> and data of some particular thresholds of a respective group are plotted. Numerals in the boxes indicate the value of chao (chao_lci – chao_hci) of distances unique, 0.005, 0.03 and 0.01 from the top in the respective group.</p

Amino acid sequences and intron positions of CDS homologs of translation elongation factor 1-α (<i>TEF1-α</i>), DNA-dependent RNA polymerase II <i>RPB140</i> (<i>RPB2</i>), and DNA-directed RNA polymerase II largest subunit (<i>RPB1</i>) from <i>Trichosporon asahii</i> JCM 2466.

<p>Numbers refer to intron positions.</p