Mycological Progress (2019) 18:1079–1099 https://doi.org/10.1007/s11557-019-01507-0 ORIGINAL ARTICLE On Protomerulius and Heterochaetella (Auriculariales, Basidiomycota) Viacheslav Spirin1,2 & Vera Malysheva3 & Otto Miettinen1 & Josef Vlasák4 & Renato Lúcio Mendes Alvarenga5 & Tatiana Baptista Gibertoni5 & Leif Ryvarden6 & Karl-Henrik Larsson2 Received: 3 June 2019 /Revised: 16 July 2019 /Accepted: 23 July 2019 # The Author(s) 2019 Abstract The taxonomy of Protomerulius and Heterochaetella is revised based on DNA data and morphological evidence, and their type species, P. brasiliensis and H. dubia, are proved to be congeneric. As a consequence, H. dubia and related species, of which four are described as new, are placed in Protomerulius. Heterochaete microspora is also combined in Protomerulius, and the genus concept is redefined to encompass effused species with smooth or spiny hymenophore and monomitic hyphal structure. Psilochaete multifora, gen. and spec. nov. is a distant relative of Protomerulius spp. found in Norway. Heterochaetella cystidiophora is re-collected in Brazil and placed in the synonyms of Heterochaete sanctae-catharinae. This species does not belong to the Protomerulius–Heterochaetella lineage, and it is transferred to Metulochaete,gen.nov. Keywords Heterobasidiomycetes . Phylogeny . Polypores . Taxonomy . New taxa Introduction Friedrichsen (1977) published a list of Möller’s collections preserved at the herbarium in Hamburg. Afterwards, Protomerulis brasiliensis Möller was described from Brazil as Bandoni et al. (1982) studied one of the authentic collections a poroid fungus with septate basidia (Möller 1895). This was labelled as P. brasiliensis and published a detailed description the first record of a species with poroid hymenophore among as well as microscopic drawings. Because Protomerulius is the so-called jelly fungi (heterobasidiomycetes). Both the much older than other generic names for poroid genus and the species remained almost forgotten until heterobasidiomycetes (i.e., Elmerina Bres., Aporpium Bondartsev & Singer and Protodaedalea Imazeki), the resur- rection of Protomerulius caused a lot of discussion concerning Responsible editor: Yu-Cheng Dai generic rearrangement of the group (Setliff & Ryvarden 1982, Ryvarden 1991, Reid 1992). However, no satisfactory solu- * Viacheslav Spirin tion for splitting this complex was proposed. [email protected] Weiss and Oberwinkler (2001) included some modern col- lections of Protomerulius spp. in the phylogenetic study of the 1 Finnish Museum of Natural History, University of Helsinki, PO Box Auriculariales. According to their results, Protomerulius 7, 00014 Helsinki, Finland forms a strongly supported clade together with corticioid 2 Natural History Museum, University of Oslo, P.O. Box 1172, (Heterochaetella brachyspora Luck-Allen), hydnoid Blindern, 0318 Oslo, Norway (Protohydnum piceicola Kühner), and clavarioid 3 Komarov Botanical Institute, Russian Academy of Sciences, Prof. (Tremellodendropsis sp.) taxa, and it is not closely related to Popova str. 2, 197376 St., Petersburg, Russia the other poroid representatives of the order. While the latter 4 Biology Centre, Academy of Sciences of the Czech Republic, ones (i.e., Aporpium, Elmerina,andProtodaedalea), as well š Č ě Brani ovská 31, 37005 eské Bud jovice, Czech Republic as Protohydnum were rather extensively studied in the last 5 Departamento de Micologia, Centro de Biociências, Universidade years (Miettinen et al. 2012,Zhou&Dai2013, Sotome Federal de Pernambuco, Av. da Engenharia s/n, et al. 2014,Vlasáketal.2016, Malysheva et al. 2018), only Recife, Pernambuco 50740-570, Brazil 6 a few new sequences were generated for Protomerulius and Department of Biosciences, University of Oslo, P.O. Box 1066, Heterochaetella, and therefore, their relationships stayed Blindern, 0316 Oslo, Norway 1080 Mycol Progress (2019) 18:1079–1099 unresolved. In the present study, we revise generic and using the Q-INS-i option for nrITS and nrITS+nrLSU species-level taxonomy of Protomerulius and associated taxa, datasets. and provide new information about Heterochaetella Phylogenetic reconstructions were performed with max- cystidiophora Lowy. imum likelihood (ML) and Bayesian inference (BI) analy- ses. Before the analyses, the best-fit substitution model was estimated separately for all alignments based on the Akaike information criterion (AIC) using FindModel Web Material and methods server (http://www.hiv.lanl.gov/content/sequence/ findmodel/findmodel.html). GTR model was chosen for Morphological study Type specimens and collections from all datasets. ML analysis was run on RAxML server, v.0. herbaria BO, BPI, CWU, GB, H, HBG, K, LE, MAN, NY, 6.0 (http://raxml-ng.vital-it.ch/#/) with one hundred rapid O, PC, TAAM, TRTC, URM, as well as from the private bootstrap replicates. BI analysis was performed with herbarium of author JV were studied. Herbarium acronyms MrBayes 3.2.5 software (Ronquist et al. 2012), for two are given according to Thiers (2019). Microscopic routine independent runs, each with 5 million generations for follows Miettinen et al. (2018). All measurements were made concatenated dataset (nrITS+nrLSU) and 10 million gen- from microscopic slides mounted in Cotton Blue, using phase erations for nrITS and tef1 datasets, under described model contrast and oil immersion lens (Leitz Diaplan microscope, × and four chains with sampling every 100 generations. To 1250 amplification). The following abbreviations are used in check for convergence of MCMC analyses and to get esti- morphological descriptions and species key for Protomerulius mates of the posterior distribution of parameter values, spp.: L, basidiospore length; W, basidiospore width; Q’, Tracer v1.7.1 was used (Rambaut et al. 2018). We accepted length/width ratio; Q, length/width ratio; n,numberofmea- the result where the ESS (effective sample size) was above surements per specimens measured. 200 and the PSRF (potential scale reduction factor) was close to 1. Newly generated sequences have been deposit- Molecular techniques In total, 44 specimens were selected ed in GenBank with corresponding accession numbers for molecular sampling (Table 1). For most specimens, (Table 1). The alignments have been deposited in DNA was extracted from small fragments of dried TreeBASE (S23966, S23967). basidiocarps using the NucleoSpin Plant II Kit (Macherey-Nagel GmbH & Co. KG) according to the man- ufacturer’s protocols. The following primers were used for Results amplification and sequencing: ITS1F-ITS4 (White et al. 1990; Gardes & Bruns 1993) for the ITS1-5.8S-ITS2 frag- Three datasets were prepared for the present study: (1) nrITS + ment; JS1-LR5 (Vilgalys & Hester 1990; Landvik 1996) nrLSU dataset used to reconstruct a general topology of the for part of nrLSU region; EF1-983F and EF1-1567R for Auriculariales, with special emphasis on Protomerulius and approximately 500–600 bp of tef1 (Rehner & Buckley Heterochaetella spp., (2) nrITS and (3) tef1 datasets 2005). PCR products were purified applying the GeneJET supporting species concepts in the genus Protomerulius as Gel Extraction Kit (Thermo Scientific, Thermo Fisher redefined below. Scientific Inc., MA, USA). Sequencing was performed with an ABI model 3130 Genetic Analyzer (Applied 1. nrITS + nrLSU dataset. The final alignment contained Biosystems, CA, USA). Raw data were edited and assem- 1646 characters (including gaps). The overall topologies bled in MEGA X (Kumar et al. 2018). Molecular studies of of the ML and BI trees were nearly identical and con- some specimens were carried out at the center for collec- firmed that Protomerulius and most Heterochaetella tive use of scientific equipment “Cellular and molecular spp. (including the genus type, Heterochaetella dubia technology of studying plants and fungi” (Komarov (Bourdot & Galzin) Bourdot & Galzin) belong to one Botanical Institute, Russian Academy of Sciences, St. strongly supported clade, while H. cystidiophora and a Petersburg), and a few poroid specimens were sequenced still unnamed taxon with a certain morphological similar- as described in Miettinen et al. (2018). ity to the H. dubia complex form their own lineages with- in the order (Fig. 1). Phylogenetic analyses For this study, 35 nrITS, 30 nrLSU, and 11 tef1 sequences were generated (Table 1). In addition to the A) Protomerulius/Heterochaetella lineage (pp = 1, bs = newly generated sequences, 47 nrITS and 45 nrLSU were 95) is uncovered as a sister clade of Hyalodon V. retrieved from the GenBank database (www.ncbi.nlm.nih. Malysheva & Spirin (typified with Protohydnum gov/genbank/). Sequences were aligned with the MAFFT piceicola Kuehner ex Bourdot). This outcome agrees version 7 Web tool (http://mafft.cbrc.jp/alignment/server/) with the earlier studies of the Auriculariales (Weiss Mycol Progress (2019) 18:1079–1099 1081 Table 1 DNA sequences generated for the present study Species Specimen/ Place of origin Host GenBank number herbarium (ISO code) nrLSU nrITS tef1 Metulochaete sanctae-catharinae AM 0678 (URC) BR-AC Hardwood MK480575 MK484065 – Protomerulius brachysporus PV 2600 (H) FI Picea abies MK480547 MK484033 MK497236 P. b rac hy sp or us LR 23195 (O) MX Conifer MK480548 MK484038 – P. b rac hy sp or us O F293193 NO P. abies MK480550 MK484036 – P. b rac hy sp or us TAAM 102746 RU-PRI Abies nephrolepis – MK484037 – P. b rac hy sp or us JN 7643 (O) SE P. abies – MK484035 – P. b rac hy sp or us VS 8351c (H) USA-WA Thuja plicata MK480549 MK484034 – Protomerulius commotus VS 11097