MycoKeys 33: | 45-| 60 (202 | ) er-reviewed open-access journal doi: 10.3897/mycokeys.83.69909 < MycoKkeys https://mycokeys.pensoft. net Launched to accelerate biodiversity research Morphological and phylogenetic evidence for recognition of two new species of Hyphoderma (Basidiomycota) from southern China, with a key to all Chinese Hyphoderma Qian-Xin Guan'?, Yi-Fei Li?, Chang-Lin Zhao'?? | Key Laboratory for Forest Resources Conservation and Utilization in the Southwest Mountains of China, Ministry of Education, Southwest Forestry University, Kunming 650224, China 2 Yunnan Academy of Bio- diversity, Southwest Forestry University, Kunming 650224, China 3 College of Biodiversity Conservation, Southwest Forestry University, Kunming 650224, China Corresponding author: Chang-Lin Zhao (fungi@swfu.edu.cn) Academic editor: R. Henrik Nilsson | Received 9 June 2021 | Accepted 28 August 2021 | Published 20 September 2021 Citation: Guan Q-X, Li Y-F, Zhao C-L (2021) Morphological and phylogenetic evidence for recognition of two new species of Hyphoderma (Basidiomycota) from southern China, with a key to all Chinese Hyphoderma. MycoKeys 83: 145-160. https://doi.org/10.3897/mycokeys.83.69909 Abstract Wood-inhabiting fungi play crucial roles as decomposers in forest ecosystems and, in this study, two new wood-inhabiting corticioid fungi, Hyphoderma puerense and H. tenuissimum spp. nov., are proposed, based on a combination of morphological features and molecular evidence. Hyphoderma puerense is chat- acterised by effused basidiomata with smooth to floccose hymenial surface, a monomitic hyphal system with clamped generative hyphae and ellipsoid basidiospores. Hyphoderma tenuissimum is characterised by resupinate basidiomata with tuberculate to minutely-grandinioid hymenial surface, septate cystidia and cylindrical to allantoid basidiospores. Sequences of ITS and nLSU rRNA markers of the studied samples were generated and phylogenetic analyses were performed with Maximum Likelihood, maximum parsi- mony and Bayesian Inference methods. These analyses showed that the two new species clustered into Hyphoderma, in which H. puerense grouped with H. moniliforme and H. tenuissimum formed a singleton lineage. In addition, an identification key to Chinese Hyphoderma is provided. Keywords Corticioid fungi, diversity, Hyphodermataceae, molecular phylogeny, taxonomy, Yunnan Province Copyright Qian-Xin Guan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 146 Qian-Xin Guan et al. / MycoKeys 83: 145-160 (2021) Introduction Fungi are eukaryotic microorganisms that play fundamental ecological roles as decom- posers and mutualists of plants and animals. They drive carbon cycling in forest soils, mediate mineral nutrition of plants and alleviate carbon limitations of other soil organ- isms (Tedersoo et al. 2014). Fungi form an ecologically important branch of the tree of life, based on their distinct and diverse characters (James et al. 2020). Hyphoderma Wallr. was typified by H. setigerum (Fr.) Donk (Donk 1957) and the genus is characterised by resupinate to effuse-reflexed basidiomata of ceraceous consistency and a smooth to tuberculate or hydnoid hymenophore. Hyphoderma species are character- ised by a monomitic (rarely dimitic) hyphal structure with clamp connections on genera- tive hyphae, presence of cystidia or not, suburniform to subcylindrical to cylindrical basid- ia and ellipsoid to subglobose, smooth, thin-walled basidiospores (Wallroth 1833; Bernic- chia and Gorjén 2010). Currently, about 105 species have been accepted in Hyphoderma worldwide (Donk 1957; Nakasone 2008; Wu et al. 2010; Baltazar et al. 2016; Martin et al. 2018; Guan and Zhao 2021a, 2021b; Ma et al. 2021). Index Fungorum (http://www. in- dexfungorum.org; accessed on 16 July 2021) and MycoBank (https://www.mycobank.org; accessed on 16 July 2021) register 199 specific and infraspecific names in Hyphoderma. Hyphoderma has been studied using molecular data, particularly the internal tran- scribed spacer (ITS) region and the large subunit nuclear ribosomal RNA gene (nLSU). Larsson (2007) showed that H. obtusum J. Erikss. and H. setigerum clustered into the Meruliaceae Rea and formed a sister taxon to Hypochnicium polonense (Bres.) A. Strid. Tel- leria et al. (2012) proposed a new species, Hyphoderma macaronesicum Telleria, M. Due- has, Beltran-Tej., Rodr.-Armas & M.P. Martin and then discussed the relationships with the closely-related taxa in Hyphoderma. Research into the Hyphoderma setigerum complex showed that 1. pinicola Yurch. & Sheng H. Wu represented a fifth species in this complex (Yurchenko and Wu 2014b). A revised family-level classification of the Polyporales re- vealed that four Hyphoderma species grouped into the residual polyporoid clade, belong- ing to Hyphodermataceae in that they grouped with three related genera in Meripilaceae: Meripilus P. Karst., Physisporinus P. Karst. and Rigidoporus Murrill (Justo et al. 2017). In this study, two undescribed species of corticioid fungi from forest ecosystems were collected in the Yunnan Province, China. We present morphological and molecu- lar phylogenetic evidence that support the recognition of two new species in Hypho- derma, based on the nuclear ribosomal internal transcribed spacer region (ITS1, 5.8S and ITS2) and the nuclear ribosomal nLSU (28S) gene. Materials and methods Morphology The studied specimens are deposited at the Herbarium of Southwest Forestry University (SWEC), Kunming, Yunnan Province, PR. China. Macromorphological descriptions are based on field notes and photos captured in the field and lab. Colour terminology fol- Two new Hyphoderma species 147 lows Petersen (Petersen 1996). Micromorphological data were obtained from the dried specimens when observed under a light microscope following Dai (2012). The following abbreviations are used: KOH = 5% potassium hydroxide water solution, CB = Cotton Blue, CB— = acyanophilous, IKI = Melzer’s Reagent, NKI- = both inamyloid and index- trinoid, L = mean spore length (arithmetic average for all spores), W = mean spore width (arithmetic average for all spores), Q = variation in the L/W ratios between the specimens studied and n = a/b (number of spores (a) measured from given number (b) of specimens). Molecular phylogeny The CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd, Beijing) was used to obtain genomic DNA from the dried specimens following the manufacturer's instructions (as done in Zhao and Wu 2017). The nuclear riboso- mal ITS region was amplified with the primers ITS5 and ITS4 (White et al. 1990). The nuclear ribosomal LSU gene was amplified with the primers LROR and LR7 (Vil- galys and Hester 1990; Rehner and Samuels 1994). The PCR procedure for ITS was as follows: initial denaturation at 95 °C for 3 min followed by 35 cycles at 94 °C for 40 s, 58 °C for 45 s and 72 °C for 1 min and a final extension of 72 °C for 10 min. The PCR procedure for nLSU was as follows: initial denaturation at 94 °C for 1 min followed by 35 cycles at 94 °C for 30 s, 48 °C for 1 min and 72 °C for 1.5 min and a final exten- sion of 72 °C for 10 min. The PCR products were purified and sequenced at Kunming Tsingke Biological Technology Limited Company, Kunming, Yunnan Province, PR. China. All newly-generated sequences were deposited in NCBI GenBank (Table 1). The sequences were aligned in MAFFT version 7 (Katoh et al. 2019) using the G- INS-i strategy. The alignment was adjusted manually using AliView version 1.27 (Lars- son 2014). Each dataset was aligned separately at first and then the ITS1, 5.88, ITS2 and nLSU regions were combined with Mesquite version 3.51. The combined dataset was deposited in TreeBASE (submission ID 28564). Climacocystis borealis (Fr.) Kotl. and Pouzar and Diplomitoporus crustulinus (Bres.) Domanski were selected as outgroup (Fig. 1) as inspired by a previous study (Justo et al. 2017). Maximum parsimony analysis in PAUP* version 4.0a169 (http://phylosolu- tions.com/paup-test/) was applied to the combined ITS1+5.8S+ITS2+nLSU data- set. All characters were equally weighted and gaps were treated as missing data. Trees were inferred using the heuristic search option with TBR branch swapping and 1,000 random sequence additions. Max-trees were set to 5,000, branches of zero length were collapsed and all parsimonious trees were saved. Clade robustness was assessed using bootstrap (BT) analysis with 1,000 pseudoreplicates (Felsenstein 1985). Descriptive tree statistics — tree length (TL), composite consistency index (CI), composite retention index (RI), composite rescaled consistency index (RC) and composite homoplasy index (HI) — were calculated for each maximum parsi- monious tree generated. The combined dataset was also analysed using Maximum Likelihood (ML) in RAxML-HPC2 through the CIPRES Science Gateway (Miller et al. 2012). Branch support (BS) for the ML analysis was determined by 1,000 bootstrap pseudoreplicates. 148 Qian-Xin Guan et al. / MycoKeys 83: 145-160 (2021) Table |. List of species, specimens and GenBank accession numbers of sequences used in this study. Species name Specimen No. GenBank accession No. References ITS LSU Climacocystis borealis FD-31 KP135308 KP135210 Justo et al. (2017) Diplomitoporus crustulinus FD-137 KP135299 KP135211 Justo et al. (2017) Hyphoderma amoenum USO 286622 HE577030 — Telleria et al. (2012) H. assimile CBS 125852 MH863808 MH875272 Vu et al. (2019) H. cremeoalbum NH 11538 DQ677492 DQ677492 Larsson (2007) H. crystallinum CLZhao 9338 MW917161 MW913414 Guan and Zhao (2021a) CLZhao 9374 MW917162 MW913415 Guan and Zhao (2021a) CLZhao 10224 MW917163 MW9 13416 Guan and Zhao (2021a) CLZhao 11723 MW917164 MW913417 Guan and Zhao (2021a) CLZhao 15841 MW917165 MW913418 Guan and Zhao (2021a) CLZhao 18459 MW917166 MW913419 Guan and Zhao (2021a) H. definitum GEL 2898 — AJ406509 Yurchenko and Wu (2014) NH 12266 DQ677493 DQ677493 Larsson (2007) H. fissuratum CLZhao 6731 MT1791331 MT791335 Ma etal. (2021) CLZhao 6726 MT791330 M1791334 Ma etal. (2021) H. floccosum CLZhao 17129 MW301683 MW293733 Guan and Zhao (2021b) CLZhao 17296 MW301686 MW293736 Guan and Zhao (2021b) CLZhao 16492 MW301688 MW293734 Guan and Zhao (2021b) CLZhao 17215 MW301687 MW293735 Guan and Zhao (2021b) H. granuliferum KHL 12561 JN710545 JN710545 Yurchenko and Wu (2014) H. incrustatum KHL 6685 — AY586668 Yurchenko and Wu (2014) H. litschaueri NH 7603 DQ677496 DQ677496 Larsson (2007) FP-101740-Sp KP135295 KP135219 Floudas and Hibbett (2015) H.. macaronesicum MA:Fungi: 16099 HE577027 — Yurchenko and Wu (2014) TFC:Mic.15981 HE577028 — Yurchenko and Wu (2014) H. medioburiense NH 10950 DQ677497 DQ677497 Larsson (2007) Hf. membranaceum CLZhao 5844 MW917167 MW913420 Guan and Zhao (2021a) CLZhao 6971 MW917168 MW913421 Guan and Zhao (2021a) H. microporoides CLZhao 6857 MW917169 MW913422 Guan and Zhao (2021a) CLZhao 8695 MW917170 MW913422 Guan and Zhao (2021a) H. moniliforme Wu 0211-42 KC928282 — Yurchenko and Wu (2015) Wu 0211-46 KC928284 KC928285 Yurchenko and Wu (2015) H.. mopanshanense CLZhao 6498 MT791329 MT791333 Ma et al. (2021) CLZhao 6493 MT791328 MT1791332 Ma et al. (2021) H.. nemorale TNM F3931 KJ885183 KJ885184 Yurchenko and Wu (2015) Wau 9508-14 KC928280 KC928281 Yurchenko and Wu (2015) H.. nudicephalum Wu 9307-29 AJ534269 — Nilsson et al. (2003) Wu 9508-225 AJ534268 = Nilsson et al. (2003) H. obtusiforme KHL 1464 JN572909 — Yurchenko and Wu (2014) KHL 11105 JN572910 — Yurchenko and Wu (2014) H. obtusum JS 17804 — AY586670 Yurchenko and Wu (2014) H. occidentale KHL 8469 —_ AY586674 Yurchenko and Wu (2014) KHL 8477 DQ677499 DQ677499 Larsson (2007) H. paramacaronesicum MA:Fungi:87736 KC984399 — Martin et al. (2018) MA:Fungi:87737 KC984405 — Martin et al. (2018) H. pinicola Wu 0108-32 KJ885181 KJ885182 Yurchenko and Wu (2014) Wu 0108-36 KC928278 KC928279 Yurchenko and Wu (2014) H. prosopidis E09/58-9 HE577029 — Yurchenko and Wu (2015) Hi. puerense CLZhao 9476* MW443045 — Present study CLZhao 9583 MW443046 MW443051 Present study H. roseocremeum NH 10545 = AY586672 Yurchenko and Wu (2014) H. setigerum FCUG 1200 AJ534273 — Nilsson et al. (2003) H. setigerum FCUG 1688 AJ534272 _ Nilsson et al. (2003) H. sinense CLZhao 7963 MW301679 MW293730 Guan and Zhao (2021b) CLZhao 17811 MW301682 MW293732 Guan and Zhao (2021b) CLZhao 7981 MW301680 MW293731 Guan and Zhao (2021b) Hyphoderma sp. KUC20121102-21 KJ668522 — Unpublished KUC11052 KJ714002 — Jang et al. (2015) Wu 0311-25 KR868735 — Unpublished Wau 0310-6 KR868736 — Unpublished Wu 0808-87 KR868737 — Unpublished GEL3689 DQ340327 — Unpublished H. subsetigerum Wu 9304-18 AJ534277 — Nilsson et al. (2003) Wu 9202-15 AJ534278 — Nilsson et al. (2003) Two new Hyphoderma species Species name Specimen No. GenBank accession No. References ITS LSU H. subsetigerum HHB11620 GQ409521 — Yurchenko and Wu (2014) CFMR MJL1536 GQ409522 — Yurchenko and Wu (2014) H. tenuissimum CLZhao 6930 MW443047 MW443052 Present study CLZhao 7003 MW443048 MW443053 Present study CLZhao 7221* MW443049 MW443054 Present study CLZhao 16210 MW443050 MW443055 Present study 7. transiens NH 12304 DQ677504 DQ677504 Larsson (2007) H. variolosum CBS 734.91 MH862320 MH873992 Vu et al. (2019) CBS 735.91 MH862321 MH873993 Vu et al. (2019) Hypochnicium erikssonii NH 9635 — DQ677508 Larsson (2007) H1. geogenium NH 10910 — DQ677509 Larsson (2007) MA-Fungi 48308 FN552534 JN939576 Telleria et al. (2010) H. michelii MA-Fungi 79155 NR119742 NG060635 Telleria et al. (2010) H. punctulatum FP101698sp KY948827 KY948860 Justo et al. (2017) H. sphaerosporum RLG15138sp KY948803 KY948861 Justo et al. (2017) H. wakefieldiae MA-Fungi 7675 FN552531 JN939577 Telleria et al. (2010) Physisporinus subcrocatus Dai 15917 KY131870 KY131926 Wu et al. (2017) P subcrocatus Dai 12800 KY131869 KY131925 Wu et al. (2017) P tibeticus Cui 9588 KY131873 KY131929 Wu et al. (2017) Cui 9518 KY131872 KY131928 Wu et al. (2017) Rigidoporus eminens Dai 17200 MT279690 MT279911 Wu et al. (2017) R. undatus Miettinen-13591 KY948731 KY948870 Justo et al. (2017) 149 New species is shown in bold; * type material. MrModeltest 2.3 (Nylander 2004) was used to determine the best-fit evolution model for each dataset (ITS1+5.8S+ITS2+nLSU) for Bayesian Inference (BI). BI was calculated with MrBayes version 3.2.7a (Ronquist et al. 2012). Four Markov chains were run for two runs from random starting trees for 3 million generations (Fig. 1). The first 25% of all generations was discarded as burn-in. A majority rule consensus tree was computed from the remaining trees. Branches were considered as significantly sup- ported if they received a maximum likelihood bootstrap support value (BS) of > 70%, a maximum parsimony bootstrap support value (BT) of > 70% or a Bayesian posterior probability (BPP) of > 0.95. Results Molecular phylogeny The ITS1+5.88+ITS2+nLSU dataset comprised sequences from 86 fungal specimens representing 46 taxa. The dataset had an aligned length of 2,034 characters, of which 1,360 characters were constant, 131 were variable and parsimony-uninformative and 543 (35%) were parsimony-informative. Maximum parsimony analysis yielded 108 equally parsimonious trees (TL = 3,317, CI = 0.3361, HI = 0.6946, RI = 0.7051 and RC = 0.2370). The best model of nucleotide evolution for the ITS1+5.8S+ITS2+nLSU dataset estimated and applied in the Bayesian analysis was found to be GITR+I+G. Bayesian analysis and ML analysis resulted in a similar topology as in the MP analysis. The Bayesian analysis had an average standard deviation of split frequencies = 0.008952 (BI) and the effective sample size (ESS) across the two runs is double the average ESS (avg. ESS) = 1,771. The Bayesian tree is shown here (Fig. 1). 150 Qian-Xin Guan et al. / MycoKeys 83: 145-160 (2021) 99/98/ 1 .00. 94/87/1.00. q Hyphoderma subs 100/91/0.95 | | Hyphoderma subs. 99/99/1.00)s. | Hyphoderma seti Hyphoderma setiger 80/79) Hyphoderma pin. Hyphoderma pinicola -/81/- 100/100/1.00 ; Hyphoderma mopanshanens Hyphoderma mopanshanense Hyphoderma nudicephalum Wu 95' 99/97/1.00" Hyphoderma nudicephalum Wu 93\ SSL — Hyphoderma granuliferum KHL 1 Hyphoderma incrustatum KHL 6685 96/95/1.00 fyphoderma macaronesicum MA:Fun, Hyphoderma prosapidis E09/58-9 100/100/1.00 - Alyphi Hyphoderma amoenum USO 2 97/9810.95 + Hyphoderma subtes Hyphoderma Hyphoderma assimile ( Hyphoderma roseocremeum NH 10 89/92/1.00] | yphoderma fissuratum CLZhao 67 Hyphoderma fissuratum CLZhao Hyphoderma obtusum JS 17804 Hyphoderma erystallinum Cl Hyphoderma erystallinum CL Hyphoderma crystallinum C) 1o0/100/1.00 || Zvphoderma erystallinum CLZha0 Ayphederma crystallinum CLZhao | Hyphoderma crystallinum CL. 100/1/00/1.00r Hyphoderma variolosum CBS Hyphoderma variolosum CBS 73: 100/98/1.00 | Hyphoderma obtusiforme KHL 11 Hyphoderma obtusiforme KHL 1 100/100/1.00 Hyphoderma nemorale Wu 950! Hyphoderma nemorale TNM F39 100/100/1.00_- Hyphoderma microporoides CLZhat Hyphoderma microporoides CLZhao- Hyphoderma cremeoalbum NH 11538 -/68/-_ ~~ Hyphoderma definitum NH 12266 -/70!-| © Hyphoderma definitum GEL 2898 FHyphoderma occidentale KHL 8477 93192099 | Fyphadermatceedenalen aire Hyphoderma sp. GEL3689 Hyphoderma sp. Wu 0311-25 Hyphoderma sp. Wu 0310-6 Hyphoderma sp. Wu 0808-87 98/100/1.00 70/92t- Hyphoderma puerense | 7 100/100/1.00 Hyphoderma puerense C13 Hyphoderma moniliforme Wu 0211-42 -/80/1.00 | '00/100/1.00 - Hyphoderma moniliforme Wu 021146 100{100/1.00 _; Hyphoderma litschaueri FP-101740-Sp HAyphoderma litschaueri NH 7603 99/96/1.00 100/100/0.98 Physisporinus tib 100/100/1.00 Physisporinus tibeticus Cui 100/91/1.00 Physisporinus subcrocatus Dai 1. 100/100/1.00 100/100/1.00 Physisporinus subcrocatus _ Rigidoporus eminens Dai 17: 100/100/1.00 Rigidoporus undatus Miettinen 96/67/1.00 Hypochnicium erikssonii NH 96. 82/85/0.95 Hypochnicium wakefieldiae MA-Fung 95/59/1.00 Hypochnicium punctulatum FP101698sp 100/100/1.00 ; Hypochnicium geogenium NH 10910 199/72/1.00 Hypochnicium geogenium MA-Fungi 4 T/61/- oe Hypochnicium sphaerosporum Hypochnicium michelii MA Fungi 79155 | Climacocystis borealis FD-31 Di iplomitoporus crustulinus FD-137 50 Figure |. Maximum parsimony strict consensus tree illustrating the phylogeny of the two new species and related species in Hyphoderma, based on ITS1+5.88+ITS2+nLSU sequences. Branches are labelled with maximum likelihood bootstrap values > 70%, parsimony bootstrap values > 50% and Bayesian posterior probabilities > 0.95, respectively. Two new Hyphoderma species 154 The phylogram inferred from ITS1+5.8S+ITS2+nLSU sequences (Fig. 1) highlights the two undescribed species in Hyphoderma; H. puerense asa sister to H. mo- niliforme and H. tenuissimum that forms an independent monophyletic lineage (100% parsimony bootstrap support, 100% likelihood bootstrap support and 1.00 BPP). Taxonomy Hyphoderma puerense C.L. Zhao & Q.X. Guan, sp. nov. MycoBank No: 838411 Figs 2, 3 Holotype. China. Yunnan Province, Puer, Jingdong County, Huilianghe Village, GPS co-ordinates 24°04'45"N, 100°56'32"E, altitude 1246 m a.s.l., on fallen angiosperm branch, leg. C.L. Zhao, 4 January 2019, CLZhao 9476 (SWFC). Etymology. puerense (Lat.): referring to the locality (Puer) of the specimens. Figure 2. Basidiomata of Hyphoderma puerense (holotype). Scale bars: 2 cm (a); 1 mm (b). 152 Qian-Xin Guan et al. / MycoKeys 83: 145-160 (2021) | C Figure 3. Microscopic structures of Hyphoderma puerense (holotype) a basidiospores b basidia and ba- sidioles ¢ cystidia d a section of hymenium. Scale bars: 5 um (a); 10 um (b=d). Description. Basidioma annual, resupinate, adnate, byssoid, without odour and taste when fresh, up to 15 cm long, 3 cm wide, 100—260 um thick. Hymenial surface smooth to floccose, cream when fresh, cream to slightly buff on drying. Margin sterile, thinning out, narrow, cream. Hyphal system monomitic, generative hyphae with clamps, colourless, thick- walled, frequently branched, interwoven, 2.5—4.5 ym in diameter; IKI-, CB-; tissues unchanged in KOH; subhymenial hyphae densely covered by crystals. Cystidia tubular, encrusted with small crystals, 25-97 x 5.5—9.5 um. Basidia clavate to subcylindrical, slightly constricted in the middle to somewhat sinuous, with 4 sterigmata and a basal clamp, 20-30 x 4.5—6 um. Basidiospores ellipsoid, colourless, thin-walled, smooth, IKI-, CB-, (5.5—)6-7.5(— 8) x 3-4.5(—5) um, L = 6.53 pm, W = 3.71 um, Q = 1.73-1.79 (n = 60/2). Habitat and ecology. Climate of the sample collection site is subtropical monsoon climate area, the forest type is evergreen angiosperm forest and samples were collected on fallen angiosperm branches. Two new Hyphoderma species lie %) Additional specimens examined. China. Yunnan Province, Puer, Jingdong County, Huilianghe Village, GPS co-ordinates 24°04'45"N, 100°56'32"E, altitude 1246 ma.s.l., on fallen angiosperm branch, leg. C.L. Zhao, 5 January 2019, CLZhao 9583 (SWFC). Hyphoderma tenuissimum C.L. Zhao & Q.X. Guan, sp. nov. MycoBank No: 838412 Figs 4, 5 Holotype. China. Yunnan Province, Chuxiong, Zixishan Forestry Park, GPS co-ordi- nates 25°01'26"N, 101°24'37"E, altitude 2313 ma.s.l., on fallen angiosperm branch, leg. C.L. Zhao, 1 July 2018, CLZhao 7221 (SWFC). Etymology. tenuissimum (Lat.): referring to the thin basidiomata. 154 Qian-Xin Guan et al. / MycoKeys 83: 145-160 (2021) Figure 5. Microscopic structures of Hyphoderma tenuissimum (holotype) a basidiospores b basidia and basidioles € cystidia d a section of hymenium. Scale bars: 10 um (a=d). Description. Basidioma annual, resupinate, adnate, membranaceous when fresh, hard membranaceous upon drying, up to 20 cm long, 3 cm wide, 30-100 um thick. Hymenial surface tuberculate to minutely-grandinioid, slightly buff when fresh, buff upon drying, cracking. Margin sterile, slightly buff, 1 mm wide. Two new Hyphoderma species 155 Hyphal system monomitic, generative hyphae with clamps, colourless, thick-walled, fre- quently branched, interwoven, 3—5 um in diameter, IKI-, CB-; tissues unchanged in KOH. Cystidia large, cylindrical, with 4-12 clamped septa, with abundant encrustations, 50-220 x 6.5-13 pm. Basidia clavate to subcylindrical, constricted, somewhat sinuous, with 4 sterigmata and a basal clamp connection, 17-31 x 4.5-8 um. Basidiospores cylindrical, colourless, thin-walled, smooth, with oil drops inside, IKI-, CB-, 7-10.5(-11) x 3-4.5(-5) um, L = 8.75 um, W = 4.15 um, Q = 2.02-2.18 (n = 120/4). Habitat and ecology. Climate of the sample collection site is subtropical monsoon climate area, the forest type is evergreen angiosperm forest and samples were collected on fallen angiosperm branches. Additional specimens examined. China. Yunnan Province, Chuxiong, Zixishan National Forestry Park, GPS co-ordinates 25°01'26"N, 101°24'37"E, altitude 2263 m a.s.l., on fallen angiosperm branch, leg. C.L. Zhao, 1 July 2018, CLZhao 6930, CLZhao 7003 (SWFC); Wenshan, Pingba Town, Wenshan National Nature Reserve, GPS co-ordinates 23°18'19"N, 104°42'47"E, altitude 1976 m a.s.l., on fallen angio- sperm branch, leg. C.L. Zhao, 25 July 2019, CLZhao 16210 (SWFC). Discussion In the present study, two new species, Hyphoderma puerense and H. tenuissimum are described, based on phylogenetic analyses and morphological characters. Phylogenetically, the two new taxa were found to belong to Hyphoderma, in which H. puerense forms a sister species to H. moniliforme and H. tenuissimum forms an inde- pendent monophyletic lineage (100% BS, 100% BP and 1.00 BPP). Morphologically, Hyphoderma puerense is similar to H. obtusiforme J. Erikss. & A. Strid and H. obtusum in having a smooth hymenium, non-septate cylindrical cystidia and ellipsoid basidiospores. However, H. obtusiforme differs from H. puer- ense by both larger basidia (30-40 x 8—9 um) and basidiospores (10-14 x 5—7 um; Eriksson and Ryvarden 1975). Hyphoderma obtusum also differs from H. puerense by larger basidia (30-35 x 6-8 um) and basidiospores (8-9 x 5—6.5 pm; Eriksson 1958). Hyphoderma puerense is similar to H. roseocremeum (Bres.) Donk in hav- ing smooth hymenium and non-septate cylindrical cystidia. However, Hyphoderma roseocremeum differs through the presence of larger basidiospores (8—12 x 3-4 um; Bernicchia and Gorjén 2010). Morphologically, Hyphoderma tenuissimum is similar to H. floccosum C.L. Zhao & Q.X. Guan, H. mopanshanense, H. nudicephalum Gilb. & M. Blackw., H. pinicola, H. setigerum and H. subsetigerum Sheng H. Wu in having septocystidia and cylindri- cal basidiospores. However, Hyphoderma floccosum differs from H. tenuissimum by having a floccose hymenial surface and tubular cystidia (Guan and Zhao 2021b); H.. mopanshanense is separated from H. tenuissimum by having porulose to pilose hymenial surface and smaller basidia (15—18.5 x 3-4.5 um; Ma et al. 2021); H. nu- 156 Qian-Xin Guan et al. / MycoKeys 83: 145-160 (2021) dicephalum differs from H. tenuissimum in the nature of the septocystidial apex (lack- ing encrustation; swollen up to 14 um; Gilbertson and Blackwell 1988); H. pinicola is separated from H. tenuissimum by having basidia with two sterigmata and larger basidiospores (13-16 x 4—4.5 um; Yurchenko and Wu 2014b); HZ. setigerum differs by having a combination of thin basidiomata with very long septocystidia (Bernic- chia and Gorjén 2010); and H. subsetigerum differs from H. tenuissimum by having narrower basidia (20-30 x 4.5—5.5 um) and smaller basidiospores (6-8 x 2.8-3.2 um; Wu 1997). Nilsson et al. (2003) highlighted the phylogeography of Hyphoderma setigerum (Basidiomycota) in the Northern Hemisphere in a study based on molecular analysis, morphological studies and crossing tests. Nine preliminary taxa were shown to exist inside the H. setigerum complex; in the present study, H. tenuissimum belongs to the H. setigerum complex, based on the morphological character of long septocystidia and phylogenetic evidence. A previous study indicated the importance of vicariance in the evolution of this species complex (Nilsson et al. 2003) and our study shows that the specimens of H. tenuissimum are collected in Zixishan National Forestry Park (GPS co-ordinates 25°01'26"N, 101°24'37"E), Chuxiong, Yunnan Province, China, which is distinct from H. setigerum s. str. (Norway: Oppland and Finland: Pohjois- Hame). The present samples of H. subsetigerum and H. nudicephalum were collected in Yunnan Province, China, but neither of these taxa groups together closely with H. tenuissimum (Fig. 1). In the current phylogenetic tree, two partially annotated GenBank sequences (KJ668522 and KJ714002) of Hyphoderma sp. (South Korea) cluster closely with four se- quences of the new species Hyphoderma tenuissimum, although whether they really belong to this species remains to be assessed. It is certainly conceivable that they do, which would mean that Hyphoderma tenuissimum has been collected and sequenced at least six times in Asia. Regarding the new taxon 1. puerense (Fig. 1), four partially annotated GenBank sequences (KR868735, KR868736, KR868737 and DQ340327) form a reasonably well- supported clade together with our two specimens of 1. puerense. We interpret this to mean that all six taxa represent H. puerense. All of the samples in this clade are from Asia, which supports the point of the importance of vicariance in the evolution in this genus. Key to 30 accepted species of Hyphoderma in China ft GE SECA SCIUE a scn scart Rea Pout tte iNest nr ta leached Rom ul deh Re ec dae che Date cece nates 2 Ste Oy SHIGE ROSE. t hls eta Gta Leno tir ohh etd Cs tra eet elee ee Ane 5 2. “Wldvmenialsuictacesoran iin Gig f...0. sachet euilrestdon eat yleges vance tease H. acystidiatum aie “iyivteniall strriace stmOothiy.t2. «4, 8ek. 2d cet tee ea ce 3 By pBasidiospores:-l0 Senin nslen ote at walaren cata nedetnat Reine ssouloune H. densum = BBasidiasporessaal 0 hi purat iON Oth. oi xignoserinc eta teoneseser a taugaieuoletaatsenteignutealantte 4 4 Hymenophore cracked; basidiospores > 8.5 um in length ............. H., fissuratum — Hymenophore uncracked; basidiospores < 8.5 um in length .......... H. sibiricum a PEumen@ pore smmoethipes cht... Abts oath Se tu Bhs Rrra neech eee amr N ee du! 6 — Hymenophore tuberculate, porulose, grandinioid or odontoid........ eee 15 olNI AN \9 Two new Hyphoderma species 157 AW Ty pes-Ok Cy sticlla PECSeIit x Pe esi8 fries osea Geet wee OP: Sl Ne Beco aee 7, PONE Ty PE OL Cy Stl aT ESCM AD. eases a acaba tok ors canned nes eo ntltn iaaeatena ces 8 Moniliforim. cystidia abseitts.csicccceonescccetesticetscraanisevbonteesots dey. H. microcystidium IMiorillinarimmeysticiiast Present tie, Sct) .c teciug chance stints sie t oeid et atee we chloe als H. sinense FelvATie nO PGE MMCEAG IER tok 86) Ssa 8. oh a Ln Sain NED Onc MR da ee ating amanminanereenhar 9 Fiyiienophore cracked? 0 We). 0 ats cote San tne, ee mee Mn rea 11 Basictos pores a rime LEMP tiv: tees, sxsewaheeowebnes date bssbo.coAivpeuels H. definitum Basidiospores call wiiciia- lene x... feu se. slaru sande case cast dahodoaliirdasiet uaetiewtedemantialins 10 Basidiospores > ‘8.5: puniain’ lem eth concen acvocestit easensetettaeee antes H. microporoides Basidiospores: < i825" parisitmleniotht x. cscssssaccesttt ai sestseensitheectnetetaants H. puerense Gy sticarim On ULEG GEN. «,.05 3.0.08 |..crokteent oneal Me aha ea ee eae 12 Cy SUI ATC CECA le 0d neste teesiedi pene eurareeh teronesay ale em erent maint cmereas Rural 13 Basidiospotesi> 9 aim in lenethr acer ze.c caso accste cstegee cienevhase orapavaalte H. litschaueri Basidiospétes’<-9" ui in len Gune.. .scssesaadectescanntecthesateneas ac teptea H. moniliforme Basidiospores ellipsoid < 10 um in length oe eeeeeeereeeee H. rimulosum Basidiospores cylindrical > 10 um in length ole cseeseeseeeeeceeeeseeneees 14 Basicios Pores etl 2 WM AME NOt irs wsAondsmmssovdeeranoervatubarespeharnassieentd H. cremeum Basithtosporesi- Ac Sa rmiinrwidthigs.shstece.o..s5o0 feces ances agie rotegeaca: H. formosanum Basidiospores larger 7—10.5 x 3—4.5 PM... cece eseeeeeeeees H. tenuissimum Basidiospores smaller 6-8 x 2.8-3.2 UM ....eeeeecseeeseseeeeseeeees H. subsetigerum Cysticiacalewo bypesee usher tesco eM le eae OD Seas cata btece tenet ioe 20 Cystidia or Onesiy pe wale heevs cola d ok eealec te Baer tee RM Son folie ee J Pate Macs 23 Septate-Cystidia absent s...0:...cacpess chee tee cst mused eee pence tere 21 SEP EALe Gy sil Glia’ DLESEM Eg. se. ctemebeks cha sbite es ath et ease BiB one ne Sedl dy Ueee halen se esha 22 Basidiospores 4 um in Width oo... eee eee eeeeeeeseeeeeeeeeees FH. crystallinum Basidia 2-sterigmata, basidiospores > 13 um in length... H. pinicola Basidia 4-sterigmata, basidiospores < 13 um in length... H. floccosum SEPLALE CY SUG EA GTEC Dre cos ce cececeeev cqneovteroan ergrvse cacevsnwendvuvves ov teteg soe snccen tr cacvesvied 24 Sep fale ey sblCMaeaDSeIEDU A, Bt bat at facia th De ane AMM Ata 30 e Aubtng Abbe tea. nates 25 Hymenophore porulose to pilose, basidia < 5 um in width ....H. mopanshanense Hymenophore tuberculate, basidia > 5 wm in width oo. H. setigerum Fiyinenopheresorulose Went... 00h. sateen ee eee Ramet H. obtusiforme Fiymenephere,tuberculate;-colltenlose sii s2.us. 2c sesesest basen eeal te: nant ctaie te 26 Cystidia = 13 Onpiis Wan lenolth, «ut, Yeancesevouep fieanedt ones vatectestaav ees tases H. cremeoalbum Cystidia >/50 suiririn Neneh cn.ecs eettsant mandate Aces ohessustadousshhdaentaauhenueteies. 27 Bastelia 2) AU) piel eRe tit 2.ce dances Stobeee hontiat Ns, bo panaas case engsa eRe. wats LNs as 28 ASICs 2.9 Onur VEE Caeser mete tiremindves ict apeeoe cer dvedsumivesden dmedsenebmesunmbnlsnamindixass 29 158 Qian-Xin Guan et al. / MycoKeys 83: 145-160 (2021) 28 Hymenophore cracking, cystidia < 10 wm in width... H. medioburiense — Hymenophore not cracking, cystidia > 10 wm in width we. H clavatum 29 )\Flyinenopmore; colle iMlOse ac. -5 cccascvocer ny ase eek ook cence tec x dcane eee H. nemorale =; “Beiymenopbose-rapereu late. 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