Mycol Progress DOI 10.1007/s11557-013-0935-1

ORIGINAL ARTICLE

Three new species of Hyphodontia with peg-like hyphal aggregations Eugene Yurchenko & Sheng-Hua Wu

Received: 3 August 2013 / Revised: 15 September 2013 / Accepted: 22 September 2013 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2013

Abstract Descriptions, illustrations and phylogenetic analyses for three new taxa of Hyphodontia (Basidiomycota) are provided. Hyphodontia astrocystidiata sp. nov. was collected from warm-temperate mountain forests of Taiwan; H. microfasciculata sp. nov. was found in subtropical Taiwan, southern China, and northern Vietnam; H. vietnamensis sp. nov. was described from northern Vietnam. All three species have sterile projecting peg-like structures, consisting of encrusted hyphae. Hyphodontia astrocystidiata is characterized by Resinicium-like astrocystidia on subicular hyphae and astrocystidia-like hyphal ends in hymenium. Hyphodontia microfasciculata is distinguished by its small-sized ellipsoid spores and fine emergent fascicles composed of thin-walled, flexuous hyphae. The features of H. vietnamensis are subulate cystidia, cylindrical spores, and projecting, densely arranged slender clusters of acuminate hyphae. The morphology of crystal deposits on projecting hyphae in these three species was studied by means of scanning electron microscopy. Molecular phylogeny analyses were performed by maximum parsimony and Bayesian methods, both based on sequences of the internal transcribed spacer region of nuclear ribosomal DNA. The phylograms from both analyses confirm independent specific status of H. microfasciculata and H. vietnamensis. The former species sits in a subclade with Hyphodontia sambuci , the latter species is in another subclade with H. crustosa. Hyphodontia astrocystidiata is placed close to Schizopora paradoxa, but the two species are distinct in terms of morphology. E. Yurchenko (*) Department of Biotechnology, Paleski State University, Dnyaprouskai flatylii str. 23, BY-225710 Pinsk, Belarus e-mail: [email protected] S.
Keywords Corticioid fungi . ITS . Schizoporaceae . SEM . Taxonomy

Introduction Hyphodontia J. Erikss., a member of the Hymenochaetales, or hymenochaetoid clade of homobasidiomycetes (Larsson 2007), is a ubiquitous corticioid genus with a large number of species. Its species occur in all natural zones with forests, and cause white rot of both small-sized and largesized dead wood mostly found lying on the ground. The morphological concept of the genus in its broad sense was outlined by e.g. Eriksson and Ryvarden (1976), Langer (1994), Maekawa (1994), and Vesterholt (1997). According to Hjortstam and Ryvarden (2009), Hyphodontia s. l. could be split into 13 genera, of which the most species-rich ones are Xylodon and Kneiffiella. Nevertheless, due to a lack of rDNA sequences for many taxa, the current molecular grounds are not enough to define all the derivative genera clearly, and in this paper we adopt a broad concept of Hyphodontia s. l. The studies of the genus in southeastern Asia had a predominant focus on Taiwan, and 22 new species were described from there (Lin and Chen 1990; Wu 1990, 2000, 2001, 2006; Xiong et al. 2009, 2010; Yurchenko and Wu 2013), whereas 50 species were recorded for the whole China (Dai 2011; Lee and Langer 2012; Yurchenko and Wu 2013). The present work gives the descriptions of three new species of Hyphodontia s. l. from Taiwan, mainland China and Vietnam, each based on 2–15 collections. Sterile aculei observed in these species are not similar microscopically with those described as true pegs, e. g. in the genus Epithele (Wang et al. 2010), and thus we apply to them the term ‘peg-like hyphal aggregations’ or ‘peg-like structures’.

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Materials and methods

Alignment and phylogenetic analysis

Morphology study

The data matrix for phylogenetic analysis included eight sequences obtained in this study and 46 sequences retrieved from GenBank. One or two sequences for each species of Hyphodontia s. l., available in GenBank, were included in the data set (Table 1). Species concepts and nomenclature are retained by us mostly such as they are held in GenBank records. Fibrodontia gossypina, earlier treated in Hyphodontia , but actually a member of the trechisporoid clade (Larsson 2007), was chosen as the outgroup. Sequences were aligned by MAFFT v.6 software (Katoh et al. 2009) on the web server (http://mafft.cbrc.jp/ alignment/server), with Q-INS-i strategy. Before this, a preliminary alignment was done using BioEdit v.7.0.4.1 software (Hall 1999), and large subunit (LSU) rDNA segments were removed manually to provide a length of each sequence not exceeding 900 nucleotides. The final data matrix of the aligned sequences was prepared in BioEdit and deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/ TB2:S14470). Two separate phylogenetic analyses were performed. Heuristic parsimony search was carried out in PAUP* 4.0b10 (Swofford 2002), with 100 random addition sequence replicates and other options (including TBR branch-swapping algorithm) using the program’s default settings. All transformations were considered unordered and equally weighted. Gaps were treated as missing data. Relative robustness of clades was assessed by bootstrapping (Hillis and Bull 1993) in PAUP*, with 100 bootstrap replicates and other options as the program’s default settings. Bayesian inference of phylogeny was performed with MrBayes v.3.2.1 (Ronquist and Huelsenbeck 2003) for the same data set and outgroup. MrModeltest 2.3 (Nylander 2004) with the Akaike information criterion (Posada and Buckley 2004) was used to assess the best-fit model of nucleotide evolution for the whole ITS region, as well for ITS1, ITS2, and 5.8S subregions separately. The suggested models were implemented in partitioned Bayesian phylogenetic analysis. Eight Metropolis-coupled Markov chain Monte Carlo chains with temperature 0.2 were initiated, and these were run for 1 M generations, in four independent runs, with tree and parameter sampling every 100 generations. The initial burn-in was set to 0.5. A majority-rule consensus phylogram was computed from the remaining 5,000 trees.

The herbarium material collected in Taiwan in 1991–1994 and 2011, southern China in 1997, Vietnam in 1998, and stored in TNM (some duplicates in MSK; collection acronyms follow Index Herbariorum, http://sweetgum.nybg.org/ih), were examined. The collector’s number is provided in the text for each specimen, together with the reference Museum number (TNM F). The descriptions are based on dried basidioma specimens. Microscopic measurements and observation of the color of hyphae and other structures were carried out in 3 % KOH water solution. Spore dimensions were determined on the spores taken from fruit body sections or squash preparations. Incrustations on hyphae and cystidia were studied in Melzer’s reagent (Mz’s). Spore wall amyloidity was checked in Mz’s, and cyanophil reaction was tested in cotton blue-lactophenol solution. Photographs of basidioma surfaces were captured by digital camera Nikon Coolpix P6000 through an ocular of Zeiss Stemi SV11 stereomicroscope. For electron microscopic study, basidiomata pieces were attached to aluminium stubs, kept overnight at +40 °C in drying oven and coated by ca. 300 Å thick platinumpalladium layer by sputtering in RF IB-2 ion coater. Stubs were examined and images obtained in a scanning electron microscope Hitachi SU 1510, operating at accelerating voltage of 15 kV.

DNA extraction, amplification and sequencing The analysis of phylogenetic relations for the new species was based on nuclear ribosomal DNA (rDNA) sequence data, derived from the internal transcribed spacer (ITS) region, including internal transcribed spacers 1, 2, and 5.8S gene. DNA was obtained from mycelia after cultivation of isolates, taken from the TNM fungal culture collection (H. arguta and H. astrocystidiata), or from herbarium specimens—dry basidioma pieces about 1–1.5 cm2 in size, removed with a razor blade from the substratum (H. microfasciculata and H. vietnamensis). Total genome DNA was extracted with Plant Genomic DNA Miniprep Extraction Kit (Viogene, Taiwan), according to the manufacturer’s protocol. Before extraction, the material was frozen by liquid nitrogen and ground into fine powder. The ITS region was amplified using primer pair ITS1/ITS4 (White et al. 1990), and PCR products were purified with PCR-M Clean Up System (Viogene). Nucleotide sequences were determined on ABI 3730 DNA Analyzer (Applied Biosystems, USA) with ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems).

Results Taxonomy Hyphodontia astrocystidiata Yurchenko & Sheng H. Wu sp. nov. Figs. 1, 2 and 9a

Mycol Progress Table 1 Data on the taxa and specimens used in phylogenetic study Speciesa

Isolate / voucher specimen No.

GenBank accession number for ITS1-5.8S-ITS2

Fibrodontia gossypina Parmasto Hyphodontia abieticola (Bourdot & Galzin) J. Erikss. H. abieticola H. alutacea (Fr. : Fr.) J. Erikss.

GEL 5042 (AFTOL-ID 599) GEL 2924 / KHL 12498 (GB) GEL 2284

DQ249274 DQ340332 DQ873601 DQ340340

H. alutacea H. alutaria (Burt) J. Erikss. H. alutaria H. arguta (Fr. : Fr.) J. Erikss. H. aspera (Fr.) J. Erikss. H. astrocystidiata H. barba-jovis (Bull. : Fr.) J. Erikss. H. breviseta (P. Karst.) J. Erikss. H. cineracea (Bourdot & Galzin) J. Erikss. & Ryvarden H. cineracea H. crustosa (Pers. : Fr.) J. Erikss. H. crustosa H. crustosa H. crustosa H. floccosa (Bourdot & Galzin) J. Erikss. H. hastata (Litsch.) J. Erikss. H. hastata H. heterocystidiata H.X. Xiong et al.

GEL 2937 / KHL 11889 (GB) / KHL 11978 (GB) Wu 0806-44 / TNM F22588 2004b / Nilsson s. n. (GB) Wu 9211-71 / TNM F24764 / KHL 11730 (GB) / KHL 12386 (GB) GEL 4875 GEL 4958 GEL 2325 GEL 5336 GEL 5360 / KHL 11731 (GB) / Berglund 150–02 (GB) GEL 2143 GEL 3124 Wu 9209-27 / TNM F24726

DQ340338 DQ873603 EU118631 JN571548 DQ873606 JN129972 DQ873609 DQ873612 DQ340335 DQ340336 DQ340313 DQ340314 DQ340315 DQ873614 DQ873618 DQ340323 DQ340311 JX175045

H. heterocystidiata H. juniperi (Bourdot & Galzin) J. Erikss. & Hjortstam H. juniperi H. microfasciculata H. mollis Sheng H. Wu H. nespori (Bres.) J. Erikss. & Hjortstam H. nespori H. niemelaei Sheng H. Wu H. nothofagi (G. Cunn.) E. Langer H. nothofagi H. pallidula (Bres.) J. Erikss. H. palmae (Rick) E. Langer H. pruni (Lasch) Svrček H. radula (Pers. : Fr.) E. Langer & Vesterh. H. radula H. rimosissima (Peck) Gilb. H. sambuci (Pers. : Fr.) J. Erikss. H. sambuci H. subalutacea (P. Karst.) J. Erikss.

Wu 9312-20 / TNM F22246 GEL 4940 Wu 0910-95 / TNM F23863 TNM F24757 (uncultured) Wu 0808-32 / TNM F22659 GEL 3309 / Nordon 030915 (GB) GEL 4998 ICMP 13842 / PDD 91630 GEL 2097 GEL 3456 / Ryberg 021018 (GB) ICMP 13832 / PDD 91616 / Ryberg 021031 (GB) GEL 3376 GEL 3400 GEL 2196

JX175046 DQ340316 JX175047 JN129976 JX175043 DQ340307 DQ873622 EU583422 AF145583 GQ411524 DQ340317 DQ340333 DQ873624 AF145581 GQ411525 DQ873627 DQ340325 DQ340326 DQ340341

H. aff. subalutacea H. tropica Sheng H. Wu H. tropica H. vietnamensis

GEL 2142 ICMP 13835 ICMP 13837 / TNM F9073 (uncultured) 16187Tell / MA-Fungi 73256 1DETLAG / MA-Fungi 5758 / KHL 12261 (GB)

DQ340334 AF145586 AF145587

Lagarobasidium calongei M. Dueñas et al. L. detriticum (Bourdot) Jülich Palifer verecundus (G. Cunn.) Stalpers & P.K. Buchanan

JX175044 FM876212 FM876211 DQ873642

Mycol Progress Table 1 (continued) Speciesa

Isolate / voucher specimen No.

GenBank accession number for ITS1-5.8S-ITS2

Rogersella griseliniae (G. Cunn.) Stalpers Schizopora flavipora (Berk. & M.A. Curtis) Ryvarden S. flavipora S. paradoxa (Schrad. : Fr.) Donk S. paradoxa S. paradoxa

/ KHL 12971 (GB) FCUG 1053 ICMP 13836 FCUG 1517 FCUG 2425 Miettinen 7978

DQ873651 AF145575 AF145585 AF145572 AF145571 FN907912

a

Emboldened taxa indicate sequences obtained in this study

MycoBank no.: MB 800644 Diagnosis. Hymenial surface minutely odontioid; aculei сomposed of thin-walled hyphae, apically encrusted by acute crystals; subicular hyphae with astrocystidia; basidiospores ellipsoid or broadly ellipsoid, 5–6 μm long. Holotype. Taiwan, Yilan County, Taipingshan, 2000 m asl., on twig of Rhododendron formosanum Hemsl., 19.XI.1992, leg. S.H. Wu, Wu 9211-71 (TNM F24764; isotype in MSK). Etymology. ‘Astrocystidiata’ refers to the presence of cystidia with stellate cap of crystals. Basidioma effused, from 2–6 mm to about 10 cm long, softmembranaceous, 70–130(150) μm thick excluding aculei. Hymenial surface cream-colored, minutely odontioid due to the presence of broadly conical to cylindrical, peg-like hyphal aggregations, 25–80 μm high, 25–70 μm broad at base, evenly distributed, 11–16/mm; basidioma between peg-like projections porulose-reticulate. Margin abrupt or slightly byssoid. Hyphal system monomitic, all hyphae clamped, (2)2.5– 4(5) μm diam. Subiculum loose, thin to moderately thick. Subicular hyphae moderately branched, colorless, thin-walled to thick-walled, naked or scarcely encrusted. Subhymenial hyphae moderately branched, fairly densely arranged, thinwalled, colorless (but in mass pale yellow), sparsely encrusted. Cystidia or cystidium-like structures of several types: (1) astrocystidia, as lateral branches on subicular hyphae, scattered

Fig. 1 Macromorphology of Hyphodontia astrocystidiata (holotype): basidioma in central (left) and in marginal part (right). Bars 1 mm

to abundant with stem 5–7.5×1–1.5 μm, apically slightly swollen, with a stellate crystalline cap 4.5–8.5 μm diam; (2) encrusted hyphal ends and astrocystidia in hymenium fairly numerous, 3–4 μm wide, thin-walled, with crystalline cap reaching 7.5–9 μm diam; (3) slightly moniliform (with 2 constrictions) and lecythiform cystidia in hymenium occasional, 18–23×5–5.5 μm; (4) bladder-like cystidia occasional, up to 10 μm wide, in hymenium or in peg-like projections. Hyphae in peg-like projections flexuous, apically blunt, occasionally subacute, generally clamped and occasionally simple-septate, about 3.5 μm diam, colorless, with thin to slightly thickened walls, above encrusted by acute crystals. Basidia utriform, with 1(2) constriction, 15–20×4–5 μm, colorless, thin-walled, with four subulate sterigmata (2.5–5.5×0.7 μm). Basidiospores ellipsoid or broadly ellipsoid, 5–6×3.7–4.2 μm, colorless, thin-walled, smooth, with a small apiculus, negative in Mz’s, slightly cyanophilous. Additional specimen examined. Taiwan, Yilan County, Taipingshan, 1900 m asl., on twig of Rhododendron formosanum, 19.XI.1992, leg. S.H. Wu, Wu 9211-66 (TNM F24763). Distribution. The collections are known from warmtemperate, northeast Taiwan. Remarks. The shape of crystals on projecting hyphae in H. astrocystidiata is partly similar to that on immersed

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Fig. 2 Micromorphology of Hyphodontia astrocystidiata (holotype). a, b Vertical sections through basidioma. c Astrocystidia on subicular hyphae (in Mz’s). d Astrocystidia (in KOH). e Peg-like cluster of projecting hyphae. f Apices of projecting hyphae. g Encrusted hyphal

apices from hymenium. h Capitate and subcapitate cystidioles from hymenium. i Bladder-like cystidia. j Basidia. k Basidiospores in KOH. l Basidiospores in Mz’s. Bars a =100 μm, b =50 μm, e, h, i =10 μm, c, d, f, g, j, k, l =5 μm

astrocystidia. Astrocystidia on subicular hyphae in this species have typical morphology characteristic of the genus Resinicium Parmasto, and were never observed in Hyphodontia before. However, hyphae morphology, basidia shape, and absence of coarse crystalline aggregations in the subiculum are typical to Hyphodontia s. l., not to Resinicium. In the latter genus the subiculum is thin, hyphae are often narrow, thin-walled, partly agglutinated and obscured by crystalline masses, basidia

semipedunculate, cylindrical to clavate, and spores are never subglobose in described species (Hjortstam and Melo 1997; Nakasone 2007). Hyphodontia microfasciculata Yurchenko & Sheng H. Wu sp. nov. Figs. 3, 4 and 9b MycoBank no.: MB 800645 Diagnosis. Hymenial surface minutely odontioid due to small emerging fascicles of flexuous hyphae, encrusted by more or less

Mycol Progress Fig. 3 Macromorphology of Hyphodontia microfasciculata (holotype): basidioma in central (left) and in marginal part (right). Bars 1 mm

isodiametric crystals; capitate cystidia in hymenium scattered, 3.4–5 μm wide; basidiospores ellipsoid, 4–5×3–3.5 μm. Holotype. Taiwan, Yilan County, Fushan Botanical Garden, 600 m asl., on fallen twig of angiosperm, 8.VIII.1991, leg. S.H. Wu, Wu 910808-56 (TNM F24757; isotype in MSK). Etymology. Specific epithet refers to the characteristic minute peg-like fascicles of hyphae. Basidioma effused, from 0.2–3 to 10 and more cm long, membranaceous, 60–100 mm thick excluding hymenial surface projections. Hymenial surface cream-colored, warted to minutely odontioid due to the presence of peg-like hyphal aggregations 40–55 μm long and 20–45 μm broad at base, evenly distributed, 8–9/mm; basidioma between them almost continuous or slightly porulose, slightly cracking. Margin thinning out. Hyphal system monomitic, all hyphae clamped, colorless. Subicular hyphae moderately branched, loosely arranged, (1.5)2–3 μm diam, thin-walled to slightly thick-walled, colorless, naked or poorly encrusted. Subhymenial hyphae richly branched, of fairly dense arrangement, 2–4 μm diam, thinwalled, non-encrusted. Peg-like projections consisting of flexuous hyphae 2.5–3.5 μm wide, with clamped septa, apically blunt or slightly subcapitate, colorless, thin-walled above to slightly thick-walled in lower part, loosely to richly encrusted by unequally-sized, more or less isodiametric crystals, from 0.5–1 to 2.5–4 μm in size. Cystidia scattered, capitate, situated in hymenium and subhymenium, often curved, 15–18×3.5–4 μm, colorless, thin-walled, non-encrusted. Hymenium scarcely encrusted. Basidia shortly utriform to suburniform, somewhat flexuous, 14–15.5(18.5)×(3.5)4– 4.5 μm, colorless, thin-walled, non-encrusted, with four subulate sterigmata about 3–4×0.5 μm. Basidiospores ellipsoid, 3.8–5.2×3–3.5 μm, thin-walled, colorless, usually with an amorphous inclusion, smooth, with indistinct apiculus, negative in Mz’s, slightly cyanophilous. Additional specimens examined . China , Yunnan, Hsishuangpanna, Manglun Nature Reserve, 750 m asl., on branch of angiosperm, 17.VIII.1997, leg. S.H. Wu and S.Z. Chen, Wu 9708-235 (TNM F7873). Taiwan , Kaohsiung

County: Liukuei, Shanping, 750 m asl., on dead branch of angiosperm species, 7.XI.1991, leg. S.H. Wu, Wu 911107-49 (TNM F24758); on dead branch of angiosperm species, 7.XI.1991, leg. S.H. Wu, Wu 911107-66 (TNM F24759); Miaoli County: Sanyi, SE foot of Sanjiao Mt., 330 m asl., broad-leaved evergreen forest, on fallen twigs, 17.VI.2011, leg. E.O. Yurchenko, EYu 110417-15 (TNM F24775); Nantou County: Yushan National Park, Nanhsi Forest Road, 1600 m asl., on dead branch of angiosperm species, 6.X.1992, leg. S.H. Wu, Wu 9210-9 (TNM F24762); 1850 m asl., on dead branch of angiosperm species, 13.X.1993, leg. S.H. Wu and S.Z. Chen, Wu 9310-27 (TNM F24770); 2200 m asl., on dead branch of angiosperm species, 14.X.1993, leg. S.H. Wu and S.Z. Chen, Wu 9310-119 (TNM F24771); Tsuifeng, 2300 m asl., on branch of angiosperm, 21.IX.1993, leg. S.H. Wu, Wu 9309-5 (TNM F24741); Taipei County: Kungliao, 200 m asl., on dead branches of angiosperm species, 25.VII.1991, leg. S.H. Wu, Wu 910725-6 (TNM F24755), Wu 910725-9 (TNM F24756), 25.VII.1991, leg. Y.F. Lin, Lin 580 (TNM F24772); Yangmingshan, 600 m asl., on dead branch of angiosperm species, 7.III.1991, leg. S.H. Wu, Wu 910307-2 (TNM F24754); 600 m asl., on dead branch of angiosperm species, 20.XI.1992, leg. S.H. Wu, Wu 9211-135 (TNM F24765). Vietnam, Phu Tho Province, Ba Vi National Park, 1200 m asl., on branch of angiosperm, 3.VII.1998, leg. S.H. Wu and S.Z. Chen, Wu 9807-37 (TNM F9025). Distribution. Hyphodontia microfasciculata is known from Taiwan, China (the south of Yunnan Province, near the border to Laos), and northern Vietnam. The known records were done between 21 and 25°N. The species was collected at the altitudes from 200 to 2300 m, in the belts of broadleaf subtropical to temperate mixed coniferous montane forests. Remarks. Hyphodontia microfasciculata resembles Pteridomyces capitatus Boidin & Gilles [Hyphodontia capitata (Boidin & Gilles) Hjortstam] in the morphology of projecting hyphae and cystidia, but in contrast with the genus Pteridomyces, subicular hyphae in H. microfasciculata have thickened walls and hyphae in the fascicles are encrusted. This species is also morphologically close to H. candidissima

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Fig. 4 Hyphodontia microfasciculata (holotype). a, b Vertical sections through basidioma. c Subicular hyphae. d Fascicle of encrusted projecting hyphae. e Capitate cystidia. f Basidia. g Basidiospores. Bars a =100 μm, b =50 μm, c, d =10 μm, e, f, g =5 μm

(Berk. & M.A. Curtis) E. Langer, but the latter has acute hymenial cystidia, larger spores, and lack of capitate cystidia. Hyphodontia serpentiformis E. Langer resembles H. microfasciculata, but according to the original diagnosis, the former has both thick-walled hyphae and tubular tramal cystidia, basidia slightly thick-walled at base, and larger spores (4.5–5×3.5–4 μm) with a large guttula inside (Langer et al. 1992). Hyphodontia vietnamensis Yurchenko & Sheng H. Wu sp. nov. Figs. 5, 6 and 9c

MycoBank no.: MB 563212 Diagnosis . Hymenial surface greyish, with minute, evenly distributed peg-like structures, consisting of richly encrusted, conglutinate hyphae; hymenial cystidia numerous, irregularly subulate; basidiospores cylindrical, (5)6–6.5(7)× 2.5–3 μm. Holotype. Vietnam, Ha Tay Province, Ba Vi National Park, 650 m asl., on dead corticated twig of angiosperm species, 4.VI.1998, leg. S.H. Wu and S.Z. Chen, Wu 9807-88 (TNM F9073; isotype in MSK).

Mycol Progress Fig. 5 Macromorphology of Hyphodontia vietnamensis (holotype): basidioma in central (left) and in marginal part (right). Bars 1 mm

Etymology. ‘Vietnamensis’ refers to the country where the species was collected. Basidioma effused, subceraceous. Hymenial surface whitish or pale greyish, very finely aculeate, due to the presence of peg-like hyphal aggregations, 40–50 μm tall, 20–35 μm diam at base, evenly distributed, 11–15/mm, penicillate and wider apically due to somewhat diverging clusters of hyphae; between sterile aculei basidioma very thin, minutely porulose. Margin abrupt to diffuse and then up to about 1 mm broad. Hyphal system monomitic, all hyphae with clamps, colorless. Subiculum very thin, of subhorizontal or disordered hyphae. Subicular hyphae moderately branched, thin-walled to slightly thick-walled, 1.2–2.5 μm diam, colorless. Coarse crystals, up to 8 μm across (observed in Mz’s), common throughout basidioma thickness. Subhymenium not clearly differentiated from subiculum. Peg-like projections consisting of wavy, thin-walled, conglutinate, apically acute to obtuse hyphae, 2–3 μm diam, richly encrusted by crystals up to 6 μm across. Cystidia in hymenium, subulate, often flexuous and somewhat constricted, 15–25×3–4.5 μm, smooth or slightly encrusted, apically often with minute, little differentiated capitulus. Basidia utriform to suburniform, rarely almost cylindrical, with 1–2 slight constrictions, 17–20×3.5–4.5 μm, colorless, thin-walled, often somewhat encrusted at base; sterigmata (2)4, when mature subulate, up to 4.5×0.5 μm, immature cylindrical, blunt, about 3×1–1.5 μm. Basidiospores cylindrical, (5)6–6.5(7)×2.5–3 μm, colorless, thin-walled, smooth, with minute apiculus, sometimes glued together in tetrads, Mz’s-negative, acyanophilous. Additional specimens examined. Vietnam, Ha Noi, Tam Dao National Park, 1050 m asl., on twigs of angiosperm, 30.VI.1998, leg. S.H. Wu and S.Z. Chen, Wu 9806-93 (TNM F8977), Wu 9806-94 (TNM F8978), Wu 9806-97 (TNM F8981). Distribution. So far known from Vietnam only. Remarks. Hyphodontia vietnamensis resembles H. crustosa in cystidia and spore morphology, but differs from the latter by sterile aculei (peg-like projections), thinner basidioma between

the aculei, shorter basidia that are not repetitive, and slightly smaller spores. Molecular phylogeny The length of DNA fragments, obtained in this work with primers ITS1/ITS4, was from 605 to 708 bp. The final alignment for 54 sequences consists of 599 positions including introduced gaps; 268 positions of these were constant, 85 were variable but parsimony uninformative and 246 (41 %) were parsimony informative. Maximum parsimony (MP) analysis yielded 22 equally most parsimonious trees (score of the best tree=1424, length of the rooted tree=1625, consistency index=0.3711, homoplasy index=0.6289). Figure 7 shows one of the most parsimonious trees together with branch support, as estimated via bootstrap method. MrModeltest suggested HKY+G, SYM+G and GTR+G as the best-fitting models of nucleotide evolution for ITS1 (positions 1–73 in the data set), 5.8S (positions 74–194) and ITS2 (positions 195–599), respectively. The best model for the ITS region in the whole and for the joint ITS1+ITS2 sequences was GTR+I+G. In Bayesian analysis, the average standard deviation of split frequencies reached 0.011 after 1 M generations. Stationary phase in cold chain likelihood values distribution was reached well before the burn-in threshold was imposed. Chain mixing was found to be satisfactory. Figure 8 shows the 50 % majority-rule consensus phylogram from the Bayesian analysis. All three new species occurred in the ingroup, together with other representatives of Hyphodontia s. l. The ingroup with respect to the outgroup was found to be monophyletic in MP analysis and monophyletic except for Lagarobasidium in the Bayesian analysis. Little bootstrap support for the deeper nodes was found in MP analysis. The phylograms derived from both MP and Bayesian analyses demonstrate very similar topology in respect of terminal branches. The results of both MP and Bayesian analyses confirm independent specific status of the new taxa, supported by the local

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Fig. 6 Micromorphology of Hyphodontia vietnamensis (holotype). a, b Vertical sections through basidioma. c Subicular hyphae. d Separate aculeus (peg-like structure). e Hypha from peg-like aggregation.

f Fragment of hymenium and subhymenium in vertical section. g Cystidia. h Basidioles. i Basidia. j Spores. Bars a =100 μm, b–e =10 μm, f–j =5 μm

tree topology and branch length. Hyphodontia microfasciculata joins with H. sambuci in a common subclade with robust bootstrap proportion (BS=98 %) and high Bayesian posterion

probability (PP=1.00). According to MP analysis, the number of supposed nucleotide changes between H. microfasciculata and H. sambuci is significant (46). Hyphodontia vietnamensis forms

Mycol Progress Fig. 7 One of 22 most parsimonious trees for Hyphodontia s. l. species, based on ITS data set. The number of estimated nucleotide substitutions is denoted above branches; the numbers below each bifurcation denote the per cent of bootstrap replicates (only values > 50 % are shown); names of the taxa with sequences obtained in this study are given in bold

one clade with H. crustosa and a specimen of H. juniperi from China (BS=82 %, PP=0.99). The phylogenetic distance measured in nucleotide changes between H. vietnamensis and H. crustosa is also significant (48). Hyphodontia astrocystidiata occurs in a well-supported clade (BS=94 %, PP=0.88) with Schizopora paradoxa and shows a short distance from the latter taxon (eight steps on MP phylogram; Fig. 7). Additionally, the phylogenetic position of two East Asian species, H. heterocystidiata and H. mollis, was elucidated for the first time in this study. Both species belong to a clade consisting of taxa classified in Xylodon, Schizopora and Palifer (Figs. 7 and 8). Hyphodontia heterocystidiata constitute an isolated phylogenetic lineage within this clade; H. mollis was found to be most phylogenetically close to H. nespori.

light microscope, the size and shape of the crystals were different between these taxa. The differences were confirmed by scanning electron microscopy (Fig. 9). We found a variation in morphology of separate crystals and their druses within a species, like variation in morphology of other structures (e.g. cystidia or basidia). However, there was a prevalent crystal morphological type or pattern for a species. In H. astrocystidiata (Fig. 9a) crystals were fairly large, mostly of narrow triangular shape, and collected in rosette-like structures. In H. microfasciculata (Fig. 9b), crystals were much smaller, mostly rhomboid, often with somewhat rounded corners, and collected in chaotic aggregations. In H. vietnamensis (Fig. 9c), crystals were middle-sized, of near cuboid shape, and often arranged in small globose aggregations.

Crystal morphology and species characterization

Discussion

All three species described here have fairly rich crystalline deposits on projecting hyphae. Examined primarily under a

The three new species described above have hyphal morphology and texture, shape of clamp connections and basidiospore

Mycol Progress Fig. 8 Majority-rule consensus phylogram for Hyphodontia s. l. species, obtained from Bayesian analysis of ITS data set. A. Lagarobasidium clade. B. Kneiffiella-Alutaceodontia clade. C. Hyphodontia clade. D. Hastodontia clade. E. XylodonLyomyces-Rogersella clade. F. Xylodon-Schizopora-Palifer clade. Bayesian posterior probability values for the clades are denoted above the branches; branches in bold indicate PP=1.00; branch length reflects estimated number of changes per site; names of the taxa with sequences obtained in this study are given in bold

morphology that are appropriate to Hyphodontia s. l. A discrepancy is that in Hyphodontia, hymenophore aculei are fertile, at least at their bases. Our hypothesis accepted before doing phylogenetic research was that all three species belong to Hyphodontia. This hypothesis was confirmed by MP and Bayesian analyses.

Phylograms obtained in MP and Bayesian analyses show a short distance between H. astrocystidiata and Schizopora paradoxa. However, significant morphological differences between them provide additional arguments that H. astrocystidiata is a distinct species. There are no minutely aculeate species, or species with peg-like projections,

Fig. 9 Scanning electron microphotographs of the apices of peg-like hyphal aggregations, covered by differently shaped crystals, in three Hyphodontia species. a H. astrocystidiata (holotype). b H. microfasciculata (holotype). c H. vietnamensis (holotype). Bars 5 μm

Mycol Progress

combined in the genus Schizopora. Hymenial astrocystidia are present only in Sch. cystidiata David & Rajchenb., but they are absent on subicular hyphae (Langer 1994). However, spores and occasional bladder-like cystidia in H. astrocystidiata are similar with those in Schizopora species. The relation of H. microfasciculata to H. sambuci, based on molecular characters, is partly in agreement with morphology and it is supported by the presence of capitate cystidia in hymenium and spore shape. But in contrast with H. sambuci, spores in H. microfasciculata are thinwalled, hyphae are mostly thin-walled, and subacute hymenial cystidia are absent. The phylogenetic position of H. vietnamensis shows no discrepancy with morphology. Based on all the main characters (hyphae, basidia, spores), this species can be referred to the crustosa-juniperi group. Separated position of H. juniperi GEL 4940 and H. juniperi Wu 0910-95 on the phylograms is most likely explained by a geographical differentiation within this taxon. GEL 4940 was collected in Réunion Island, far from the main species range of H. juniperi (E. Langer, pers. comm.). As seen from the phylogram based on Bayesian inference, Hyphodontia s. l. splits into several well-distinguished clades (Fig. 8). Three of them coincide with the derivative genera Lagarobasidium (A), Hyphodontia s. str. (C) and Hastodontia (D). In the MP phylogram, there are clades equal to the clades C, D, E and F, obtained in Bayesian analysis, but they have low bootstrap support. Probability support for Kneiffiella-Alutaceodontia clade (B) is weak (PP=0.57). The genera Xylodon , Schizopora , Palifer, Lyomyces and Rogersella are mixed within clades E and F, and thus have not phylogenetic support in our study. At the same time, only clades E and F are robustly supported (PP=0.99–1.00). All three new species are characterized by a common feature—the presence of sterile, peg-like hyphal aggregations of encrusted hyphae, projecting beyond hymenium. However, the species occur in the two different clades, E and F, and in the two subclades within E. Thus, peg-like hyphal aggregations must be considered as a paraphyletic character. The ingroup splitting obtained in this study is generally in agreement with the results of molecular phylogeny analyses involving Hyphodontia spp. and carried out by previous authors, namely: MP analysis, based on 5.8S and nucLSU (Larsson et al. 2004); parsimony ratchet analysis, based on nucLSU (Binder et al. 2005); Bayesian analysis, based on 5.8S and nucLSU (Larsson et al. 2006; Larsson 2007). Today the sequence data of rDNA are available for less than one-third of 95 species accepted in Hyphodontia in Index Fungorum (http://www. indexfungorum.org/Names/Names.asp). A splitting of the genus should wait until a denser molecular sampling has been done, resulting in well-supported subclades.

Acknowledgments The work was financially supported by National Science Council of the Republic of China (grants No. NSC 98-2621-B178-002-MY3 and NSC 100-2811-B-178-001). The authors are much indebted to David Mitchel (Northern Ireland Fungus Group, Belfast) for improvement of the English. We are acknowledged to the two anonymous reviewers for critical consideration of the manuscript. Our thanks go to Ms S.Z. Chen (TNM herbarium) for help in the management of the collections studied, and to Ms S.K. Hu (Department of Geology, National Museum of Natural Science) for assistance in obtaining SEM photographs.

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