C Nova Hedwigia Vol. 103 (2016) Issue 3–4, 339–347 published online May 24, 2016; published in print November 2016 Article Polyporus minutosquamosus sp. nov. from tropical rainforests in French Guiana with a key to neotropical species of Polyporus (Polyporaceae, Basidiomycota) Kadri Runnel1* and Leif Ryvarden2 1 Department of Zoology, Institute of Ecology and Earth Sciences, University of Tartu, Vanemuise 46, EE-51014 Tartu, Estonia 2 Institute of Biological Sciences, University of Oslo, Blindern, N-0371 Oslo, Norway With 3 figures and 1 table Abstract: Tropical rainforests present high biodiversity, including high numbers of unknown fungal species. During a fungal survey in continuous near-natural rainforests in natural reserve of Kaw, French Guiana, we collected a remarkable polypore, characterized by a lateral stipe and numerous small black scales on its flabelliform pileus. It is here described as Polyporus minutosquamosus, nova species. In phylogenetic trees, based on combined ITS and partial nLSU rDNA sequences, it is nested within the Polyporus sensu lato clade, but does not belong to any of the four subclades distinguished within this clade. A key to species of genus Polyporus in tropical America is provided. Key words: Basidiomycetes; Polyporaceae; ribosomal DNA; taxonomy; wood inhabiting fungi. Introduction The tropical rain forests are known for their biodiversity, including high number of endemic and still unknown species. Fungi is to date a relatively little-studied group in such forests, illustrated by the fact that the highest proportion of new fungal species are found in the tropics (Hawksworth 2001). We conducted a field survey in continuous near-natural lowland equatorial evergreen rain forests in natural reserve of Kaw, French Guiana, focusing on polyporous fungi: a morphological group of wood inhabiting basidiomycetes that hold key roles in forests as pathogens and wood decomposers (Rayner & Boddy 1988). During this survey we collected a remarkable fungus, characterized by lateral stipe and numerous small black scales on its flabelliform pileus, presented here as a new species in genus Polyporus. *Corresponding author: kadri.runnel@ut.ee © 2016 J. Cramer in Gebr. Borntraeger Verlagsbuchhandlung, Stuttgart, Germany. DOI: 10.1127/nova_hedwigia/2016/0354 339 eschweizerbart_xxx www.borntraeger-cramer.de 0029-5035/2016/0354 $ 2.25 Polyporus Fr. ex Adans. is the type genus of the Polyporaceae. It is a cosmopolitan genus, including ecologically diverse species that mostly grow on dead wood and cause white rot. The common morphological traits for the genus are stipitate to substipitate basidiocarps, a dimitic hyphal system, thick-walled and arboriform skeleto-binding hyphae, and thin-walled, smooth and cylindrical to subellipsoid basidiospores with negative reaction in Melzer’s reagent (Gilbertson & Ryvarden 1987; Núñez & Ryvarden 1995). Núñez & Ryvarden (1995) divided species in Polyporus sensu lato into six groups by their macromorphological characters: Admirabilis, Dendropolyporus [=Dendropolyporus (Pouzar) Jülich], Favolus (=Favolus Fr.), Polyporellus (=Polyporellus P.Karst.), Melanopus (=Melanopus Pat.) and Polyporus. The recent phylogenetic analyses based on molecular data have proven that the genus is polyphyletic, and divided into four to six major clades that besides species of Polyporus also comprise species from several allied genera, e.g. Datronia and Lentinus (Sotome et al. 2008, Dai et al. 2014). Recently, Sotome et al (2013) proposed one of these clades as a new genus Neofavolus Sotome & T.Hatt.; and revised genus Favolus Fr. However, the recent studies also show that the phylogenetic clades within Polyporus sensu lato do not entirely correspond to the morphological groups (Sotome et al. 2008, Dai et al. 2014). In the present paper we describe and illustrate the newly collected specimen as Polyporus minutosquamosus and explore its affinity in the genus by both analysing its morphological and molecular traits; and provide a key to species of the genus Polyporus in tropical America. Materials and methods The basidiomata were analyzed macro- (shape, colour, hymenophore) and micromorphologically (hyphal system, presence/absence and measurement of sterile structures and basidiospores). Color descriptions were given according to Munsell System (1977). Sections were mounted in 3% KOH solution for micromorphological study (Ryvarden 1991) and examined in a phase contrast microscope. In presenting the variation of spore size, the whole range is given in parentheses and the 90% range is given without parentheses. The voucher specimen is deposited in TU, with isotype in O. DNA was extracted from dried basidiocarps and the ITS and nLSU rDNA regions were sequenced applying the protocols described by Tamm & Põldmaa (2013). The sequences were edited and combined for the phylogenetic analyses with Sequencher 5.1 (Gene Codes, Ann Arbor, MI, USA). As a base for the phylogenetic analyses, the original dataset of combined ITS and nLSU sequences of Polyporus sensu lato by Dai et al. (2014), with Trametes orientalis (Yasuda) Imazeki and Pycnoporus cinnabarinus (Jasq.) P.Karst. as outgroups, was used. The combined ITS and nLSU sequence of Polyporus minutosquamosus and all publicly avalilable rDNA ITS and nLSU sequences representing its closely related species and not yet being represented, were added to the dataset. The resulting data was re-aligned with Mafft 7 online version (Katoh & Toh 2008). Further, the preliminary set of Maximum Likelihood (ML) analyses was run (resulting trees not shown), to determine the clades phylogenetically farther from Polyporus minutosquamosus. These clades (Favolus, Neofavolus, Melanopus and Polyporellus) were reduced in number of representing species. Besides, all the remaining species were reduced to maximum three representing sequences. Data for the studied specimens and the GenBank accession numbers of their ITS and nLSU sequences are presented in Table 1. The final dataset was re-aligned with Mafft 7 online, and the resulting alignment was edited manually using GeneDoc 2.6.0.3. Gblocks (Castresana 2000) was used to select those regions of the sequences that were confidently aligned for analysis. The alignments before and after Gblocks analyses were stored in TreeBASE (http://www.treebase.org; accession number S17625). RaxML- 340 eschweizerbart_xxx Table 1. Data of ITS and nLSU sequences used in the phylogenetic analysis Species Voucher no. Country of origin GenBank no. ITS nLSU Datronia mollis (Sommerf.) Donk Favolus brasiliensis (Fr.) Fr. F. emerici (Berk. ex Cooke) Imazeki Neofavolus alveolaris (DC.) Sotome & T.Hatt. N. cremeoalbidus Sotome & T.Hatt. Polyporus arcularius (Batsch) Fr. P. austroandinus Rajchenb. & Y.C.Dai P. austroandinus P. badius (Pers.) Schwein. P. badius P. badius P. brumalis (Pers.) Fr. P. dictyopus Mont. P. guianensis Mont. P. guianensis P. hapalopus H.J.Xue & L.W.Zhou P. leprieurii Mont. P. melanopus (Pers.) Fr. P. melanopus P. minutosquamosus Runnel & Ryvarden P. squamosus (Huds.) Fr. P. squamosus P. squamosus P. subvarius C.J.Yu & Y.C.Dai P. subvarius P. tricholoma Mont. P. tricholoma P. tuberaster (Jacq.) Fr. P. tuberaster P. tuberaster P. varius (Pers.) Fr. P. varius P. varius P. virgatus Berk. & M.A.Curtis P. virgatus Pycnoporus cinnabarinus (Jacq.) P.Karst. Trametes orientalis (Yasuda) Imazeki WD 794 INPA 241452 WD 2379 Japan Brazil Japan AB587623 AB368063 AB735977 AB735953 AB587628 AB587619 TUMH 50002 Japan AB735969 AB735947 TUMH 50006 Japan Dai 8159 China MR 10472 Argentina MR 10701 Argentina Cui 10484 China JV 0408-12 USA JV 0409-9 Czech Wei 2948 China TENN 59385 Belize TENN 58404 Venezuela TENN 59093 Argentina Yuan 5809 China TENN 58597 Costa Rica H 6003449 Finland TENN 59326 Austria Runnel 690 French Guiana Dai 8082 China WD 2380 Japan MUCL 30721 Belgium WD 1872 Japan Yu 2 China TENN 56503 USA TENN 50439 Mexico Dai 4662 China DAOM 7997B Italy TENN 58530 Germany TENN 58216 Russia TENN 58587 USA WD 2347 Japan TENN 58908 Argentina TENN 59111 Argentina WD 741 Japan WD 1660 Japan 341 eschweizerbart_xxx AB735979 AB735956 KC572005 KC572044 AF516568 – AF516569 – KC572014 KC572052 KC572017 KC572055 KC572010 KC572048 KC572021 KC572058 AF516561 AJ487945 AF516566 AJ487948 AF516564 AJ487947 KC297219 KC297220 AF516567 AJ487949 JQ964422 KC572064 AF518759 AJ487951 KR584661 KC572030 KC572068 AB587631 AB587645 AB587630 AB368094 AB587641 AB368092 AB587632 AB587621 AB478884 AB368100 AF516551 AJ488114 KC572037 KC572074 AY218420 AF393070 AF516593 AJ488117 AF516576 AJ488120 AF516580 AJ488121 AB587636 AB368111 AF516581 AJ488122 AF516582 AJ488123 AB735965 AB735945 AB735966 AB735946 Fig. 1. Phylogenetic relationship of Polyporus minutosquamosus within Polyporus s.l, inferred from Bayesian analysis of the combined ITS and LSU rDNA dataset. Posterior probability and ML branch bootstrap scores are given in case >0.90 and/or >70 respectively; the new species is in bold script. The scale bar indicates substitutions per nucleotide position. HPC BlackBox 8.0.9 was used for Maximum likelihood (ML) analyses of combined two-gene dataset, divided in two partitions. The analyses were run wihout the invariant sites and with 1000 bootstrapping replicates. MrBayes v.3.2.1 (Ronquist et al. 2012) was used for Bayesian inference (BI) of partitioned two-gene dataset with two parallel MCMC analyses, each consisting of seven chains, initiated from random starting trees. The analyses were run for 10 000 000 generations, sampling every 1 000th generation. The first 25% of the trees were discarded as the burn-in; at this point, the average standard deviation of split frequencies had reached 0.007. Posterior probabilities (PP) were calculated from the remaining 15 002 trees. Both phylogenetic analyses were run at the CIPRES Science Gateway (Miller et al. 2010). Results The combined ITS and nLSU dataset included sequences from 37 specimens (Table 1), and 1037 characters after removing ambiguously aligned regions with Gblocks. ML and BI analyses generated nearly congruent topologies (Fig. 1), giving maximum support for the distinction of the ingroup (the Polyporus sensu lato clade). Three subclades: Favolus, Neofavolus and Polyporellus) were distinguished within ingroup in accordance with Dai et al. (2014). P. minutosquamosus sp. nova appeared unresolved within fourth supported clade, that besides this species also comprised (1) P. melanopus, P. badius and P. austroandinus, clustered with strong support as Melanopus subclade (sensu Dai et al. 2014); (2) another well-supported subclade comprising P. subvarius, Datronia 342 eschweizerbart_xxx Fig. 2. Dry basidiocarps of Polyporus minutosquamosus (holotype), bar = 5cm. mollis, P. squamosus, P. leprieurii, P. dictyopus and P. varius; and (3) P. tuberaster, P. hapalopus and P. virgatus with unresolved position. Taxonomy Polyporus minutosquamosus Runnel & Ryvarden sp. nov. Index Fungorum: IF551187 HOLOTYPE: (designated here) Runnel 690 (TU 121001, Isotype in O). FRENCH GUIANA, Roura: lowland equatorial evergreen rain forest in natural reserve of Kaw, 18 Oct 2013. Coordinates: 4.55382 N, 52.21429 W (map projection: WGS84); elevation: 250 m asl. On dead hardwood log. BASIDIOCARPS: annual, laterally stipitate; pileus flabelliform, up to 4 cm wide and 4 mm thick; upper surface cream to tan or cinnamon brown (7.5 YR 5/6–8) with numerous minute black scales or tufted agglutinated hyphae, especially at the centre, towards the margin the scales are lighter to almost hyaline at the very edge of the pileus; stipe round, lateral to eccentric, up to 1 cm long and 4 mm in diameter, ochraceous and with 343 eschweizerbart_xxx Fig. 3. Microscopic structures of Polyporus minutosquamosus (drawn from holotype): a. Basidia; b. Basidiospores; c. Binding hyphae. decurrent pores in upper part; pore surface deep ochraceous (10YR 8/3–4), pores 3–4 per mm, angular and thin walled; tubes concolorous with the pore surface, up to 1 mm deep; context homogenous, white, up to 5 mm thick at the centre (Fig. 2). HYPHAL SYSTEM: dimitic; generative hyphae with clamps, 3–8 µm wide, hyaline, rather thin walled with large lumen; those of the pileus scales, pale brown, thin-walled and 4– 10 µm wide; binding hyphae seen only in upper part of context, hyaline, dichotomously branched with long whip like thin branches, almost solid towards the apex (Fig. 3). 344 eschweizerbart_xxx BASIDIA 18–25 × 5–8 µm, clavate, tetrasterigmatic. BASIDIOSPORES: (4.0–)4.5–5.5(–5.5) × (1.5–)1.5–2.0(–2.0) µm, L=4.98µm, W=1.88 µm, Q=2.68 (n=30/1), cylindrical, smooth, thin walled, hyaline and negative in Melzers reagent. SUBSTRATA: Dead hard wood. DISTRIBUTION: Known only from the type locality. REMARKS: The striking character in this species is the minute dark brown squamules on the pileus. There are two other species with similar squamules, viz, Polyporus squamosus (Huds.) Fr. and Neofavolus alveolaris (DC.) Sotome & T.Hatt. However, the latter species are temperate in their distribution and their spores are much larger than those seen in P. minutosquamosus (14–17 × 5–6 µm and 10–13 × 4–5 µm, respectively). Further their pores are also larger than seen in the new species (1–2 per mm measured tangentially). Thus, even if there is a certain similarity in the squamulose pileus, this does not indicate a closer relationship, as also evident according to the molecular data. The squamulose pileus surface represents in this case a biological convergence with an unknown selective factor behind its establishment. Key to groups and species of Polyporus in tropical America 1a Basidiocarps fleshy, more than 1 cm thick .....................................................................Group 1 1b Basidiocarps leathery, up to 1 cm thick, with or without a black, endured cuticle at least on the stipe basis ....................................................................................................................................2 2a Basidiocarps with a black, endured cuticle at least on the stipe basis ............................Group 2 2b Basidiocarps without a black, endured cuticle on the stipe (a thin cuticle may be present in old specimens) ..................................................................................................................................3 3a Basidiocarps centrally stipitate .......................................................................................Group 3 3b Basidiocarps laterally stipitate ........................................................................................Group 4 Group 1 1a Basidiocarp developing from a buried sclerotium ......................................................................2 1b Basidiocarp growing on dead wood............................................................................................3 2a Surface cream, glabrous; pores 3–4 per mm; spores 5–6 × 3.5–4 µm .......................................... ..................................................................................P. indigenus I.J.A.Aguiar & M.A.de Sousa 2b Surface scaly, cream to brown by drying; pores 1–2 per mm; spores 9–11 × 4–5 µm ................. ...................................................................................................................... P. sapurema Möller 3a Context up to 1 cm thick; pileus with rose to purple tints .....................................P. udus Jungh. 3b Context up to 4 mm thick; pileus ochraceous to deep tan ......P. craterellus Berk. & M.A.Curtis Group 2 1a Pileus white, tan, leather coloured to light tobacco brown .........................................................2 1b Pileus dark brown, purplish or black ..........................................................................................4 2a Temperate to montane species; pores 7–9 per mm ........................................ P. varius (Pers.) Fr. 2b Tropical to subtropical species; pores larger ..............................................................................3 345 eschweizerbart_xxx 3a Pores radially arranged, 1 per mm ................................................................ P. guianensis Mont. 3b More than 4 pores per mm .............................................................................. P. leprieurii Mont. 4a Pileus velutinate ....................................................................P. nigrovelutinus Ryvarden & Iturr. 4b Pileus glabrous ............................................................................................................................5 5a Pileus infundibuliform; spores 9–12.5 5 µm long ......................................................................6 5b Pileus mostly applanate; spores shorter than 9 um .....................................................................7 6a Pileus striate pale brown becoming chestnut; pores 2–4 per mm; spores 9–12 × 4–5 um ........... ....................................................................................................P. virgatus Berk. & M.A.Curtis 6b Pileus surface whitish to beige and without radial lines or striae; pores 1–2 per mm, up to 1.5 mm long at the stipe; spores 8–10 × 3–4 um ....................................................P. puttemannsii Henn. 7a Tropical species; clamps present .................................................................... P. dictyopus Mont. 7b Montane to temperate species; simple septa present .......................... P. badius (Pers.) Schwein. Group 3 1a More than 4 pores per mm; pileus white to cream…P. tricholoma Mont. 1b Less than 4 pores per mm; pileus pale brown, often discoloured in patches...P. arcularius (Batsch) Fr. Group 4 1a Pileus dark chestnut to bay or deep vinaceous .............. P. subpurpurascens (Murrill) Ryvarden 1b Pileus white, pale vinaceous, deep tan or leather coloured ........................................................2 2a Pileus white when fresh, darker when dry, tessellate to smooth; spores 9–12 um long ............... .........................................................................................................Favolus brasiliensis (Fr.) Fr. 2b Pileus leather coloured, orange to brown, radially striate or with small hydnoid processes or squamules, at least at the base; spores shorter than 9 µm ..................................................................3 3a Pores 2–5 per mm .......................................................................................................................4 3b Pores 1–2 per mm or larger ........................................................................................................6 4a Pileus pale reddish brown with minute black squamules ............................................................. .................................................................................... P. minutosquamosus Runnel & Ryvarden 4b Pileus differently coloured and without dark squamules, but often with radial lines.................5 5a Pileus vinaceous to pale brown, smooth; pores consistently round; spores 5–6 µm long ............ .............................................................................................. P. ianthinus Gibertoni & Ryvarden 5b Pileus ochraceous smooth or with minute hyphal tufts at base; pores often slightly elongated; spores 6–8 µm long ..............................................................................P. grammocephalus Berk. 6a Pores 1–2 per mm, angular; pileus cream to tan, smooth, but often with radial lines .................. ..................................................................................................................P. philippinensis Berk. 6b Pores elongated 2–4 × 0.5–0.7 mm; pileus whitish with hydnoid protuberances .. P. biskeletalis Acknowledgements We kindly thank Dr. Kadri Põldmaa for constructive comments on the manuscript. Rasmus Puusepp (University of Tartu) performed the lab work, and Annely Tomson provided accommodation to KR during study visits to Oslo. The study was supported by the Doctoral School of Earth Sciences and Ecology, created under the auspices of European Social Fund; and Estonian Research Agency (projects ETF9051 and IUT34-7) 346 eschweizerbart_xxx References CASTRESANA, J. 2000: Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. – Mol. Biol. Evol. 17: 540–552. DAI, Y.C., H.J. XUE, J. VLASÁK, M. RAJCHENBERG, B. WANG et al. 2014: Phylogeny and global diversity of Polyporus group Melanopus (Polyporales, Basidiomycota). – Fungal Divers. 64: 133–144. doi: 10.1007/s13225-013-0248-3 GILBERTSON, R.L. & L. 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