First record of Borofutus dhakanus (Boletaceae, Leccinoideae) in Thailand

A new record of Borofutus dhakanus Hosen & Zhu L. Yang is reported from Chiang Mai Province, northern Thailand. The phylogenetic relationships of the Thai specimen were compared with those of the type species and allied genera from seven major subfamilies of Boletaceae. The trees were constructed using sequences of partial 28S rDNA combined with internal transcribed spacer (ITS) gene regions. The morphological characteristics of the fungus and its edibility are also discussed. Although many boletes have been documented from Thailand, most previous records are based only on morphological characters and there is an urgent need for taxonomic revision.


INTRODUCTION
Over the past 100 years, the diversity of macrofungi in Thailand has been investigated all over the country by foreign as well as Thai mycologists [1][2][3][4][5][6][7][8][9][10][11].The most up-to-date checklist contains 1978 species of Thai mushroom (Basidiomycetes) and was published in 2011 [12].Approximately 35 genera and 230 species of Boletales were recorded.This included 19  ).However, most reports have been based on morphological identification and they lack supporting molecular data.Some genera and species require revision with the aid of molecular techniques, alongside an examination of new collections.
During a field survey in 2010, small-to mediumsized basidiomata of a bolete with distinctly broad pores were collected from the forest area of Huai Hongkhrai Royal Development Study Center.The unique macro-and microcharacters, in combination with the molecular sequence of the large subunit ribosomal deoxyribonucleic acid (28S rDNA) and the internal transcribed spacer (ITS) gene region, indicate that the fungus is representative of the genus Borofutus Hosen & Zhu L. Yang, which was recently introduced as a new genus in the Boletaceae [13].The type species of the genus, Borofutus dhakanus Hosen & Zhu L. Yang, is known only from the Dhaka division of Bhawal National Park in Bangladesh.Description, illustrations, phylogenetic analysis within the Boletaceae as well as comparison of the nucleotide sequence alignment with the allied taxa are presented herein.

Fungal collection and morphological identification
Specimens of boletes were collected during early rainy season (July) from the dry dipterocarp forest area of Huai Hongkhrai Royal Development Study Center, Chiang Mai, Thailand.Macroscopic characters were described from fresh basidiomata, and documented by photographs.Codes of color follow Kornerup and Wanscher [14].Basidiomes were sectioned using a sharp razor blade and revived in lactoglycerol.One hundred basidiospores were measured and their dimensions are presented as n=number of measured spores, L m =average length, W m =average width, E=length/width ratio range, and Q=average of length/width ratio from all spores measured.The amyloid reaction was examined by Melzer's reagent.Dried material of the fungus was deposited at the Research Laboratory for Excellence in Sustainable Development of Biological Resources, Department of Biology, Faculty of Science, Chiang Mai University (SDBR) and the Biology Department's Herbarium of Chiang Mai University (CMUB).

DNA extraction
A small piece of fruit body, cut from the flesh section between the pileus and stipe, was placed into a 1.5-ml centrifuge tube containing 300 µl 2× cetyltrimethylammonium bromide (CTAB) buffer and stored at -20°C for subsequent DNA extraction using a modified procedure of Doyle [15].The small piece of tissue was ground with a mini pestle and sterilized quartz sand (200 mg).The extraction buffer was adjusted to 600 µl and placed in a 60°C incubator for 30 min, with gentle swirling.The solution was then extracted until no interface was visible with an equal volume of chloroform:isoamyl alcohol (24:1) at 13000 rpm for 30 min, two or three times.The supernatant phase containing the DNA was precipitated by the addition of 2.5 volumes of absolute ethanol and kept at -20°C overnight.The DNA pellet was washed with 70% ethanol twice, dried under vacuum, resuspended in TE buffer (1 mM EDTA, 10mM Tris-HCl, pH 8) and mixed with RNase A (1 mg ml -1 ).Genomic DNA was checked by electrophoresis on 1% agarose gels stained with ethidium bromide and visualized under UV illuminator.

Sequence alignment and phylogenetic analysis
The 28S rDNA and ITS sequences of the bolete CMUB39815 were compared with those available in the GenBank database.The sequences were 99% matches with all sequences of the same gene region of Borofutus dhakanus (28S rDNA: JQ928615-JQ928617, NG042663, ITS: JQ928606, JQ928607, NR120117).Both gene region sequences of CMUB39815 and all those from GenBank (B.dhakanus, 99% and Spongiforma spp., 94%) were checked in detail by multiple base pair alignment.In addition, the sequence datasets reported by Hosen et al. [13] and some other allied genera in the family Boletaceae were retrieved from GenBank and combined with our own sequences to analyze their phylogenetic relationship.
A total of 24 bolete taxa were included in the final dataset analyses (Table 1).Nucleotide sequences of the 28S rDNA and ITS gene regions were initially aligned and combined using BioEdit 7.2.5 [18] and/or Clustal X 1.83 with default parameter settings [19].The alignments were then manually optimized using BioEdit.Maximum likelihood (ML), maximum parsimony (MP) and neighbor-joining (NJ) analyses were conducted using MEGA6 [20] and PAUP*4.0b10[21].Tree searches were carried out using the heuristic method with a random stepwise addition and tree bisection and reconstruction branch-swapping algorithm.

Molecular studies
Comparison of the highest percentage matched 28S rDNA sequence of the bolete CMUB39815 with three sequences of the type species of Borofutus dhakanus [13] (99%: JQ928615-JQ928617) and Spongiforma spp.(94%: S. squarepantsii: HQ724509, HQ724510 and S. thailandica: EU865108, NG042464) from Gen-Bank showed that a good quality nucleotide sequence of CMUB39815 (910 bases) is longer than those of B. dhakanus JQ928617 (856), JQ928615 (846) and JQ928616 (804) with 54, 64 and 106 bases, respec-tively.In addition, all are similar throughout their sequence alignment and only at a few positions the bases are not the same.The differentiation between the sequences of B. dhakanus and the two species of Spongiforma were also investigated and the results are summarized in Table 2 and Fig. 2.
The combined dataset consisted of 851 and 652 nucleotides (including gaps) for 28S and ITS, respectively.In this alignment, 463 characters were constant, while 1040 characters were variable, of which 1343 were informative.For both datasets, phylogenetic trees generated from ML, MP and NJ analyses were almost identical, with minimal variation of the statistical support values.Phylogenetic trees generated from both 28S (data not shown) and the combined datasets (Fig. 3) showed that Borofutus formed an independent clade in the family Boletaceae, clearly separated by genetic distance, and clustered with the gasteroid bolete Spongiforma.The current phylogenetic relationship study of B. dhakanus CMUB39815 agrees well with the molecular analyses reported by Hosen et al. [13] and Wu et al. [22].These results inply that Borofutus is a member of Boletaceae with 99% bootstrap support in ML (Fig. 3) and it is sister to Spongiforma with high bootstrap support (100%).
However, the ITS sequences of some other species of Austroboletus, Porphyrellus and Zangia, as well as those of Retiboletus spp.(Leccinoideae), need to be combined with 28S sequences to resolve their phylogenetic placement within subfamilies.Analysis of some other gene regions such as atp6, tef1-α, rpb 1, rpb 2 and/or the small subunit (18S) might be needed to clarify their genetic lineage within the complex species of Boletaceae as well as at a higher taxonomic level [13,22,[23][24][25][26][27].
Multiple base pairing of the ITS sequence of Borofutus dhakanus CMUB39815 (800 bases) with those of three taxa, as reported by Hosen et al. [13], was observed: B. dhakanus JQ928606 (800), JQ928607 (745) and NR120117 (746) (Fig. 4).There is one site on the nucleotide sequence alignment that is shown as a deletion/insertion region at position 714-720.
The general morphological features combined with molecular evidence from the Thai specimen (CMUB39815) show that it is a best fit to B. dhakanus, which was described by Hosen et al. [13].This species is characterized by a small-to medium-sized fruit body with grayish brown to cocoa brown pileus; subdecurrent hymenophore that changes from cream to golden brown, with broad and nearly hexagonal pores; basidiospores that are purple to purplish red in water [13] or brown-violet to purplish red in lactoglycerol (this study), ornamented with irregular to regular shallow pits; cystidia lageniform and thickwalled.The Thai specimens have a slightly smaller pileus (25-50 mm vs. 30-65 mm) and the basidiospores are slightly shorter but equal in width (10.3-12.0×4.8-5.9 µm Q=2.0 vs. 10-14×4.5-6.5 μm, Q=2.3).The Thai specimen was found on red clay soil in a forest dominated by various species of Shorea and Dipterocarpus, features similar to the collection site of the holotype.However, the type specimen was reported as a mycorrhizal bolete associated with Shorea robusta.
Tropical forests support a wide diversity of macrofungi, often including ectomycorrhizal fungi, which play a major role in nutrient cycling by mobilizing nitrogen and phosphorus [28].Additionally, many species are also edible, for example, Boletus edulis, B. griseus, B. nobilis, Phlebopus portentosus, Tylopilus balloui and T. virens.Edible mushrooms are not only appreciated for their texture, flavor and nutritional properties, but also for their bioactive compounds that have been demonstrated to possess antitumor, antimicrobial and other activities [29][30][31][32].The present study also found that B. dhakanus is an edible species, an attribute not mentioned by Hosen et al. [13].It is collected for cooking and for sale in northeastern  Thailand (Fig. 1F and G).Further collections of B. dhakanus are needed for additional studies on its edibility as well as nutrition and content of bioactive compounds.

CONCLUSION
Basidiocarps of a small-to medium-sized bolete were collected during the rainy season (July, 2010) from dry dipterocarp forest areas of Huai Hongkhrai Royal Development Study Center in Chiang Mai Province.Based on both morphological and molecular analyses (28S rDNA and ITS sequences) this specimen (CMUB39815) was confidently identified as Borofutus dhakanus.

Fig. 2 .
Fig. 2. Multiple base pair indels in 28S rDNA sequence of Borofutus dhakanus CMUB39815 (910 bases), which is very similar to a sequence of JQ928615 but different from those of two Spongiforma species.Numbers refer to the positions in the alignment (260-360 and 810-840 are not shown because they are the same bases).

Fig. 4 .
Fig. 4. Multiple base pairs of ITS sequences of Borofutus dhakanus CMUB39815, JQ928606, JQ928607 and NR120117.Numbers refer to the position in the alignment from position 700-807 (1-700 are not shown because they are the same bases).

Fig. 3 .
Fig. 3. Phylogenetic relationship among representative specimens of Boletaceae (Leccinoideae) and Borofutus dhakanus CMUB39815 inferred from a combined 28S rDNA and ITS dataset analyzed by ML method with the Kimura 2-parameter model.There was a total of 1343 positions in the final dataset (1503 bases including gaps).

Table 1
Specimens used in the ITS and 28S rDNA phylogenetic study and their GenBank accession numbers.

Table 2
The 28S rDNA sequence length of Borofutus dhakanus CMUB39815, HKAS73785, HKAS73789 and HKAS73792 compared with those of two Spongiforma species.