Mycologia, 106(2), 2014, pp. 323–327. DOI: 10.3852/13-154 # 2014 by The Mycological Society of America, Lawrence, KS 66044-8897
Phylogenetic relationships of Hydnum peroxydatum support the synonymy of Hydnochaete with Hymenochaete (Hymenochaetaceae, Agaricomycetes) Juliano M. Baltazar1
classified with genera of hymenochaetoid fungi with smooth (e.g. Hymenochaete Le´v.), poroid (e.g. Phellinus Que´l. s. l.) and poroid to cyclolamellate (e.g. Cyclomyces Kunze ex Fr.) hymenophores, mainly based on the xanthochroic reaction and presence of setae in many species (Patouillard 1900; Corner 1948, 1991; Donk 1964; Ju¨lich 1981; Ryvarden 2004). Phylogenetic studies have shown that several Hydnochaete species belong to Hymenochaete s. str., a monophyletic genus that also includes some Cyclomyces taxa, including its type species Cyclomyces fuscus Kunze ex Fr. (Wagner and Fischer 2002, He and Dai 2012, He and Li 2013, Parmasto et al. 2014). Several studies also show that Hymenochaete tabacina (Sowerby : Fr.) Le´v. and some other species of Cyclomyces, Hydnochaete and Hymenochaete s. l. form a clade independent from Hymenochaete s. str. and are currently accepted in Pseudochaete T. Wagner & M. Fisch. (Wagner and Fischer 2002, He and Dai 2012, He and Li 2013, Parmasto et al. 2014). Here Hymenochaete s. str. refers to those species placed in the same clade of the type species of Hymenochaete, that is Hymenochaete rubiginosa (Dicks. : Fr.) Le´v., while Hymenochaete s. l. also includes species of Pseudochaete. The synonym of Cyclomyces with Hymenochaete would create an undesirable nomenclatural situation because Cyclomyces is an older name but has considerable fewer specific names than Hymenochaete (five in Cyclomyces against ca. 110 in Hymenochaete). Thus, Fischer and Wagner (2001) proposed the rejection of Cyclomyces in favor of Hymenochaete, which was approved by the 17th International Botanical Congress (McNeill et al. 2006). Wagner and Fischer (2002) also had to propose a new name for C. fuscus (type species of Cyclomyces) when combined in Hymenochaete (viz. Hymenochaete cyclolamellata T. Wagner & M. Fisch.) because of the existence of the binomial Hymenochaete fusca P. Karst. The phylogenetic analyses carried out by Wagner and Fischer (2002), He and Dai (2012), He and Li (2013) and Parmasto et al. (2014) sampled many species, including the type species of Cyclomyces, Hymenochaete and Pseudochaete. However, the phylogenetic placement of Hyd. peroxydata, type species of Hydnochaete, remained unknown. The assessment of its phylogenetic placement is required to verify the current taxonomic status of Hydnochaete. The aims of this study were to address the phylogenetic relationships
Programa de Po´s-Graduac¸a˜o em Botaˆnica, Departamento de Botaˆnica, Universidade Federal do Rio Grande do Sul, Av. Bento Gonc¸alves 9500, Porto Alegre, CEP 91501-970, Rio Grande do Sul, Brazil
Marı´a Bele´n Pildain Sergio P. Gorjo´n Centro Forestal CIEFAP, C.C. 14, 9200 Esquel, Chubut, Argentina
Rosa Mara B. da Silveira Programa de Po´s-Graduac¸a˜o em Botaˆnica, Departamento de Botaˆnica, Universidade Federal do Rio Grande do Sul, Av. Bento Gonc¸alves 9500, Porto Alegre, CEP 91501-970, Rio Grande do Sul, Brazil
Mario Rajchenberg Centro Forestal CIEFAP, C.C. 14, 9200 Esquel, Chubut, Argentina
Abstract: A combined dataset of rDNA ITS and LSU sequences was used to infer the phylogenetic relationships of Hydnochaete peroxydata (; Hydnum peroxydatum), the type species of Hydnochaete. The species was retrieved nested within the Hymenochaete s. str. clade; therefore Hydnochaete is regarded as a synonym of Hymenochaete and the new combination Hymenochaete peroxydata is proposed. Key words: Basidiomycota, corticioid fungi, Cyclomyces, Hymenochaetales, ITS and LSU rDNA, xanthochroic fungi INTRODUCTION Bresadola (1896) described Hydnochaete Bres., with the new species Hydnochaete badia Bres. as type species, based on collections from southern Brazil, and characterized by a hydnoid hymenophore and presence of setae. Dennis (1970) regarded Hyd. badia as a synonym of Hydnum peroxydatum Berk. ex Cooke, a name based on a collection from Venezuela, and proposed the binomial Hydnochaete peroxydata (Berk. ex Cooke) Dennis. Hydnochaete in its traditional sense now comprises 10 species, mainly found in warm, subtropical and tropical areas (Ryvarden 1982, Parmasto and Wu 2005, Dai and Li 2010). It commonly has been Submitted 13 May 2013; accepted for publication 15 Aug 2013. 1 Corresponding author, E-mail: [email protected]
323
324 TABLE I.
MYCOLOGIA Specimens presented in this study with GenBank accession numbers for the ITS and LSU sequences GenBank accession nos. Voucher/strain nos.
LSU
ITS
Reference
Hymenochaete acanthophysata Hymenochaete cruenta Hymenochaete cyclolamellata Hymenochaete duportii Hymenochaete floridea Hymenochaete hydnoides Hymenochaete murina Hymenochaete paucisetigera Hymenochaete peroxydata Hymenochaete peroxydata Hymenochaete peroxydata Hymenochaete rheicolor Hymenochaete rubiginosa Hymenochaete sphaericola Hymenochaete xerantica Pseudochaete corrugata Pseudochaete lamellata Pseudochaete tabacina Pseudochaete tabacinoides
CBS 925.26 He766 Cui7393 AFTOL-ID 666 He536 He245 He569 Cui7845 J.M. Baltazar 1819 J.M. Baltazar 2056, CIEFAPcc 409 J.M. Baltazar 2102, CIEFAPcc 411 He503 He1049 He303 Cui9209 He761 Cui7629 He810 Cui10428
AF385144 JQ279681 JQ279629 AY635770 JQ279683 JQ279680 JQ716412 JQ279644 KF371647 KF371648 KF371649 JQ279632 JQ279667 JQ279684 JQ279635 JQ279621 JQ279617 JQ279626 JQ279618
– JQ279595 JQ279513 DQ404386 JQ279597 JQ279590 JQ716406 JQ279560 KF371644 KF371645 KF371646 JQ279530 JQ716407 JQ279599 JQ279519 JQ279606 JQ279603 JQ279611 JQ279604
Wagner and Fischer (2002) He and Dai (2012) He and Dai (2012) – He and Dai (2012) He and Dai (2012) He and Li (2013) He and Dai (2012) this study this study this study He and Dai (2012) He and Li (2013) He and Dai (2012) He and Dai (2012) He and Dai (2012) He and Dai (2012) He and Dai (2012) He and Dai (2012)
Outgroups Fomitopsis pinicola Trametes villosa
AFTOL-ID 770 FP71974R
AY684164 JN164810
AY854083 JN164969
– Justo and Hibbett (2011)
Species
a
Newly sequenced strains are indicated in boldface; – information not available.
of Hyd. peroxydata based on the analysis of a two-gene dataset containing nuc-LSU and ITS rDNA gene sequences and to provide the necessary taxonomic treatment. MATERIALS AND METHODS Cultures and herbarium specimens.— Cultures for DNA extraction were obtained from fresh spore prints and kept at the authors’ institutional culture collections (CIEFAPcc and Laborato´rio de Micologia/BOT/UFRGS). They were grown on malt extract agar (MEA) in the dark at 25 C and stored in sterile distilled water following Burdsall and Dorworth (1994). Voucher specimens were deposited at Herbaria ICN and Centro de Investigacio´ n y Extensio´n Forestal Andino Patago´ nico (CIEFAP, Esquel, Argentina). Data about vouchers are given in the taxonomic treatment. DNA extraction and PCR conditions.— For DNA extractions, strains were cultured in malt peptone broth with 10% (v/v) of malt extract (Merck) and 0.1% (w/v) Bacto peptone (Difco), 2 mL medium in 15 mL tubes. The cultures were incubated at 25 C for 5 d in darkness. Total DNA was extracted with the UltraCleanTM Microbial DNA Isolation Kit (MO BIO laboratories Inc., USA) according to the manufacturer’s instructions. rDNAs ITS (including ITS1, 5.8S and ITS2) and nucLSU regions were amplified with universal primers ITS5-LR21 and LR0R-LR5 respectively
(R. Vilgalys lab webpage at http://www.botany.duke.edu/ fungi/mycolab). PCR reaction mixtures for amplification of both regions were modified from Rajchenberg et al. (2011) in a final reaction volume of 50 mL with 100–500 ng DNA. PCR reactions were performed in a thermal cycler (My CyclerTM, BioRad) and the thermal-cycling program was the same described in Rajchenberg et al. (2011). The amplified fragments were purified and sequenced on an ABI 3700 automated sequencer (Perkin-Elmer, USA) at the DNA Synthesis and Sequencing Facility (Macrogen, Korea). The same primers were used for amplification and sequencing. (Sequences generated in this study were submitted to GenBank and accession numbers are given in TABLE I.) Phylogenetic analyses.—DNA sequences generated in this study were manually edited with BioEdit 7.1.3.0 (Hall 1999), and additional sequences for the ingroup and outgroup, based on studies of Wagner and Fischer (2002) and He and Dai (2012), were retrieved from the GenBank nucleotide database. ITS sequences were not available for Hymenochaete acanthophysata J.C. Le´ger. Sequence alignments were automatically performed on MUSCLE 3.8.31 (Edgar 2004) and manually checked on MEGA 5.10 (Tamura et al. 2011). Alignments are available from TreeBASE (http://purl.org/ phylo/treebase/phylows/study/TB2:S14475). The two DNA regions first were analyzed independently (data not shown), and because there were no major topological conflicts the data were combined into a single matrix for subsequent analyses. Phylogenetic analyses were conducted
BALTAZAR ET AL.: HYDNUM PEROXYDATUM
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FIG. 1. Phylogenetic relationships of Hymenochaete s.l. inferred with a combined dataset (rDNA ITS and LSU) under maximum parsimony (MB) and Bayesian inference (BI) analyses. Tree topology is based on one of the three equally most parsimonious trees. Support values for internal nodes are given on the branches as bootstrap/posterior probability (BS/PP). Fully supported nodes (BS 100/PP 1.0) are indicated by black circles. Incongruous topologies between MP and BI analyses are indicated with an asterisk instead of the PP value. Species names in boldface indicate the type species of Cyclomyces (Hym. cyclolamellata), Hydnochaete (Hym. peroxydata), Hymenochaete (Hym. rubiginosa) and Pseudochaete (P. tabacina). Species names are followed by symbols indicating hymenophore types: §5 smooth, ¤5 hydnoid, # 5 poroid to cyclolamellate. for the two loci combined dataset under maximum parsimony (MP) and Bayesian inference (BI) criteria. Fomitopsis pinicola (Sw. : Fr.) P. Karst. and Trametes villosa (Sw. : Fr.) Kreisel were used as outgroup species. MP analysis was performed in PAUP* 4.0b10 (Swofford 2002) with gaps treated as missing characters, equal weighting of characters and transformations, heuristic searches (TBR and MULTREES options on) with random addition of sequences (1000 replicates), and MAXTREES set to auto-increase. Nodal support was tested with bootstrap (BS) of 1000 replicates using the heuristic search option (TBR and MULTREES options on) and 10 random addition sequences. Bayesian analysis was conducted in MrBayes 3.2.1 (Ronquist et al. 2012). Models of evolution were identified for each dataset using jModelTest 2.1.2 (Darriba et al. 2012) under selection AIC, resulting in the model TPM2uf + I + G for ITS and TPM3uf + I + G for LSU. BI posterior probabilities (PP) were estimated for 107 generations by
running four chains and sampling a tree each 105 generations, and the first 5% trees from each run were discarded as burn-in. Burn-in was determined with Tracer 1.5 (http://tree.bio.ed.ac.uk/software/tracer/) to analyze MrBayes output files.
RESULTS The combined dataset (ITS and LSU) included 21 taxa and a total of 1670 characters, of which 1067 were constant, 161 were variable and parsimony uninformative, and 442 were parsimony informative. MP analysis resulted in three equally most parsimonious trees (Tree length 5 1229; CI 5 0.6373; RI 5 0.7785; RC 5 0.5384), one of which is presented (FIG. 1). Six new sequences were generated (TABLE I). The monophyly of the ingroup is fully supported with both MP and BI analyses and only two
326
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topological incongruencies between the MP and BI analyses were observed (FIG. 1, SUPPLEMENTARY FIG. 1). Those nodes are weakly supported with MP (BS , 80) and not supported with BI analysis (PP , 0.85). The ingroup taxa pertained to two major strongly supported clades: Hymenochaete s. str. (13 species) and Pseudochaete (four species). From a total of 17 clades identified, 10 were fully supported (BS 100/PP 1.0), five received moderate to high support (BS . 80; PP . 0.94), and two terminal clades were not supported (FIG. 1). The segregation of Cyclomyces and Hydnochaete as independent from Hymenochaete was not supported by our analyses because the type species of these three genera were placed in the same clade (Hymenochaete s. str.) with full support (FIG. 1). Furthermore, monophyletic groups of species with different hymenophoral types were observed—for example, Hymenochaete rheicolor (Mont.) Le´v. (smooth) with Hym. cyclolamellata (concentrically lamellate to poroid) and Hym. xerantica (Berk.) S.H. He & Y.C. Dai (poroid); Hym. rubiginosa (smooth) with Hym. paucisetigera (Parmasto & Sheng H. Wu) S.H. He & Y.C. Dai (hydnoid). Hymenochaete s. str. and Pseudochaete each include at least three hymenophoral types, according to the results presented here. The three Hyd. peroxydata specimens sampled clustered with full support with a sister clade formed by Hym. duportii (Pat.) T. Wagner & M. Fisch. and Hym. hydnoides T. Wagner & M. Fisch. The entire group is nested in the Hymenochaete s. str. clade. TAXONOMY Hymenochaete Le´v., Ann. Sci. Nat., Bot. Ser. III 5:150, 1846, nom. cons., non Hymenochaeta P. Beauv. ex T. Lestib. 1819 (Cyperaceae). 5 Cyclomyces Kunze ex Fr., Linnaea 5:512, 1830, nom. rej. 5 Hydnochaete Bres., Hedwigia 35(5):287, 1896, syn. nov., non Hydnochaete Peck 1897.
Hymenochaete peroxydata (Berk. ex Cooke) Baltazar, Gorjo´n & Rajchenb., comb. nov. MycoBank MB804370 ; Hydnum peroxydatum Berk. ex Cooke, Grevillea 20(93):1, 1891 (basionym). ; Hydnochaete peroxydata (Berk. ex Cooke) Dennis, Kew Bull. Addit. Ser. 3:105, 1970. 5 Hydnochaete badia Bres., Hedwigia 35:287, 1896. Specimens examined: BRAZIL. RIO GRANDE DO SUL: Riozinho. On dead hardwood, 10 Apr 2010, J.M. Baltazar 1819 (ICN, CIEFAP); Sa˜o Francisco de Paula, PROMATAPUC. On dead hardwood, 26 Jun 2010, J.M. Baltazar 2056 (ICN, CIEFAP); SANTA CATARINA: Blumenau. 1894, A. Mo¨ller nu 211 (S, lectotype of Hydnochaete badia); Blumenau. A. Mo¨ller nu 268 (S, paratype of Hydnochaete badia);
A. Mo¨ller nu 801 (S, paratype of Hydnochaete badia); Blumenau, A. Mo¨ ller (S); Floriano´ polis, Unidade de Conservac¸a˜o Ambiental Desterro (UCAD). On dead hardwood, 17 Sep 2010, J.M. Baltazar 2102 (ICN, CIEFAP).
A monograph of Hymenochaete by Le´ger (1998) presented a morphological treatment of species with smooth hymenophore. Ryvarden (1982) monographed Hydnochaete with descriptions and drawings of all the accepted taxa. DISCUSSION Results of the present study are similar to those of Wagner and Fischer (2002), He and Dai (2012), He and Li (2013) and Parmasto et al. (2014). Sequences of two nuclear rDNA regions were used to infer the phylogenetic placement of Hyd. peroxydata, the type species of Hydnochaete. Before the present study, the phylogenetic relationships of Hymenochaete and allied genera using a combined dataset of ITS and LSU sequences were investigated only by He and Li (2013). Specimens of Hyd. peroxydata are nested within the Hymenochaete s. str. clade with full support, and two other hydnoid species (Hym. duportii and Hym. hydnoides) form a sister group, but they are also closely related to species with a smooth hymenophore such as Hym. acanthophysata and Hym. murina Bres. Hymenochaete is accepted as monophyletic and includes species with resupinate, pileate sessile or stipitate basidiomes, with smooth, fissured or verrucose, hydnoid, poroid or concentrically lamellate hymenophores, monomitic hyphal system, simple septate hyphae, presence or absence of setae, and hyaline basidiospores. Pseudochaete also includes species with smooth, hydnoid and poroid to concentrically lamellate hymenophores, and there is no evident morphological feature to separate it from Hymenochaete s. str. Pseudochaete tabacina (Sowerby : Fr.) T. Wagner & M. Fisch., type species of the genus, presents a holocenocytic nuclear behavior (Wagner and Fischer 2002). However, data on the nuclear behavior of other species currently accepted in Pseudochaete and of most species of Hymenochaete s. str. is lacking, and the importance of this feature to segregate these genera remains unknown. Because Hymenochaete s. str. and Pseudochaete can be separated based on molecular data only, the generic placement of 70–80 species of Hymenochaete s. l. not yet included in phylogenetic analyses remains unknown (Parmasto et al. 2014). ACKNOWLEDGMENTS The authors are grateful for the help of Olivier Chauveau and Larissa Trierveiler Pereira for contributions on a
BALTAZAR ET AL.: HYDNUM PEROXYDATUM preliminary draft of the manuscript and suggestions on the phylogenetic analyses. Loreta Branda˜o de Freitas and Patrı´cia Valente da Silva also made suggestions for these analyses. JMB has doctoral scholarships from CNPq (GD 141495/2010-3) and CAPES (PDSE—proceeding 9715/118). CAPES (Brazil) and MINCyT (Argentina) are thanked by financial support (Bilateral cooperation CAPES/MINCyT Rede 003/11). MBP and MR are researchers of CONICET (Argentina).
LITERATURE CITED Bresadola G. 1896. Fungi Brasilienses lecti a cl. Dr. Alfredo Mo¨ller. Hedwigia 35:276–302. Burdsall HH, Dorworth EB. 1994. Preserving cultures of wooddecaying Basidiomycotina using sterile distilled water in cryovials. Mycologia 86:275–280, doi:10.2307/3760650 Corner EJH. 1948. Asterodon, a clue to the morphology of fungus fruit bodies: with notes on Asterostroma and Asterostromella. Trans Br Mycol Soc 31:234–245, doi:10.1016/S0007-1536(48)80005-7 ———. 1991. Ad Polyporaceas VII: the Xanthochroic polypores. Beih Nova Hedwigia 101:1–175. Dai Y-C, Li H-J. 2010. Notes on Hydnochaete (Hymenochaetales) with a seta-less new species discovered in China. Mycotaxon 111:481–487, doi:10.5248/111.481 Darriba D, Taboada GL, Doallo R, Posada D. 2012. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9:772–772, doi:10.1038/ nmeth.2109 Dennis RWG. 1970. Fungus flora of Venezuela and adjacent countries. Kew Bull Add Ser 3:1–531. Donk MA. 1964. A conspectus of the families of Aphyllophorales. Persoonia 3:199–324. Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797, doi:10.1093/nar/gkh340 Fischer M, Wagner T. 2001. (1497) Proposal to conserve Hymenochaete Le´ v. nom. cons. (Hymenochaetales, Basidiomycetes), against an additional name, Cyclomyces Fr. Taxon 50:1185–1186, doi:10.2307/1224741 Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98. He S-H, Dai Y-C. 2012. Taxonomy and phylogeny of Hymenochaete and allied genera of Hymenochaetaceae (Basidiomycota) in China. Fungal Divers 56:77–93, doi:10.1007/s13225-012-0174-9 ———, Li H-J. 2013. Pseudochaete latesetosa and P. subrigidula spp. nov. (Hymenochaetales, Basidiomycota) from China based on morphological and molecular
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characters. Mycol Progr 12:331–339, doi:10.1007/ s11557-012-0838-6 Ju¨lich W. 1981. Higher taxa of Basidiomycetes. Bibl Mycol 85:1–485. Justo A, Hibbett DS. 2011. Phylogenetic classification of Trametes (Basidiomycota, Polyporales) based on a fivemarker dataset. Taxon 60:1567–1583. Le´ger J-C. 1998. Le genre Hymenochaete Le´veille´. Bibl Mycol 171:1–319. McNeill J, Barrie FR, Burdet HM, Demoulin V, Hawksworth DL, Marhold K, Nicolson DH, Prado J, Silva PC, Skog JE, et al. 2006. International Code of Botanical Nomenclature (Vienna Code) adopted by the 17th International Botanical Congress Vienna, Austria, Jul 2005. Regnum Vegetabile 146:I–XVIII, 1–568. Parmasto E, Saar I, Larsson E, Rummo S. 2014. Phylogenetic taxonomy of Hymenochaete and related genera (Hymenochaetales). Mycol Progr 13:55–64, doi:10.1007/ s11557-013-0891-9 ———, Wu S-H. 2005. Hydnochaete paucisetigera, a new species of Hymenochaetales. Mycotaxon 91:461–463. Patouillard NT. 1900. Essai taxonomique sur les familles et les genres des Hyme´nomyce`tes. Lons-Le-Saunier, France: Lucien Declume. 184 p. Rajchenberg M, Gorjo´ n SP, Pildain MB. 2011. The phylogenetic disposition of Antrodia s.l. (Polyporales, Basidiomycota) from Patagonia, Argentina. Austral Syst Bot 24:111–120, doi:10.1071/SB11003 Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61:539–42, doi:10.1093/sysbio/sys029 Ryvarden L. 1982. The genus Hydnochaete Bres. (Hymenochaetaceae). Mycotaxon 15:425–447. ———. 2004. Neotropical polypores 1. Introduction, Ganodermataceae & Hymenochaetaceae. Synop Fungorum 19:1–229. Swofford DL. 2002. PAUP* 4: phylogenetic analysis using parsimony (*and other methods). Sunderland, Massachusetts: Sinauer Associates. p 1–142. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA 5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance and maximum parsimony methods. Mol Biol Evol 28:2731–2739, doi:10.1093/molbev/msr121 Wagner T, Fischer M. 2002. Classification and phylogenetic relationships of Hymenochaete and allied genera of the Hymenochaetales, inferred from rDNA sequence data and nuclear behavior of vegetative mycelium. Mycol Progr 1:93–104, doi:10.1007/s11557-006-0008-9
Phylogenetic relationships of Hydnum peroxydatum support the synonymy of Hydnochaete with Hymenochaete (Hymenochaetaceae, Agaricomycetes) Juliano M. Baltazar1
classified with genera of hymenochaetoid fungi with smooth (e.g. Hymenochaete Le´v.), poroid (e.g. Phellinus Que´l. s. l.) and poroid to cyclolamellate (e.g. Cyclomyces Kunze ex Fr.) hymenophores, mainly based on the xanthochroic reaction and presence of setae in many species (Patouillard 1900; Corner 1948, 1991; Donk 1964; Ju¨lich 1981; Ryvarden 2004). Phylogenetic studies have shown that several Hydnochaete species belong to Hymenochaete s. str., a monophyletic genus that also includes some Cyclomyces taxa, including its type species Cyclomyces fuscus Kunze ex Fr. (Wagner and Fischer 2002, He and Dai 2012, He and Li 2013, Parmasto et al. 2014). Several studies also show that Hymenochaete tabacina (Sowerby : Fr.) Le´v. and some other species of Cyclomyces, Hydnochaete and Hymenochaete s. l. form a clade independent from Hymenochaete s. str. and are currently accepted in Pseudochaete T. Wagner & M. Fisch. (Wagner and Fischer 2002, He and Dai 2012, He and Li 2013, Parmasto et al. 2014). Here Hymenochaete s. str. refers to those species placed in the same clade of the type species of Hymenochaete, that is Hymenochaete rubiginosa (Dicks. : Fr.) Le´v., while Hymenochaete s. l. also includes species of Pseudochaete. The synonym of Cyclomyces with Hymenochaete would create an undesirable nomenclatural situation because Cyclomyces is an older name but has considerable fewer specific names than Hymenochaete (five in Cyclomyces against ca. 110 in Hymenochaete). Thus, Fischer and Wagner (2001) proposed the rejection of Cyclomyces in favor of Hymenochaete, which was approved by the 17th International Botanical Congress (McNeill et al. 2006). Wagner and Fischer (2002) also had to propose a new name for C. fuscus (type species of Cyclomyces) when combined in Hymenochaete (viz. Hymenochaete cyclolamellata T. Wagner & M. Fisch.) because of the existence of the binomial Hymenochaete fusca P. Karst. The phylogenetic analyses carried out by Wagner and Fischer (2002), He and Dai (2012), He and Li (2013) and Parmasto et al. (2014) sampled many species, including the type species of Cyclomyces, Hymenochaete and Pseudochaete. However, the phylogenetic placement of Hyd. peroxydata, type species of Hydnochaete, remained unknown. The assessment of its phylogenetic placement is required to verify the current taxonomic status of Hydnochaete. The aims of this study were to address the phylogenetic relationships
Programa de Po´s-Graduac¸a˜o em Botaˆnica, Departamento de Botaˆnica, Universidade Federal do Rio Grande do Sul, Av. Bento Gonc¸alves 9500, Porto Alegre, CEP 91501-970, Rio Grande do Sul, Brazil
Marı´a Bele´n Pildain Sergio P. Gorjo´n Centro Forestal CIEFAP, C.C. 14, 9200 Esquel, Chubut, Argentina
Rosa Mara B. da Silveira Programa de Po´s-Graduac¸a˜o em Botaˆnica, Departamento de Botaˆnica, Universidade Federal do Rio Grande do Sul, Av. Bento Gonc¸alves 9500, Porto Alegre, CEP 91501-970, Rio Grande do Sul, Brazil
Mario Rajchenberg Centro Forestal CIEFAP, C.C. 14, 9200 Esquel, Chubut, Argentina
Abstract: A combined dataset of rDNA ITS and LSU sequences was used to infer the phylogenetic relationships of Hydnochaete peroxydata (; Hydnum peroxydatum), the type species of Hydnochaete. The species was retrieved nested within the Hymenochaete s. str. clade; therefore Hydnochaete is regarded as a synonym of Hymenochaete and the new combination Hymenochaete peroxydata is proposed. Key words: Basidiomycota, corticioid fungi, Cyclomyces, Hymenochaetales, ITS and LSU rDNA, xanthochroic fungi INTRODUCTION Bresadola (1896) described Hydnochaete Bres., with the new species Hydnochaete badia Bres. as type species, based on collections from southern Brazil, and characterized by a hydnoid hymenophore and presence of setae. Dennis (1970) regarded Hyd. badia as a synonym of Hydnum peroxydatum Berk. ex Cooke, a name based on a collection from Venezuela, and proposed the binomial Hydnochaete peroxydata (Berk. ex Cooke) Dennis. Hydnochaete in its traditional sense now comprises 10 species, mainly found in warm, subtropical and tropical areas (Ryvarden 1982, Parmasto and Wu 2005, Dai and Li 2010). It commonly has been Submitted 13 May 2013; accepted for publication 15 Aug 2013. 1 Corresponding author, E-mail: [email protected]
323
324 TABLE I.
MYCOLOGIA Specimens presented in this study with GenBank accession numbers for the ITS and LSU sequences GenBank accession nos. Voucher/strain nos.
LSU
ITS
Reference
Hymenochaete acanthophysata Hymenochaete cruenta Hymenochaete cyclolamellata Hymenochaete duportii Hymenochaete floridea Hymenochaete hydnoides Hymenochaete murina Hymenochaete paucisetigera Hymenochaete peroxydata Hymenochaete peroxydata Hymenochaete peroxydata Hymenochaete rheicolor Hymenochaete rubiginosa Hymenochaete sphaericola Hymenochaete xerantica Pseudochaete corrugata Pseudochaete lamellata Pseudochaete tabacina Pseudochaete tabacinoides
CBS 925.26 He766 Cui7393 AFTOL-ID 666 He536 He245 He569 Cui7845 J.M. Baltazar 1819 J.M. Baltazar 2056, CIEFAPcc 409 J.M. Baltazar 2102, CIEFAPcc 411 He503 He1049 He303 Cui9209 He761 Cui7629 He810 Cui10428
AF385144 JQ279681 JQ279629 AY635770 JQ279683 JQ279680 JQ716412 JQ279644 KF371647 KF371648 KF371649 JQ279632 JQ279667 JQ279684 JQ279635 JQ279621 JQ279617 JQ279626 JQ279618
– JQ279595 JQ279513 DQ404386 JQ279597 JQ279590 JQ716406 JQ279560 KF371644 KF371645 KF371646 JQ279530 JQ716407 JQ279599 JQ279519 JQ279606 JQ279603 JQ279611 JQ279604
Wagner and Fischer (2002) He and Dai (2012) He and Dai (2012) – He and Dai (2012) He and Dai (2012) He and Li (2013) He and Dai (2012) this study this study this study He and Dai (2012) He and Li (2013) He and Dai (2012) He and Dai (2012) He and Dai (2012) He and Dai (2012) He and Dai (2012) He and Dai (2012)
Outgroups Fomitopsis pinicola Trametes villosa
AFTOL-ID 770 FP71974R
AY684164 JN164810
AY854083 JN164969
– Justo and Hibbett (2011)
Species
a
Newly sequenced strains are indicated in boldface; – information not available.
of Hyd. peroxydata based on the analysis of a two-gene dataset containing nuc-LSU and ITS rDNA gene sequences and to provide the necessary taxonomic treatment. MATERIALS AND METHODS Cultures and herbarium specimens.— Cultures for DNA extraction were obtained from fresh spore prints and kept at the authors’ institutional culture collections (CIEFAPcc and Laborato´rio de Micologia/BOT/UFRGS). They were grown on malt extract agar (MEA) in the dark at 25 C and stored in sterile distilled water following Burdsall and Dorworth (1994). Voucher specimens were deposited at Herbaria ICN and Centro de Investigacio´ n y Extensio´n Forestal Andino Patago´ nico (CIEFAP, Esquel, Argentina). Data about vouchers are given in the taxonomic treatment. DNA extraction and PCR conditions.— For DNA extractions, strains were cultured in malt peptone broth with 10% (v/v) of malt extract (Merck) and 0.1% (w/v) Bacto peptone (Difco), 2 mL medium in 15 mL tubes. The cultures were incubated at 25 C for 5 d in darkness. Total DNA was extracted with the UltraCleanTM Microbial DNA Isolation Kit (MO BIO laboratories Inc., USA) according to the manufacturer’s instructions. rDNAs ITS (including ITS1, 5.8S and ITS2) and nucLSU regions were amplified with universal primers ITS5-LR21 and LR0R-LR5 respectively
(R. Vilgalys lab webpage at http://www.botany.duke.edu/ fungi/mycolab). PCR reaction mixtures for amplification of both regions were modified from Rajchenberg et al. (2011) in a final reaction volume of 50 mL with 100–500 ng DNA. PCR reactions were performed in a thermal cycler (My CyclerTM, BioRad) and the thermal-cycling program was the same described in Rajchenberg et al. (2011). The amplified fragments were purified and sequenced on an ABI 3700 automated sequencer (Perkin-Elmer, USA) at the DNA Synthesis and Sequencing Facility (Macrogen, Korea). The same primers were used for amplification and sequencing. (Sequences generated in this study were submitted to GenBank and accession numbers are given in TABLE I.) Phylogenetic analyses.—DNA sequences generated in this study were manually edited with BioEdit 7.1.3.0 (Hall 1999), and additional sequences for the ingroup and outgroup, based on studies of Wagner and Fischer (2002) and He and Dai (2012), were retrieved from the GenBank nucleotide database. ITS sequences were not available for Hymenochaete acanthophysata J.C. Le´ger. Sequence alignments were automatically performed on MUSCLE 3.8.31 (Edgar 2004) and manually checked on MEGA 5.10 (Tamura et al. 2011). Alignments are available from TreeBASE (http://purl.org/ phylo/treebase/phylows/study/TB2:S14475). The two DNA regions first were analyzed independently (data not shown), and because there were no major topological conflicts the data were combined into a single matrix for subsequent analyses. Phylogenetic analyses were conducted
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FIG. 1. Phylogenetic relationships of Hymenochaete s.l. inferred with a combined dataset (rDNA ITS and LSU) under maximum parsimony (MB) and Bayesian inference (BI) analyses. Tree topology is based on one of the three equally most parsimonious trees. Support values for internal nodes are given on the branches as bootstrap/posterior probability (BS/PP). Fully supported nodes (BS 100/PP 1.0) are indicated by black circles. Incongruous topologies between MP and BI analyses are indicated with an asterisk instead of the PP value. Species names in boldface indicate the type species of Cyclomyces (Hym. cyclolamellata), Hydnochaete (Hym. peroxydata), Hymenochaete (Hym. rubiginosa) and Pseudochaete (P. tabacina). Species names are followed by symbols indicating hymenophore types: §5 smooth, ¤5 hydnoid, # 5 poroid to cyclolamellate. for the two loci combined dataset under maximum parsimony (MP) and Bayesian inference (BI) criteria. Fomitopsis pinicola (Sw. : Fr.) P. Karst. and Trametes villosa (Sw. : Fr.) Kreisel were used as outgroup species. MP analysis was performed in PAUP* 4.0b10 (Swofford 2002) with gaps treated as missing characters, equal weighting of characters and transformations, heuristic searches (TBR and MULTREES options on) with random addition of sequences (1000 replicates), and MAXTREES set to auto-increase. Nodal support was tested with bootstrap (BS) of 1000 replicates using the heuristic search option (TBR and MULTREES options on) and 10 random addition sequences. Bayesian analysis was conducted in MrBayes 3.2.1 (Ronquist et al. 2012). Models of evolution were identified for each dataset using jModelTest 2.1.2 (Darriba et al. 2012) under selection AIC, resulting in the model TPM2uf + I + G for ITS and TPM3uf + I + G for LSU. BI posterior probabilities (PP) were estimated for 107 generations by
running four chains and sampling a tree each 105 generations, and the first 5% trees from each run were discarded as burn-in. Burn-in was determined with Tracer 1.5 (http://tree.bio.ed.ac.uk/software/tracer/) to analyze MrBayes output files.
RESULTS The combined dataset (ITS and LSU) included 21 taxa and a total of 1670 characters, of which 1067 were constant, 161 were variable and parsimony uninformative, and 442 were parsimony informative. MP analysis resulted in three equally most parsimonious trees (Tree length 5 1229; CI 5 0.6373; RI 5 0.7785; RC 5 0.5384), one of which is presented (FIG. 1). Six new sequences were generated (TABLE I). The monophyly of the ingroup is fully supported with both MP and BI analyses and only two
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topological incongruencies between the MP and BI analyses were observed (FIG. 1, SUPPLEMENTARY FIG. 1). Those nodes are weakly supported with MP (BS , 80) and not supported with BI analysis (PP , 0.85). The ingroup taxa pertained to two major strongly supported clades: Hymenochaete s. str. (13 species) and Pseudochaete (four species). From a total of 17 clades identified, 10 were fully supported (BS 100/PP 1.0), five received moderate to high support (BS . 80; PP . 0.94), and two terminal clades were not supported (FIG. 1). The segregation of Cyclomyces and Hydnochaete as independent from Hymenochaete was not supported by our analyses because the type species of these three genera were placed in the same clade (Hymenochaete s. str.) with full support (FIG. 1). Furthermore, monophyletic groups of species with different hymenophoral types were observed—for example, Hymenochaete rheicolor (Mont.) Le´v. (smooth) with Hym. cyclolamellata (concentrically lamellate to poroid) and Hym. xerantica (Berk.) S.H. He & Y.C. Dai (poroid); Hym. rubiginosa (smooth) with Hym. paucisetigera (Parmasto & Sheng H. Wu) S.H. He & Y.C. Dai (hydnoid). Hymenochaete s. str. and Pseudochaete each include at least three hymenophoral types, according to the results presented here. The three Hyd. peroxydata specimens sampled clustered with full support with a sister clade formed by Hym. duportii (Pat.) T. Wagner & M. Fisch. and Hym. hydnoides T. Wagner & M. Fisch. The entire group is nested in the Hymenochaete s. str. clade. TAXONOMY Hymenochaete Le´v., Ann. Sci. Nat., Bot. Ser. III 5:150, 1846, nom. cons., non Hymenochaeta P. Beauv. ex T. Lestib. 1819 (Cyperaceae). 5 Cyclomyces Kunze ex Fr., Linnaea 5:512, 1830, nom. rej. 5 Hydnochaete Bres., Hedwigia 35(5):287, 1896, syn. nov., non Hydnochaete Peck 1897.
Hymenochaete peroxydata (Berk. ex Cooke) Baltazar, Gorjo´n & Rajchenb., comb. nov. MycoBank MB804370 ; Hydnum peroxydatum Berk. ex Cooke, Grevillea 20(93):1, 1891 (basionym). ; Hydnochaete peroxydata (Berk. ex Cooke) Dennis, Kew Bull. Addit. Ser. 3:105, 1970. 5 Hydnochaete badia Bres., Hedwigia 35:287, 1896. Specimens examined: BRAZIL. RIO GRANDE DO SUL: Riozinho. On dead hardwood, 10 Apr 2010, J.M. Baltazar 1819 (ICN, CIEFAP); Sa˜o Francisco de Paula, PROMATAPUC. On dead hardwood, 26 Jun 2010, J.M. Baltazar 2056 (ICN, CIEFAP); SANTA CATARINA: Blumenau. 1894, A. Mo¨ller nu 211 (S, lectotype of Hydnochaete badia); Blumenau. A. Mo¨ller nu 268 (S, paratype of Hydnochaete badia);
A. Mo¨ller nu 801 (S, paratype of Hydnochaete badia); Blumenau, A. Mo¨ ller (S); Floriano´ polis, Unidade de Conservac¸a˜o Ambiental Desterro (UCAD). On dead hardwood, 17 Sep 2010, J.M. Baltazar 2102 (ICN, CIEFAP).
A monograph of Hymenochaete by Le´ger (1998) presented a morphological treatment of species with smooth hymenophore. Ryvarden (1982) monographed Hydnochaete with descriptions and drawings of all the accepted taxa. DISCUSSION Results of the present study are similar to those of Wagner and Fischer (2002), He and Dai (2012), He and Li (2013) and Parmasto et al. (2014). Sequences of two nuclear rDNA regions were used to infer the phylogenetic placement of Hyd. peroxydata, the type species of Hydnochaete. Before the present study, the phylogenetic relationships of Hymenochaete and allied genera using a combined dataset of ITS and LSU sequences were investigated only by He and Li (2013). Specimens of Hyd. peroxydata are nested within the Hymenochaete s. str. clade with full support, and two other hydnoid species (Hym. duportii and Hym. hydnoides) form a sister group, but they are also closely related to species with a smooth hymenophore such as Hym. acanthophysata and Hym. murina Bres. Hymenochaete is accepted as monophyletic and includes species with resupinate, pileate sessile or stipitate basidiomes, with smooth, fissured or verrucose, hydnoid, poroid or concentrically lamellate hymenophores, monomitic hyphal system, simple septate hyphae, presence or absence of setae, and hyaline basidiospores. Pseudochaete also includes species with smooth, hydnoid and poroid to concentrically lamellate hymenophores, and there is no evident morphological feature to separate it from Hymenochaete s. str. Pseudochaete tabacina (Sowerby : Fr.) T. Wagner & M. Fisch., type species of the genus, presents a holocenocytic nuclear behavior (Wagner and Fischer 2002). However, data on the nuclear behavior of other species currently accepted in Pseudochaete and of most species of Hymenochaete s. str. is lacking, and the importance of this feature to segregate these genera remains unknown. Because Hymenochaete s. str. and Pseudochaete can be separated based on molecular data only, the generic placement of 70–80 species of Hymenochaete s. l. not yet included in phylogenetic analyses remains unknown (Parmasto et al. 2014). ACKNOWLEDGMENTS The authors are grateful for the help of Olivier Chauveau and Larissa Trierveiler Pereira for contributions on a
BALTAZAR ET AL.: HYDNUM PEROXYDATUM preliminary draft of the manuscript and suggestions on the phylogenetic analyses. Loreta Branda˜o de Freitas and Patrı´cia Valente da Silva also made suggestions for these analyses. JMB has doctoral scholarships from CNPq (GD 141495/2010-3) and CAPES (PDSE—proceeding 9715/118). CAPES (Brazil) and MINCyT (Argentina) are thanked by financial support (Bilateral cooperation CAPES/MINCyT Rede 003/11). MBP and MR are researchers of CONICET (Argentina).
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