Spongiforma thailandica, apical pore, KOH, basidiospores, Thailand, reddish brown, DED, Spongiforma, Tropical Forest Science, Applied Biosystems, R. Halling, Binder, Thailand Desjardin, molecular sequences, Star Lab Co., Ltd., molecular phylogenetics, herbarium specimens, Khao Yai National Park, Peninsular Malaysia, Journal of Bacteriology, Journal of Tropical Forest Science, Fungal Diversity, Ectomycorrhizal fungi, Dipterocarpus gracilis Blume, phylogenetic analyses, Sinauer Associates, Research and Development, smooth surface, D.E. Desjardin, Gymnopaxillus nudus Claridge, tree topologies, confidence values, Spongiforma Desjardin, Spongiforma thailandica Desjardin, Porphyrellus, San Francisco State University, Mahidol University, phylogenetic analysis, Clark University, morphological features, independent analyses, Dennis E. Desjardin, central Thailand, Diversity Desjardin, San Francisco, CA
Spongiforma, a new genus of gasteroid boletes from Thailand
Desjardin, D.E.1*, Binder, M.2, Roekring, S.3 and Flegel, T.4 1Department of Biology
, San Francisco State University
, 1600 Holloway Ave., San Francisco, CA 94132 2Department of Biology, Clark University, 950 Main St., Worcester, MA 01601 3Asia Star Lab Co., Ltd., research and development
, 9 Soi Prachanimitr, Pradipat Road, Samsennai Phayathai, Bangkok 10400, Thailand 4Centex Shrimp, 4th Floor Chalermprakiat Bldg., Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand Desjardin, D.E., Binder, M., Roekring, S. and Flegel, T. (2009). Spongiforma, a new genus of gastroid boletes from Thailand. Fungal Diversity 37: 1-8. Based on morphological and molecular characters, Spongiforma is described as a new genus of gasteroid boletes belonging in the Boletineae. It is represented by a single species, S. thailandica, that is putatively mycorrhizal with dipterocarp trees in central Thailand. Unusual morphological features include a sponge-like, astipitate, epigeous basidiome with large exposed locules and a strong coal tar odor, and rugulose, reddish brown basidiospores with an apical pore that become smooth and violet grey in 3% potasium hydroxide solution. A description, illustrations, phylogenetic analysis
and comparison with allied taxa are presented. Key words: Agaricomycotina, Basidiomycota, Boletineae, molecular phylogenetics, taxonomy. Article Information Received 27 October 2008 Accepted 4 March 2009 Published online 1 August 2009 *Corresponding author: Dennis E. Desjardin; email: [email protected]
Materials and Methods
Although Southeast Asia
is home to numerous genera of boletes (Basidiomycota, Agaricomycotina, Boletales), few secotioid or gasteroid taxa have been reported from the region (Corner and Hawker, 1953; Corner, 1972; Watling and Lee, 1995, 1998; Lee et al., 2002; Halling et al., 2007). Recently, specimens of an unusual, epigeous, astipitate and sponge-like species were collected from Khao Yai National Park in central Thailand. Basidiomes have a rubbery-gelatinous texture, emit a strong coal tar odor, and are associated with Shorea henryana Pierre and Dipterocarpus gracilis Blume (Dipterocarpaceae) in a primary forest. A unique set of macro- and micromorphological features in combination with molecular sequences of the nuclear large subunit (nuc-lsu) gene region indicate that the organism represents a new genus in the Boletineae. A description, illustrations, phylogenetic analysis and comparison with allied taxa are presented herein.
Morphology Macromorphological data were derived from fresh specimens, whereas micromorphological data were derived from dried specimens rehydrated in ethanol followed by distilled water, 3% KOH or Melzer's reagent. Duplicate specimens are deposited in BBH and SFSU. DNA extraction, PCR and DNA sequencing Collections used in this study for molecular analyses are listed in Table 1. Genomic DNA from herbarium specimens was isolated following the phenol/chloroform procedure by Lee and Taylor (1990). The crude DNA extracts were diluted up to 1000-fold with deionized water for use as PCR templates. PCR reactions were performed for two nuclear rDNA regions using the primer combinations ITS1-F-ITS4 (ITS region including the 5.8S gene) and LR0R-LR5 (nuc-lsu). Sequences of primers used in this study have been described elsewhere (Vilgalys and Hester, 1990; White
Table 1. Isolate numbers, location, date, collector and accession numbers for fungal isolates sequenced for use in this study.
Species Chalciporus rubinellus Porphyrellus pseudoscaber Spongiforma thailandica Strobilomyces sp. Tylopilus atronicotianus
Isolate 191/81 RH8716
U.S.A., ME 8/1981
U.S.A., CA 11/15/2005 R. Hall
GenBank accession numbers
DED7873 Thailand 7/7/2005 D. Desjardin EU685113 EU685108
RH4514 Australia 2/1992
R. Halling ---
Both s.n. U.S.A., NY 9/18/2004 E. Both
et al., 1990; Gardes and Bruns, 1993). The amplifications were run in 35 cycles on a PTC200 thermal cycler (MJ Research, Waltham, Massachusetts) using the following parameters: denaturation 94єC (1 min), annealing 50єC (45 sec), extension 72єC (1.5 min). PCR products were purified using Pellet Paint (Novagen, EMB Biosciences, San Diego, California). All PCR products were sequenced using BigDye terminator sequencing 3.1 chemistry (Applied Biosystems, Foster City, California), and run on an Applied Biosystems 3130 genetic analyzer. Contiguous sequences were assembled and edited using Sequencher 4.7 (GeneCodes Corp., Ann Arbor, Michigan). Automated alignments that were generated with ClustalX (Thompson et al., 1997) were manually adjusted in MacClade 4.08 (Maddison and Maddison, 2005). Datasets and phylogenetic analyses Initial blast searches (Altschul et al., 1997) using the ITS sequence of S. thailandica as a query produced exclusively hits in the Boletaceae, however, without providing significant results at the generic rank. The newly generated nuc-lsu rDNA data were first blasted and then, along with the best blast hits, aligned into the dataset of Binder and Hibbett (2006), which was narrowed down to 85 taxa after several rounds of consecutive bootstrapping and pruning of redundant sequences. The final alignment included 933 positions and the data were analyzed using maximum-parsimony (MP), maximum-likelihood (ML) and Bayesian methods. A non-parametric MP bootstrap analysis was performed using 1000 replicates in PAUP*4.0b10 (Swofford, 2002), all characters equally weighted, one random taxon addition sequence, and tree bisection
reconnection (TBR) branch swapping. RAxML v. 2.2.3 (Stamatakis, 2006) was used to run 100 ML replicates under the GTRMIX model and, in addition, 1000 ML bootstrap replicates under the GTRCAT model. A 50% majorityrule consensus tree was built from the resulting 1000 trees in PAUP* to estimate confidence values. Finally, posterior probabilities (PP) were determined in two independent analyses by running one cold and three incrementally heated Metropolis-coupled Markov chain
Monte Carlo (MCMCMC) analyses for 3 Ч 106 generations using MrBayes v3.0b4 (Ronquist and Huelsenbeck, 2003), saving trees every 100th generation. The GTR++I model was specified as prior, assuming equal probability for all trees and unconstrained branch length. The final burn-in period determining the stationary proportion of trees saved after likelihood scores converged to a stable equilibrium was estimated using Tracer v1.4 (Rambaut and Drummond, 2007). A 50% majority-rule consensus tree was generated from the stationary trees in PAUP* to estimate confidence interval
s. The results of the analyses are summarized in Fig. 1. Results Phylogenetic analyses The complete ITS sequence obtained from Spongiforma thailandica, consisting of the two spacer regions and 5.8S gene, has a size of 682 bp. Blast searches using the entire ITS sequence and the three regions separately as queries produce only matches for the 5.8S gene, and place the species in the Boletaceae. Inferences on the nuc-lsu dataset under parsimony, likelihood, and Bayesian methods yielded highly similar tree topologies,
Fungal Diversity Fig. 1. phylogenetic relationships
of Spongiforma inferred from nuc-lsu rDNA data using RAxML. One of 100 trees is shown here (-lnL = 10299.869) and BS support values > 50% are indicated along nodes, while PP > 0.98 are marked as thickened branches. A GenBank accession number is provided for published sequences and the isolate number is provided for newly generated data. 3
suggesting that the new genus Spongiforma is the sister group of Porphyrellus. Long Branch
es produced by species in both genera seem to obscure this relationship to some degree. Nevertheless, high statistical support received from three different methods suggests that this finding is based on a true phylogenetic signal and not a result of long-branch attraction. In addition, the independent Bayesian runs under a GTR++I model, which was predetermined (Binder & Hibbett 2006) without testing the suitability of simpler models for this analysis, repeatedly and quickly converged after 230 000 generations. Thus, the clade including Spongiforma and Porphyrellus is consistently resolved in all analyses presented here (Fig. 1). Taxonomy Spongiforma Desjardin, Manf. Binder, Roekring & Flegel gen. nov. MycoBank: MB 512541 Etymology: Referring to the sponge-like basidiomes. type species
: Spongiforma thailandica Desjardin, Manf. Binder, Roekring & Flegel Basidiomata epigaea, sessilia, cerebriformia. Peridium nulla. Gleba loculis labyrinthiformibus, 2-20 mm lata, brunneis vel rubrobrunneis; columella irregularis, pyriformis, cremea-alba. Basidiosporae amygdaliformae, symmetricae, rugulosae, apiculatae cum poro, inamyloideae, cyanophileae. Basidia statismosporicae, 4-sporigera. Cystidia cylindrica vel ventricosa-rostrata. Trama glebae gelatinosae. Fibulae nulla. Basidiomes epigeous, sessile, cerebriform to sponge-like with rubbery-gelatinous texture; peridium absent; locules 2-20 mm diam., irregular in outline, lined with a smooth, greyish orange to brown or reddish brown hymenium, sterile ridges white to creamcoloured; columella poorly developed, pyriform, cream-coloured, attached to white rhizomorphs. Basidiospores brown to vinaceous brown in mass, amygdaliform, bilaterally symmetrical, rugulose, with a central apiculus and small apical pore, reddish brown in water, violet grey in 3% KOH, inamyloid, cyanophilic. Basidia statismosporic, 4-sterigmate. Cystidia common on sterile locule edges, cylindrical to ventricose-rostrate, hyaline. Tramal hyphae gelatinous, inamyloid. Clamp connections absent.
Spongiforma thailandica Desjardin, Manf. Binder, Roekring & Flegel sp. nov. (Fig. 2a-f) MycoBank: MB 512542 Etymology: Referring to the country in which the species grows. Basidiomata epigaea, sessilia, cerebriformia, 50100 Ч 40-70 mm, globosa vel ovoidea. Peridium nulla. Gleba loculis labyrinthiformibus, 2-20 mm lata, brunneis vel rubrobrunneis; columella irregularis, pyriformis, cremea-alba. Basidiosporae (9)1011.5(12.5) Ч 5.57(7.5) µm, amygdaliformae, symmetricae, rugulosae, apiculatae cum poro, inamyloideae, cyanophileae. Basidia statismosporicae, 2532 Ч 6.59.5 µm, cylindrica vel subclavata, 4-sporigera, sterigmata usque 9.5 µm. Cystidia 2548 Ч 510 µm, cylindrica vel ventricosa-rostrata. Trama glebae gelatinosae. Fibulae nulla. Holotypus hic designatus: Thailand, DED 7873 (BBH) Basidiomes (Fig. 2a-b) epigeous, 50100 mm diam Ч 4070 mm tall, astipitate, irregularly globose to ovoid, cerebriform to sponge-like, rubbery-pliant (not brittle; can be squeezed like a sponge to remove water and it will spring back to its original shape). Peridium absent. Hymenophore composed of ridges or folds delimiting empty locules; locules 215( 20) mm diam, irregular in outline, lined with a well-developed hymenium, pale greyish orange (5B3-4) to brownish grey (6C3) when young, becoming light brown (6D4-5) to brown (6E58), dark brown (6F5-8) or reddish brown (89E6-8) at maturity; sterile ridges paler, white to cream when dried; all tissues immediately deep purple to purplish black in 10% KOH. Spores brown (6E5-6) to light brown (6D5-6) or vinaceous brown (8E-F6). Columella 1015 mm tall Ч 810 mm diam (apex) Ч 34 mm diam (base), obconical to pyriform, apex lacunose, base folded, cream buff (4A3) to pale greyish orange (5B3); attached to fine, reticulate-branched, white rhizomorphs. Odor mild and pleasant when young, but soon becoming strong and unpleasant, of coal tar or burned rubber (like Tricholoma sulphureum), can be detected from 10 m distant. Peridium absent. Sterile ridges composed of erect chains of cells terminated by cystidia. Cystidia 2548 Ч 510 µm, cylindrical to subclavate or ventricose-rostrate, obtuse, hyaline or a few with pale brown cellular contents, inamyloid, thin-walled; abundant on the sterile locule edges and scattered amongst basidia near the
Fig. 2. Spongiforma thailandica. a-b. Basidiomes (DED 7485). c-d. SEM of air dried basidiospores (DED 7873, holotype). Note the distinct ornamentation and obvious apical pore. e-f. SEM of basidiospores mounted in 3% KOH (DED 7873, holotype). Note the loss of an apical pore and nearly smooth surface. -- Scale bar
s: a-b = 10 mm; c-f = 1 µm.
locule edges but absent elsewhere. Hymenophoral trama of subparallel to slightly interwoven hyphae 310 µm diam, cylindrical, branched, septa often inflated (like an elbow joint), gelatinous, hyaline, inamyloid, thinwalled. Subhymenium of inflated to vesiculose cells 920 Ч 914 µm, hyaline, inamyloid, thinwalled, non-gelatinous. Hymenial cystidia absent (except as noted above). Basidia statismosporic, 2532 Ч 6.59.5 µm, cylindrical to subclavate, 4-spored with straight
sterigmata up to 9.5 µm long, unclamped. Basidioles cylindrical to subclavate. Basidiospores (Fig. 2c-f) (9)1011.5(12.5) Ч 5.5-7( 7.5) µm [xmr = 10.711.1 Ч 5.96.5 µm, xmm = 11 ± 0.3 Ч 6.2 ± 0.4, Q = 1.52.1, Qmr = 1.7 1.8, Qmm = 1.76 ± 0.09, n = 20-25 spores per 2 specimens], amygdaliform, broadest at proximal end and gradually narrowed towards distal end, bilaterally symmetrical, with a small central apiculus, subtruncate at distal end and forming a tiny, narrow pore that may have a
small lip, walls thick (0.5 µm), thinner at the distal end, roughened to rugulose overall and reddish brown in water (Fig. 2c-d), appearing nearly smooth and violet grey in 3% KOH (Fig. 2e-f), inamyloid, cyanophilic. Clamp connections absent in all tissues. Habit and habitat: Solitary, in soil under Dipterocarpus sp. and Shorea sp. in primary forest. Known distribution: Thailand. Material examined: THAILAND, Nakorn Nayok Province, Khao Yai National Park, Princess Trail ca 2 km from Visitor Center, N14°26.142', E101°23.080', elev. ca 750 m, 9 July 2002, E. Horak, T. Flegel and D.E. Desjardin as DED 7485 (BBH, SFSU); same location, 7 July 2005, D.E. Desjardin, DED 7873 (holotype BBH, isotype SFSU). Notes: Diagnostic features of Spongiforma thailandica include the following: a relatively large, sponge-like and rubbery basidiome colored pale brownish grey to brown or reddish brown with irregular, relatively large locules lined with sporogenous tissue; no stipe but a small columella that is attached to copious, fine white rhizomorphs; a strong coal tar odor (like Tricholoma sulphureum); tissue that turns purple in 3-10% KOH; amygdaliform basidiospores with a tiny apical pore that are reddish brown and rugulose-roughened in water (Fig. 2c-d) but become violet grey, collapsed and nearly smooth in KOH (Fig. 2ef); and the absence of clamp connections. Discussion The new Thai taxon is superficially similar to Gymnopaxillus nudus Claridge, Trappe & Castellano, a species described from material associated with Eucalyptus in Australia (Claridge et al., 2001). In common with Spongiforma, Gymnopaxillus nudus forms astipitate, cerebriform, bright ferrugineous basidiomes that lack a peridium and have relatively large locules, a basal columella, white rhizomorphs and cyanophilic, bilaterally symmetric basidiospores. However, the genus Gymnopaxillus forms smooth basidiospores that are ellipsoid to subfusoid, golden yellow in KOH and lack a tiny apical pore. In addition, G. nudus differs in forming primarily hypogeous basidiomes, forms a larger and more welldeveloped columella, lacks a strong odor, lacks a purple KOH reaction, has longer
basidiospores (11-16 µm), and is mycorrhizal with Eucalyptus. Molecular data indicate that G. nudus is allied with Austropaxillus species (Jarosch 2001) and together they are sister of Serpula in the Serpulaceae, all distantly related to Spongiforma (Binder and Hibbett, unpubl. data). Our results drawn from the nuc-lsu dataset indicate that Spongiforma belongs in the Boletaceae where it is sister of Porphyrellus (with 84% MP BS and 93% RAxML BS; PP = 1.0), although on a long branch. Together, this group forms the sister clade of Strobilomyces (Fig. 1), which is resolved in all analyses but is only supported by PP = 0.98. As noted in previous studies, ITS sequences of Porphyrellus and Strobilomyces spp. are highly divergent (Lutzoni et al., 2004), and we were not able to align both taxa to the S. thailandica sequence or the latter sequence to any other taxa in the Boletaceae. Bolete species with reddish brown to vinaceous brown, rugulose to perforatepunctate basidiospores have been placed historically in a number of taxa including Boletus subgen. Tylopilus (Corner, 1972), Tylopilus subgen. Porphyrellus, Austroboletus (both Wolfe, 1979), or Porphyrellus (Singer, 1945; Wolfe and Petersen, 1978). Two Southeast Asian species with basidiospore features very similar to those of Spongiforma thailandica are currently named Austroboletus tristis (Pat. & C.F. Baker) Wolfe and A. longipes (Massee) Wolfe. All three latter species form basidiospores with rugulose to rugulose-punctate surface that turn purple to violaceous grey in KOH. SEM photos of the type specimens of A. tristis and A. longipes (Figs 1, 2 and 4, respectively in Wolfe and Petersen, 1978) clearly show ornamentation similar to that of S. thailandica (Fig. 2c-d) although an apical pore was not reported on basidiospores of any Austroboletus species. Recently collected material of A. longipes from Malaysia confirms these observations (Chan & Halling, NYBG, pers. comm.). Sequences of A. tristis and A. longipes were not available for inclusion in our analyses. It is interesting to note, however, that in our analyses (Fig. 1), Spongiforma was more closely allied with the smooth-spored Porphyrellus (P. porphyrosporus (Fr.) E.-J. Gilbert, P. pseudoscaber Secr.
ex Singer, P. sordidus (Frost) Snell) than with the rugulose-punctate-spored Austroboletus (A. mucosus (Corner) Wolfe, A. niveus (G. Stev.) Wolfe, A. gracilis (Peck) Wolfe). An apical pore at the distal end of basidiospores is a rare feature in the boletes. Thiers (1975) reported the basidiospores of Porphyrellus amylosporus A.H. Sm. as dark reddish brown, smooth and truncate with a thin-walled depression. Several Old World species of Heimioporus (= Heimiella) form basidiospores with apical pores, such as Heimiella sp. 2 as illustrated in two SEM photographs by Watling and Hollands (Figs 11-12; 1989), and H. fruticicola (Berk.) E. Horak (R. Halling, NYBG, pers. comm.). In our molecular analyses (Fig. 1), Heimioporus is distantly related to Spongiforma. Another interesting similarity is that the pileipelli of Austroboletus and Porphyrellus species is a (ixo-) trichodermium formed from chains of short, cylindrical to submoniliform cells with cylindrical to clavate terminal cells. This anatomy is similar to that of the sterile edges of the tissues that delimit the locules in Spongiforma, which may represent vestigial pileipellis (peridium) tissue. Spongiforma thailandica was first reported from Thailand as Hymenogaster sp. and illustrated with two color photographs in Ruksawong and Flegel (2001: 248). Acknowledgments This research was funded in part by National Science Foundation
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DE Desjardin, M Binder, S Roekring