Fungal Diversity (2013) 58:215–226 DOI 10.1007/s13225-012-0211-8 Borofutus, a new genus of Boletaceae from tropical Asia: phylogeny, morphology and taxonomy Md. Iqbal Hosen & Bang Feng & Gang Wu & Xue Tai Zhu & Yan Chun Li & Zhu L. Yang Received: 10 May 2012 /Accepted: 22 October 2012 /Published online: 30 October 2012 # Mushroom Research Foundation 2012 Abstract A new monotypic genus in the Boletaceae, Australia; Halling and Mueller 2003 in Costa Rica; Ortiz- Borofutus, typified by B. dhakanus, is described using morpho- Santana et al. 2007 in the Caribbean region; Halling and logical and molecular evidence. This is a putatively ectomycor- Fechner 2011 in Australia). Recently, three new genera of rhizal fungus associated with Shorea robusta. Borofutus is Boletaceae, Durianella Desjardin et al., Spongiforma characterized by the combination of the following characters: Desjardin et al., and Corneroboletus N. K. Zeng & Zhu L. basidiomata small to medium-sized; pileus grayish brown to Yang were described from tropical Southeast Asia (Desjardin et cocoa brown; hymenophore subdecurrent, cream then golden al. 2008, 2009;Zengetal.2012). Forests in this region are often brown, with broad, nearly hexagonal pores; basidiospores pur- dominated by Shorea robusta, a tree which plays an important ple to purplish red in H2O, ornamented with irregular to regular role as ectomycorrhizal partner of boletes and other groups of shallow pits; cystidia lageniform, thick-walled. Borofutus is fungi. During the last four monsoons (2009–2012), a broad sister to Spongiforma in molecular phylogenetic analyses using survey of mushrooms was conducted in Bangladesh and sev- DNA nucleotide sequences of single or multiple loci. A de- eral boletes were collected. One of the bolete species belongs to scription, line drawings, phylogenetic placement and compari- the porcini mushroom “Alloboletus” based on DNA sequence son with allied taxa are presented herein. data (Feng et al. 2012).Recently,aboleteassociatedwithS. robusta was collected and carefully examined. The broad-pores Keywords Boletes . Dipterocarpaceae . Multiple gene with subdecurrent hymenophore indicate that this bolete repre- analysis . New fungal taxon sents a new genus in the Boletaceae. In this study, we used morphological data together with DNA nucleotide sequence analysis of multiple loci including the 5.8S region of ITS, the large subunit nuclear ribosomal Introduction RNA (nrLSU), translation elongation factor 1-alpha (tef1-α), the largest subunit of RNA polymerase II (rpb1) and the second Fungi in the Boletaceae have been widely studied by fungal largest subunit of RNA polymerase II (rpb2). Both morpholog- taxonomists and molecular mycologists (Watling 1970;Smith ical and molecular phylogenetic analyses are indispensable for and Thiers 1971;BeslandBresinsky1997; Binder and Hibbett understanding the taxonomic and phylogenetic relationships 2007). However, research on samples collected from tropical among the genera within the Boletaceae (Yang 2011). This regions, such as tropical Southeast Asia, South America, and work addresses the following two objectives: (i) to compare Africa is limited (Corner 1972 in Malaysia; Heinemann and the morphological characters among Borofutus and the related Rammeloo 1987a, b in Africa; Wolfe and Bougher 1993 in genera, and (ii) to assess the phylogenetic position of Borofutus. M. I. Hosen : B. Feng : G. Wu : X. T. Zhu : Y. C. Li : Z. L. Yang (*) Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Materials and methods Kunming 650201, Yunnan, China e-mail: [email protected] Sampling M. I. Hosen : G. Wu : X. T. Zhu University of Chinese Academy of Sciences, Materials were collected by the first author from Bhawal Beijing 100049, China National Park, Gazipur, Dhaka, Bangladesh during 2009– 216 Fungal Diversity (2013) 58:215–226 2012 in forests dominated by S. robusta. Specimens exam- 1 μL of both forward and reverse primer, 2.5 μLPCR ined are deposited in the Herbarium of Kunming Institute of reaction buffer, 2.5 μLdNTP,0.3μL Taq polymerase, Botany (KUN-HKAS) of the Chinese Academy of Sciences, 1 μLBSA,and1.5μL DNA template. The final volume China and two duplicates as SHAF 1 and SHAF 2 are kept wasadjustedto25μL by adding double distilled water in SAU Herbarium of Agarics Flora (SHAF), Department of (ddH2O). PCR reactions were conducted on an ABI 2720 Plant Pathology, Faculty of Agriculture, Sher-e-Bangla Thermal Cycler (Applied Biosystems, USA) and the reaction Agricultural University, Dhaka, Bangladesh. conditions were as follows: pre-denaturation at 94 °C for 5min,thenfollowedby35cyclesofdenaturationat94°C Morphological studies for 40s (ITS), 60s for (nrLSU, rpb1, rpb2andtef1-α); anneal- ing at 50 °C (ITS) for 40s, 53 °C (nrLSU, rpb1, rpb2andtef1- The macro-morphological features were described based on α) for 60s; elongation at 72 °C (ITS) for 40s, 80s for (nrLSU, detailed field notes made from fresh basidiomata, and docu- rpb1, rpb2andtef1-α); a final elongation at 72 °C for 8 min mented by photographs. Color codes are according to was included after the cycles. PCR products were purified Kornerup and Wanscher (1981). Microscopic structures were with a Gel Extraction & PCR Purification Combo Kit (Spin- revived in 5 % KOH. Radial-vertical sections of the pileipellis column) (Bioteke, Beijing, China) and then sequenced on an and longitudinal sections of the stipitipellis were made half- ABI-3730-XL sequence analyzer (Applied Biosystems, USA) way the pileus and the stipe, respectively. All microscopic using the same primers as in the original PCR amplifications. features were drawn by free hand. The notations ‘(n/m/p)’ indicate that the measurements were made on ‘n’ basidio- DNA sequence alignments and dataset assembly spores from ‘m’ basidiomata of ‘p’ collections with a mini- mum of 20 basidiospores per basidioma. Dimensions of The nrLSU nucleotide sequences from Borofutus are com- basidiospores are presented in the following form (a) b–c pared with those submitted to GenBank database and a single (d); in which ‘b–c’ contains a minimum value of 90 % and 94 % match with the nrLSU sequence of Spongiforma thai- extreme values ‘a’ and ‘d’ are kept in parentheses. Q 0 length/ landica (DED 7873) was identified. This level is consistent width ratio derived from each basidiospore measured; Qm0Q with other congeneric comparisons in the Boletales (Binder ±SD, where Q is the average of basidiospores measurement and Hibbett 2007). Subsequently, the nrLSU sequences of and SD is the standard deviation. Basidiospores were also representatives of Boletaceae were downloaded from observed using a scanning electron microscope (SEM). Tiny GenBank and combined with our own dataset. The sequences pieces of hymenophoral fragments dried by silica gel were were aligned with MAFFT v6.8 (Katoh et al. 2005)and mounted on aluminum SEM stubs with double-sided adhesive manually optimized on BioEdit v7.0.9 (Hall 1999) using tape. The samples were coated with gold palladium (thickness default settings. To understand the relationships of our sam- 10 nm) and provided 8,600 nA current flow at 10 s to enhance ples with the remaining genera in Boletaceae,5.8S,tef1-α, conductivity. Images were obtained via a SEM (HITACHI S- rpb1 and rpb2 sequences of Boletaceae were also retrieved 4800) using a working distance of 7,700 μm and accelerating from GenBank and combined with selected nrLSU sequences. voltage of 10.0 kV. Procedures followed for the alignment and optimization of this dataset were identical to those of the nrLSU dataset. In total 47 sequences were newly generated for this study and Molecular studies deposited in GenBank including nine sequences each for ITS and tef1-α, twelve for nrLSU, ten for rpb1 and seven for rpb2 DNA extraction and PCR amplification (Table 1). The nrLSU and the combined datasets were comple- mented with selected published sequences (Binder and Fischer Genomic DNA was extracted from silica-gel dried or her- 1997; Bresinsky et al. 1999;BinderandBesl2000;Binderand barium materials using a modified Cetyltrimethylammonium Bresinsky 2002a, b;Grubishaetal.2002; Peintner et al. 2003; bromide (CTAB) procedure of Doyle and Doyle (1987). Leonardi et al. 2005; Matheny et al. 2006; Binder and Hibbett ITS1/ITS4 (White et al. 1990), LROR/LR5 (Vilgalys and 2007; Halling et al. 2007; Matheny et al. 2007; Drehmel et al. Hester 1990), ef1-983F/ef1-1567R (Rehner and Buckley 2008;Porteretal.2008; Desjardin et al. 2009, 2011;Binderet 2005), rpb1-Af/rpb1-Cr(Dentingeretal.2010)andrpb2- al. 2010; Dentinger et al. 2010;Lietal.2011; Lebel et al. 2012; 6F/rpb2-7.1R (Matheny 2005)wereusedfortheamplifi- Neves et al. 2012) and those from GenBank. To assemble the cations of ITS, nrLSU, tef1-α, rpb1 and rpb2fragments, combined dataset, nrLSU, 5.8S, tef1-α, rpb1 and rpb2 sequen- respectively. In addition, we used the three primers pair cesusedinBinderetal.2010 were retrieved and combined with ef1-BF/ef1-BR, rpb1-BF/rpb1-BR and rpb2-BF/rpb2-BR, our data. The resulting five alignments (nrLSU, 5.8S, tef1-α, newlydesignedbyG.Wu(unpublisheddata),toamplify rpb1 and rpb2) were then concatenated using Phyutility tef1-α, rpb1 and rpb2 fragments. PCR mixtures contained (Smith and Dunn 2008) for further phylogenetic analysis. Fungal Diversity (2013) 58:215–226 217 Table 1 Specimens used in phylogenetic analyses and their GenBank accession numbers Species Name Isolate/Voucher/strain Locality GenBank Accessions ITS, 5.8S nrLSU tef1-α rpb1 rpb2 Aureoboletus