A New Representative of Star-Shaped Fungi: Astraeus sirindhorniae sp. nov. from Thailand

Phu Khieo Wildlife Sanctuary (PKWS) is a major hotspot of biological diversity in Thailand but its fungal diversity has not been thouroughly explored. A two-year macrofungal study of this remote locality has resulted in the recognition of a new species of a star-shaped gasteroid fungus in the genus Astraeus. This fungus has been identified based on a morphological approach and the molecular study of five loci (LSU nrDNA, 5.8S nrDNA, RPB1, RPB2 and EF1-a). Multigene phylogenetic analysis of this new species places it basal relative to other Astraeus, providing additional evidence for the SE Asian orgin of the genus. The fungus is named in honour of Her Majesty Princess Sirindhorn on the occasion the 84th birthday of her father, who have both been supportive of natural heritage studies in Thailand.


Introduction
Tropical rain forests are important terrestrial ecosystems. They harbour tremendous biodiversity and several of them are recongized as biodiversity hotspots [1]. Most of the attention paid to this biodiversity has focused on the fauna and flora at the expense of less charasmatic organisms such as fungi. In 1991, Hawksworth [2] has estimated that the number of fungi worldwide ultimately will be around 1.5 million species with fungal diversity considered to be highest in the tropical forests. More recently the estimated number of fungal species has been estimated anywhere between 3.5-5.1 million species [3]. According to Hibbett et al. [4] the overall rate of fungal species discovered worldwide has been fairly level for the last 10 years with a range of 1000-1200 new species reported per year, in both Basidiomycota and Ascomycota but mainly in the latter. Herein a new basidiomycete is added.
The current project is part of an effort to document the diversity of EM fungi associated with a broad range of host plants at a variety of spatial scales in Phu Khieo Wildlife Sanctuary (abbrev.: PKWS) of northeastern Thailand. This project was lead by a team of biologists from Nakhon Phanom University (NPU), in collaboration with Pibulsongkram Rajabhat University (PSRU), Srinakharinwirot University (SWU), Chulalongkorn University (CU), Real Jardín Botánico (RJB-CSIC, Madrid, Spain) and Caledonian Mycological Enterprises (Scotland, UK). The PKWS is a tropical region with a relatively high concentration of ectomycorrhizal associations. It is located in Chaiyaphum province, consisting of a complex of eigth contiguous protected areas in the western part of NE Thailand, and covers and area of 4,594 square kilometers. The western Isan forest complex is the only sizeable expanse of closed forest remaining in the region. It is unique in that it is able to sustain viable populations of wildlife species requiring large home ranges (e.g. tigers and elephants) [5]. It is also important in supporting a range of IUCN Red Listed animals and birds and is essential for conserving water resources in what is otherwise a hot and dry environment [6].
The scant research on fungi for the area has been partly addressed by excursions to describe the macrofungi associated with deciduous and mixed deciduous forest with pine. During the rainy season (July-September) in both 2010 and 2011, a subepigeous, gasteroid fungus was encountered exhibiting characteristics associated with the genus Astraeus Morgan (Order Boletales, clade Sclerodermatineae in [7]) and Geastrum Pers (Order Geastrales in [8]) (Fig. 1). The goal of this study is to identify the phylogenetic placement of this fungus using sequences of LSU nrDNA, 5.8S nrDNA, RPB1, RPB2 and EF1-a, as well as compare its morphology to that of other species of star-shaped gasteroid fungi.

Fungal specimens
All necessary permits were obtained for the described field studies issued by Department of National park, wildlife & plant conservation, Bangkok, Thailand (Reference document number 0907.1/17723).
Basidiomes were collected in Phu Khieo Wildlife Sanctuary, Chaiyaphum province, Thailand, during the months of July and September, 2010 and 2011. Field characters such as peridial and glebal colours (Colour identification chart, Royal Botanic Garden, E, 1969) and textures, etc. were recorded in the field and in the laboratory. Basidiospores were mounted in Melzer's reagent [9] and examined and photographed using light microscopy at magnifications of 400-10006 (DIC BX51 Olympus). Mean spore size and range was determined by measuring the diameter of at least 30 spores. Ornamentations were described and later analysed using scanning electron microscopy (SEM). For SEM, spore samples were air-dried, mounted, and sputter-coated with gold before being scanned using a JEOL JSM-840 scanning electron microscope. Peridium structure was examined under polarization microscopy (Imager A1, Zeiss). Attempts to culture the mycelium from fresh basidiomes using a modified Melin Norkrans's medium (MMN) were unsuccessful. Specimens are deposited in BBH, E and MA-Fungi.

DNA isolation, amplification and sequencing
Genomic DNA was extracted from specimens mentioned in Table 1. DNeasy Plant Mini Kit (Qiagen) was used according to the manufacturer's instructions. Five loci were amplified: a) the partial of 59 end of nuclear ribosomal large subunit RNA gene sequences (nrLSU) with primers LR0R, LR3R, LR5, and LR7 [10]; b) the internal transcribed spacer of nuclear ribosomal DNA (ITS) with primers ITS1F and ITS4B [11]; c) the largest subunit of RNA polymerase II gene sequences (RPB1) with primers RPB1-Af (59-GAR TGY CCD GGD CAY TTY GG-39) and RPB1-Cr (59-CC NGC DAT NTC RTT RTC CAT RTA-39) [12]; d) the second largest subunit of RNA polymerase II gene sequences (RPB2) with primers RPB2-f5F (59-GAY GAY MGW GAT CAY TTY GG-39) [13] and RPB2-b7R (59-GAY TGR TTR TGR TCR GGG AAV GG-39) [14]; and e) the transcription elongation factor 1-alpha (EF1-a) with primers 983F (59-GCY CCY GGH CAY CGT GAY TTY AT-39) and 2218R (59-ATG ACA CCR ACR GCR ACR GTY TG-39) [15]. Polymerase chain reactions (PCR) contained 0.4 U Phire Hot Start II DNA Polymerase (Finnzymes, Sweden), 16 Phire Plant PCR Buffer with 1.5 mM MgCl 2 , 200 mM of each dNTP and 0.5 mM of each primer. The ITS amplification was run on an Eppendorf thermocycler (Eppendorf, Germany) using the following parameters: initial denaturation of 5 min at 98uC, followed by 40 cycles each with a denaturation step of 5 s at 98uC, annealing for 5 s at 57uC, an elongation step of 20 s at 72uC, and a final elongation step of 10 min at 72uC. The same conditions were used for nrLSU, RPB1, RPB2 and EF1-a amplification except that the annealing temperatures were 50uC, 55uC, 55uC and 57uC, respectively. Amplicons were purified using the QIAquick PCR Purification Kit (Qiagen) and then sequenced at the 1 st BASE laboratories Sdn Bhd (Malaysia). Except for RPB2 amplicon was cloned using TA cloning kit (Invitrogen) into Escherichia coli TOP10 before sequenced. Sequences were assembled and edited with BioEdit [16]. BLASTN queries with MEGABLAST option were used to compare sequences obtained against sequences in the National Center of Biotechnology Information (NCBI) nucleotide database [17]. All new sequences have been deposited on the EMBL-EBI database and their accession numbers are presented in Table 1.

Phylogenetic analysis
Two datasets were created for this study. One is a multigene dataset that examines the phylogenetic position of the gasteroid fungus from PKWS using ribosomal RNA and protein coding genes (nrLSU, the 5.8S region of the ITS, RPB1, RPB2 and EF1a). Genes missing for individual samples were coded as ''?'' in the dataset to represent missing data. A second dataset consisted of only ITS sequence data to compare this taxon against other known Astraeus species. Both datasets, consisting of original sequences, plus sequences acquired from Genbank, were aligned using MUSCLE [18] with additional manual adjustments to the alignment performed in Mesquite 2.74 [19].
For each dataset, maximum likelihood and Bayesian analyses were performed using the CIPRES web portal (http://www.phylo. org/portal2/) [20]. Maximum likelihood bootstrapping analyses was performed on each dataset with RAxML 7.2.8 [21], using the default parameters as implemented on the CIPRES NSF XSEDE resource with bootstrap statistics calculated from 1000 bootstrap replicates. Bayesian phylogenetic analyses were performed using Mr Bayes v. 3.2.1 [22] on CIPRES XSEDE resource with default parameters (Nst = 6, with 2 runs, 4 chains per run, each run searching for 1000000 generations sampling every 1000 th generation).

Nomenclature
The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.
The new taxon described herein has been submitted to MycoBank and the unique MycoBank number provided can be used to retrieve the associated taxonomic information at http:// www.mycobank.org/MycoTaxo.aspx?Link = T&Rec = .

Phylogenetic analysis
BLAST searches with megablast option were used to compare the sequences obtained (nrLSU and RPB1 around 1460 and 1310 bp, respectively) against the sequences in the National Center of Biotechnology Information (NCBI) nucleotide databases   [7]. To evaluate the phylogenetic position of the sclerodermatoid fungus from PKWS, a multigene dataset was created using nrLSU, 5.8S, RPB1, RPB2 and EF1-a genes from 80 specimens. This dataset was rooted using the Boletinellaceae (Boletellus and Phlebopus) while the genera Astraeus, Calostoma, Diplocystis, Gyroporus, Phlebopus, Scleroderma and Tremellogaster consisted of the ingroup. Maximum likelihood bootstrap (MLB) and Bayesian posterior probabilities (PP) strongly support a monophyletic placement for the sclerodermatoid fungus with Astraeus (MLB = 99%, PP = 1.0; Fig. 2). With it's inclusion in Astraeus, there is a strong sister relationship with the monotypic genus Tremellogaster (MLB = 97%, PP = 1.0) and weak support for the inclusion of these taxa, along with Diplocystus to form the Diplocystidiaceae (MLB = 71%, PP = 0.95). Sequences use for both phylogenetic datasets and their corresponding GenBank accession numbers are given in Table 1.
An ITS dataset was developed to evaluate the uniqueness of this new taxon relative to other Astraeus species. This dataset consists of 28 samples (2 outgroup samples from Gyroporus). Maximum likelihood and Bayesian phylogenetic analysis identifies eight major clades that can be recognized as species (each with MLB. 98% and PP = 1.0; Fig. 3). Five of these represent taxa already Figure 2. Maximum likelihood tree from a multigene dataset reveals the placement of Astraeus sirindhorniae within the Sclerodermatineae. 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). doi:10.1371/journal.pone.0071160.g002 Astraeus sirindhorniae Figure 3. Maximum likelihood tree from ITS dataset identifies Astraeus sirindhorniae as a distinct species of Astraeus. 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). doi:10.1371/journal.pone.0071160.g003 defined by Phosri et al. [23]. Four samples of the new Astraeus taxon form a strongly supported group distinct from the other major Astraeus clades (MLB = 100%, PP = 1.0) which we will from now on refer to as Astraeus sirindhorniae.

Diagnostic description
Basidiomycota: Boletales: Sclerodermatineae. Large, subglobose to ellipsoid, subepigeous, dry basidiomes splitting at maturity to form a non-gelatinised, exoperidium with rays that unfold into a star-shaped structure. Enclosed within the exoperidium is a pale, thin, dry, stipitate endoperidium containing a powdery gleba of date-brown to umber (Colour identification chart, Royal Botanic Garden, E, 1969), large, globose, distinctly but minutely verrucose spores ,11 mm diam. and lacking a columella.
Distribution Note. Her Royal Highness the Crown Princess of Thailand, has considered and granted for a new fungus name; A. sirindhornii. This name is a great honor and a privilege. However according to ICBN Recommendation 60C.1(b) If the personal name ends with a consonant (but not in -er), substantival epithets are formed by adding -i-(stem augmentation) plus the genitive inflection appropriate to the sex and number of the person(s) honoured (e.g. lecard-ii for Lecard (m), wilson-iae for Wilson (f), verlot-iorum for the Verlot brothers, braun-iarum for the Braun sisters, mason-iorum for Mason, father and daughter). Therefore A. sirindhornii should ending with -iae and then the epithet to be spelled; A. sirindhorniae.

Discussion
Astraeus sirindhorniae represents a new species of star-shaped gasteroid fungus which differs morphologically from many other genera of star-shaped fungi. In comparing this species, the earthstar genus, Geastrum, tends to have a well defined peristome. Myriostoma species may be distinguished by the formation of multiple irregular shaped peristomes from which spores escape. Trichaster differs in having an endoperidium that remains attached to the exoperidium after opening, then soon disintegrates leaving a powdery spore-mass suppported by a stout, persistent collumella. The endoperidium of Terrostella is thin and peels away to expose a powdery spore-mass supported by a distinct sterile base. Phialastrum has a strongly developed columella and Geasteropsis produces an extremely hard basidiome when dry.
According to Phosri et al. there are only two Astraeus species in Thailand, A. odoratus and A. asiaticus [23,24]. Astraeus odoratus is found under ecological conditions similar to those at the Phu Khieo Wildlife Sanctuary. However, A. sirindhorniae differs in its much larger basidiomes, both when immature and when its rays are fully expanded, displaying flared margins, and exposing complex layering. Astraeus sirindhorniae is further differentiated from A. odoratus through the presence of prominent rhizomorphs, a complex multi-layered exoperidium, and smaller basidiospores (range 6-11 mm). These basidiospores are also smaller than A. asiaticus spores (8.75-15.2 mm) and generally given for A. hygrometricus s. str. viz. (7.5-12 mm) [25,26,27,28,29]. The spore ornamentation of A. sirindhorniae is notable under SEM as it has moderately dense, rounded, narrow, tapered, separate tubercles, which coalesce spines in groups. In addition, A. sirindhorniae has a short stipitate, very fluffly-fibrillose endoperidium when immature, which further differentiates this taxon from other Astraeus species.
The outermost felty, scaly covering of the young basidiomes of A. sirindhorniae closely resembles that of members of Scleroderma previously placed in Veligaster. The gleba is probably not divided into tramal plates. As in A. sirindhorniae clamp-connections are absent from both the gleba and the peridium. On maturing the highly gelatinized middle layer is exposed well before the powdery gleba is revealed. In A. sirindhorniae the peridial medio-and subpeillis are not gelatinized and the hyphae are fully differentiated but otherwise the very young basidiomes are similar in primordial structure.
In the multi-gene phylogenetic analyses A. sirindhorniae, along with other Astraeus species, form a monophyletic clade with Tremellogaster, and Diplocystis (Fig. 2). This clade is recognized as the Diplocystidiaceae. The structure of the peridium in Tremellogaster is also rather complex: the outer wall consists of thickened, sclerotised hyphae; the middle layer is brown and heavily gelatinised and divided into polygonal areas of plate-like, nongelatinous tissue; and the innermost layer consisting of hyaline, thin-walled hyphae similar to those in A. sirindhorniae but posses transverse thickenings. A summary of the pertinent characters and literature references for Tremellogaster are given in Watling [30].
Members of the Sclerodermatineae form ectomycorrhizal associations with many host plants. Species of Astraeus are known to associate with ectomycorrhizal plant hosts in the Pinaceae, Betulaceae, Fagaceae, Ericaceae and Dipterocarpaceae [31]. Given its phylogenetic placement, and the fact that it is found in dipterocarp dominated forests, it is likely that A. sirindhorniae is also an ectomycorrhizal fungus. Further study into the ecology of this species is needed in order to conclusively identify possible relationships to dipterocarpacious hosts.
In the multigene phylogeny the basal position of A. sirindhorniae relative to other Astraeus taxa is interesting from a biogeographic standpoint (Fig. 2). This placement suggests a Southeast Asian origin for the genus, which is observed in many Sclerodermatineae genera [31]. However, this is complicated by the fact that the basal Diplocystidiaceae (Diplocystis and Tremellogaster) are monotypic genera whose species are described from the new world (the Caribbean and South America respectively). Further investigation into the biogeogaphic history of these taxa is necessary to understand the current distribution of new-and old-world Astraeus.

Conclusions
In summary A. sirindhorniae is morphologically distinguished from A. odoratus, A. asiaticus and A. hygrometricus s.l. by basidiome and basidiospore size, spore ornamentation and peridium structure. Phylogenetic analysis clearly resolves Astraeus sirindhorniae as a basal lineage of Astraeus, within the Diplocystidiaceae and Sclerodermatineae. This systematic relationship, in combination with its associations with dipterocarp forests, it is probable that this species is ectomycorrhizal with members of the Dipterocarpaceae. Astraeus sirindhorniae represents a new gasteroid, star-shaped fungus from Thailand. This discovery reinforces the belief that fungi represent a group of organisms with many undescribed taxa; some of which exist within the dry evergreen dipterocarp forests of SE Asia.