Maximum likelihood tree from ITS dataset identifies <i>Astraeus sirindhorniae</i> as a distinct species of <i>Astraeus</i>.

<p>Numbers above branches identify the statistics bootstrap percentages (bold text, before forward slash) and Bayesian posterior probabilities (normal text, after forward slash) for that branch. Maximum likelihood bootstraps from 1000 iterations. Bayesian posterior probabilities from 1000 iterations (1 million runs sampling every 1000^th iteration).</p

<i>Astraeus sirindhorniae</i>.

<p>Exoperidium layers (A–B). (A) exoperidial subpellis, bar = 5 µm. (B) exoperidial subpellis (innermost), bar = 10 µm. (C) rhizomorph hyphae with clamp connection (arrowhead), bar = 5 µm. (D) capillitium hyphae displaying continuous lumen (arrowhead) and basidiospore (arrow), bar = 5 µm. (E–F) spore ornamentation demonstrated coalescent spines in groups, bar = 1 µm. A–D magnification at 1,000×.</p

<i>Astraeus sirindhorniae</i>.

<p>(A) immature basidiomes, bar = 60 mm. (B) short stipitate endoperidium (arrowhead), bar = 3 mm. (C) fibrillose endoperidium (arrowhead), bar = 3 mm. (D) gleba colour become umber to date- brown when mature (arrowhead), bar = 10 mm. (E) complex outer peridium, bar = 3 mm.</p

List of specimens in this study.

<p>Notes:</p>a<p>New species described in Phosri et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071160#pone.0071160-Phosri3" target="_blank">[32]</a>: <i>A. smithii</i> and ASTRAE-86 is the holotype.</p>b<p>New species described in Phosri et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071160#pone.0071160-Phosri3" target="_blank">[32]</a>: <i>A. telleriae</i> and ASTRAE-87 is the holotype.</p><p>Specimen codes as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071160#pone-0071160-g002" target="_blank">Figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071160#pone-0071160-g003" target="_blank">3</a>. All specimens from Phu Khieo Wildlife Sanctuary abbreviated as PKWS.</p

Maximum likelihood tree from a multigene dataset reveals the placement of <i>Astraeus sirindhorniae</i> within the Sclerodermatineae.

<p>Thick vertical black bars identify root branch for the taxonomic lineage indicated by the adjacent label. Numbers above branches identify the statistics bootstrap percentages (bold text, before forward slash) and Bayesian posterior probabilities (normal text, after forward slash) for that branch. Maximum likelihood bootstraps from 1000 iterations. Bayesian posterior probabilities from 1000 iterations (1 million runs sampling every 1000^th iteration).</p

Fungi in Thailand: A Case Study of the Efficacy of an ITS Barcode for Automatically Identifying Species within the Annulohypoxylon and Hypoxylon Genera

<div><p>Thailand, a part of the Indo-Burma biodiversity hotspot, has many endemic animals and plants. Some of its fungal species are difficult to recognize and separate, complicating assessments of biodiversity. We assessed species diversity within the fungal genera <em>Annulohypoxylon</em> and <em>Hypoxylon</em>, which produce biologically active and potentially therapeutic compounds, by applying classical taxonomic methods to 552 teleomorphs collected from across Thailand. Using probability of correct identification (PCI), we also assessed the efficacy of automated species identification with a fungal barcode marker, ITS, in the model system of <em>Annulohypoxylon</em> and <em>Hypoxylon</em>. The 552 teleomorphs yielded 137 ITS sequences; in addition, we examined 128 GenBank ITS sequences, to assess biases in evaluating a DNA barcode with GenBank data. The use of multiple sequence alignment in a barcode database like BOLD raises some concerns about non-protein barcode markers like ITS, so we also compared species identification using different alignment methods. Our results suggest the following. (1) Multiple sequence alignment of ITS sequences is competitive with pairwise alignment when identifying species, so BOLD should be able to preserve its present bioinformatics workflow for species identification for ITS, and possibly therefore with at least some other non-protein barcode markers. (2) Automated species identification is insensitive to a specific choice of evolutionary distance, contributing to resolution of a current debate in DNA barcoding. (3) Statistical methods are available to address, at least partially, the possibility of expert misidentification of species. Phylogenetic trees discovered a cryptic species and strongly supported monophyletic clades for many <em>Annulohypoxylon</em> and <em>Hypoxylon</em> species, suggesting that ITS can contribute usefully to a barcode for these fungi. The PCIs here, derived solely from ITS, suggest that a fungal barcode will require secondary markers in <em>Annulohypoxylon</em> and <em>Hypoxylon</em>, however. The URL <a href="http://tinyurl.com/spouge-barcode">http://tinyurl.com/spouge-barcode</a> contains computer programs and other supplementary material relevant to this article.</p> </div

PCIs for each of four alignment types and two types of sequence distance.

<p>The error bars indicate 95% confidence intervals, as calculated by the Wilson score interval <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054529#pone.0054529-Wilson1" target="_blank">[46]</a>. The four alignment types used (indicated by different colors at the bottom) were multiple sequence alignment (which imposes an implicit pairwise global alignment on each pair of sequences), and global, semi-global, and local pairwise alignment. The two types of sequence distance used for each alignment method were alignment distance and evolutionary distance. (In fact, for a fixed alignment type and dataset, all evolutionary distances produced the same PCI as p-distance.) The green bars give the value of the barcode gap PCI.</p

Morphological characteristics of <i>Annulohypoxylon</i> and <i>Hypoxylon</i> species found in Thailand.

<p>Stromata (a–p); perithecial structure (q–s); ascospore shapes (t–z); perispore dehiscence (v, y, z). (a) <i>Annulohypoxylon stygium</i> SUT058, (b) <i>A. purpureonitens</i> H125, (c) <i>A. nitens</i> H154, (d) <i>A.</i> aff. <i>nitens</i> H099, (e) <i>Annulohypoxylon</i> sp. H213, (f) <i>Annulohypoxylon</i> sp. H255, (g) <i>Hypoxylon monticulosum</i> H188, (h) <i>H</i>. <i>lenormandii</i> H212, (i) <i>H. investiens</i> H259, (j) <i>H. perforatum</i> SUT218, (k) <i>H. duranii</i> H250, (l) <i>H. haematostroma</i> H114, (m) <i>H. crocopeplum</i> H119, (n) <i>H. pelliculosum</i> H227, (o) <i>H. diatrypeoides</i> H226, (p) <i>H. rubiginosum</i> SUT082, (q) <i>H. fendleri</i> SUT061, (r) <i>H. investiens</i> H259, (s) <i>H. haematostroma</i> H114, (t) <i>H. haematostroma</i> SUT293, (u) <i>A. stygium</i> SUT010, (v) <i>H. duranii</i> SUT284, (w) <i>H. investiens</i> SUT041, (x) <i>A. nitens</i> SUT249, (y) <i>H. monticulosum</i> SUT185 and (z) <i>A. nitens</i> SUT025.</p

<i>Scleroderma camassuense</i>.

<p>(A) Fresh basidiomata in the field, bar = 10 mm. (B) Detail of verrucose exoperidium surface, bar = 2 mm. (C) Basidioma cut away side view, bar = 2 mm. (D) Exoperidium hyphae, bar = 20 μm. (E) Basidiospores under LM, bar = 10 μm. (F) Basidiospores under SEM, bar = 2 μm.</p

<i>Scleroderma anomalosporum</i>.

<p>(A) Fresh basidiomata in the field, bar = 30 mm. (B) Detail of reticulation in exoperidium of young basidioma, bar = 2 mm. (C) Basidioma cut away side view, bar = 2 mm. (D) Exoperidium hyphae, bar = 20 μm. (E) Basidiospores under LM, bar = 10 μm. (F) Basidiospores under SEM, bar = 2 μm.</p