Novel saprobic Hermatomyces species (Hermatomycetaceae, Pleosporales) from China (Yunnan Province) and Thailand

Abstract

During our survey of the diversity of woody litter fungi in China and Thailand, three Hermatomyces species were collected from dead woody twigs of Dipterocarpus sp. (Dipterocarpaceae) and Ehretiaacuminata (Boraginaceae). Both morphology and multigene analyses revealed two taxa as new species (Hermatomycesturbinatus and H.jinghaensis) and the remaining collections as new records of H.sphaericus. Hermatomycesturbinatus is characterized by 1) dimorphic conidia, having circular to oval lenticular conidia and 2) turbinate conidia consisting of two columns with two septa composed of 2–3 cells in each column. Hermatomycesjinghaensis is characterized by dimorphic conidia, having circular to oval lenticular conidia and clavate or subcylindrical to cylindrical conidia and consisting of one or two columns with 6–8 cells in each column. Phylogenetic analyses of combined LSU, ITS, tub2, tef1-α and rpb2 sequence data supports the placement of these new taxa within Hermatomycetaceae with high statistical support.

Introduction

Over the past few decades, the number of studies using a molecular-based approach to study microfungal diversity in the greater Mekong subregion (GMS) has increased rapidly, especially on freshwater and woody litter fungi from China (Yunnan Province) and Thailand (Hapuarachchi et al. 2019; Dong et al. 2020; Li et al. 2020; Monkai et al. 2020; Wanasinghe et al. 2020, 2021; Mortimer et al. 2021). Hyde et al. (2018) reported that about 96% of fungi from Thailand are new to science. Feng and Yang (2018) estimated 104,000 fungal species currently exist in Yunnan Province, China; however, only about 6,000 are extant. Therefore, further studies need to be conducted to fill gaps in knowledge regarding the diversity, taxonomy and phylogeny of microfungi in the GMS. Supporting this obligation, we have begun to study plant-based ascomycetes in GMS. The current study accounts for hermatomyces-like ascomycetes recovered from the woody litter in China (Yunnan Province) and Thailand.

Hermatomyces was introduced by Spegazzini (1911) with H.tucumanensis as the type species. Doilom et al. (2017) accommodated Hermatomyces in Lophiotremataceae based on combined LSU, SSU, tef1-α and rpb2 sequence data. Later, Hashimoto et al. (2017) validated Hermatomycetaceae (Hermatomycetaceae Locq. 1984 was not validly published, Art. 39.1) to accommodate the genus Hermatomyces. This genus is known only by its asexual morph that is characterized by sporodochial conidiomata and dimorphic (lenticular or cylindrical) conidia of one or two types. The lenticular conidia are globose to subglobose, hyaline to pale brown peripheral cells with dark brown central cells, and the cylindrical conidia is hyaline, cylindrical to subcylindrical or turbinate and consisting of 1–4 columns of 2–12 cells (Spegazzini 1911; Tibpromma et al. 2016; Hashimoto et al. 2017; Hyde et al. 2019; Pem et al. 2019; Phukhamsakda et al. 2020).

Based on morphological comparisons and phylogenetic affinities, Koukol et al. (2018) revised Hermatomyces species and described five new species (viz. H.bifurcatus, H.constrictus, H.megasporus, H.sphaericoides and H.verrucosus) and one new combination, H.reticulatus, from Panama. Accordingly, H.chromolaenae, H.saikhuensis, H.tectonae were treated as H.sphaericus and H.subiculosus, H.chiangmaiensis, H.thailandicus were synonymized with H.reticulatus, H.krabiensis and H.indicus, respectively (Koukol et al. 2018). These are probably species complexes that need more detailed study. Subsequent studies introduced H.bauhiniae, H.biconisporus, H.clematidis, H.trangensis and H.truncates into Hermatomyces (Tibpromma et al. 2018; Hyde et al. 2019; Koukol et al. 2019; Nuankaew et al. 2019; Phukhamsakda et al. 2020). Currently, 24 species are recognized in Hermatomyces (Koukol et al. 2018, 2019; Nuankaew et al. 2019; Delgado et al. 2020; Phukhamsakda et al. 2020; Table 2).

Table 2.

Synopsis of the morphological characteristics of Hermatomyces species.

Species	Lenticular conidia size (μm)	Cylindrical / turbinate conidia feature			Host	Country	Reference
		Shape	Length × width (μm)	Number of columns (cells)
Hermatomyces amphisporus	27–36(–38) × 18–29(–31)	Cylindrical, pyriform or turbinate	30‒38 × 20‒26	2(–4) (6–12 cells)	Cyathea sp., Sabalminor	Mexico, USA	Castañeda-Ruiz and Heredia (2000); Delgado et al. (2020)
H. bauhiniae	25–36 × 15–20	Cylindrical	20–28 × 8–11	1 (2–3-septate)	Bauhinia variegata	Thailand	Hyde et al. (2019)
H. biconisporus	28–34 × 15–25	Cylindrical	32–39 × 14.5–26	1–2 (3–4 cells)	Pandanus sp.	China	Tibpromma et al. (2018)
H. bifurcatus	(24–)30–36.5(–41) × (18–)21.5–26(–28)	Cylindrical	Apex: 7–16 × 7–12 Basal: 9–14 × 13–18.5	2 (2–3 cells)	Unknown	Panama	Koukol et al. (2018)
H. chromolaenae	9.2–10.4 × 10.2–11.5	NA	NA	NA	Chromolaena odorata	Thailand	Tibpromma et al. (2017)
H. clematidis	30–45 × 24–31	Cylindrical	29–35 × 12–14	1–2 (5–6 cells)	Clematis sikkimensis	Thailand	Phukhamsakda et al. (2020)
H. constrictus	(22–)25.5–29.5(–32) × 19–23.5(–27.5)	Cylindrical	Lower cells: (20–)24–30.5(–37) × 12–17 Upper cells: (16–)20–26(–30) × 8–14	1 (2 cells)	Bauhinia cumanensis	Panama	Koukol et al. (2018)
H. dimorphus	35‒55 × 15‒20	Cylindrical	15‒40 × 10‒15	4 (7 cells)	Unknown	India	Rao and de Hoog (1986)
H. indicus	18‒30 × 11.5‒19	Turbinate	22.4‒35.4 × 11.4‒21.6	2 (6–7 cells)	Phoenix rupicola	India	Prasher and Prasher (2014)
H. iriomotensis	30–36 × 20–27	Cylindrical	20.5–33 × 7–12.5	1–2 (3–7 cells)	Unknown	Japan	Hashimoto et al. (2017)
H. jinghaensis	30–40 × 25–30	Clavate, subcylindrical	33–43 × 11–13	1–2 (6–8 cells)	Unknown	China	This study
H. krabiensis	24.3–32.5 × 12.1–21.3	Cylindrical	20.4–26.4 × 8.6–19.7	1–2 (2–3 cells)	Pandanus odorifer	Thailand	Tibpromma et al. (2016)
H. megasporus	(45–)49–56(–59) × (31–)37–46	Cylindrical	(37–)49.5–60.5(67–) × 18–28(–32)	2 ((5–)6–7(–10) cells)	Unknown	Panama	Koukol et al. (2018)
H. nabanheensis	20.2–25.1 × 16.6–20.7	Cylindrical	15.3–26.8 × 12.1–18.2	1–2 (2–3 cells)	Pandanus sp.	China	Hyde et al. (2017)
H. pandanicola	12–15.7 × 20–30.1	Cylindrical	13.2–20.6 × 8.9–11.9	2 (2 cells)	Pandanus odorifer	Thailand	Tibpromma et al. (2016)
H. reticulatus	3–40(–45) × 25–34(–41)	NA	NA	NA	Unknown	Thailand, Panama	Hyde et al. (2016); Koukol et al. (2018)
H. saikhuensis	14.2–21.4 × 11.2–19.3	NA	NA	NA	Pandanus odorifer	Thailand	Tibpromma et al. (2016)
H. sphaericoides	(20.5–)24.5–28(–31) × (20–)23–26(–29)	NA	NA	NA	Unknown	Panama	Koukol et al. (2018)
H.sphaericus (PMA 116080)	(21–)24–29(–32.5) × (18–)21–27(–31.5)	NA	NA	NA	Various host plants	Tropical or subtropical	Koukol et al. (2018)
H. sphaericus	27–29 × 26–28	NA	NA	NA	Dipterocarpus sp., Ehretiaacuminata	China, Thailand	This study
H. tectonae	(23–)26–29(–33) × (19–)23–25(–28)	Cylindrical	(27–)31–32(–35) ×(21–)23	2 (6 cells)	Tectona grandis	Thailand	Doilom et al. (2017)
H. trangensis	27.5‒35 × 25‒32.5	NA	NA	NA	Arenga pinnata	Thailand	Nuankaew et al. (2019)
H. truncates	(26–)31.5–36.5(–37) × 22–27(–30)	Cylindrical	Lower cells: 14–22.5(–28) × 8.5–14.5	1 (2–3 cells)	Averrhoa carambola	Ghana, Panama	Koukol et al. (2019)
			Upper cells: 12–18(–30) × (6–)8–12.5
H. tucumanensis	(22–)27–35 × 18–25	Obclavate or subcylindrical	(21–)23–26(–28.5) × 7–14	2 (3–6 cells)	Unknown	Panama	Koukol et al. (2018)
H. turbinatus	24–30 × 17–21	Turbinate	27–36 × 19–28	2 (2–3 cells)	Dipterocarpus sp.	Thailand	This study
H. uniseriatus	27–36 × 15.5–24	Cylindrical	19–34 × 10–12.5	1 (3–4 cells)	Smilax campestris	Argentina	Leão-Ferreira et al. (2013)
H. verrucosus	23–30(–39) × 21–29.5	NA	NA	NA	Unknown	Panama	Koukol et al. (2018)

NA: absent

Our investigation led to the discovery of three Hermatomyces species, including two novel species, on dead woody-based substrates. Morphological illustrations and multi-gene phylogenetic analyses with ML, MP and BI of combined LSU, ITS, tub2, tef1-α and rpb2 sequence data are used to confirm the phylogenetic placement of the novel species within Hermatomyces.

Materials and methods

Sample collection, examination and isolation

Woody litter samples were collected from China (Yunnan Province) during the dry season (December 2019) and Thailand (Tak Province) during the wet season (August 2019). Samples were brought to the laboratory using plastic Ziploc bags. Fungal specimens were then examined using a stereomicroscope (Olympus SZ61, China). Pure cultures were obtained via single spore isolation on potato dextrose agar (PDA) following the methods described in Senanayake et al. (2020). Cultures were incubated at 25 °C for three weeks. Micro-morphological structures were photographed using a Nikon compound microscope (Nikon ECLIPSE Ni) fitted with a Canon (EOS 600D) digital camera. Measurements were taken using the Tarosoft (R) Image Frame Work program. Figures were processed using Adobe Photoshop CS6. Type specimens were deposited in the herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (KUN-HKAS). Ex-type living cultures were deposited at the Culture Collection of Mae Fah Luang University (MFLUCC) and Kunming Institute of Botany Culture Collection (KUMCC).

DNA extraction, amplification and sequencing

DNA extraction, amplification, sequencing, sequence alignment and phylogenetic analyses follow the methods of Dissanayake et al. (2020) with the following details. Two partial rDNA genes and three protein coding genes were used in our study, including internal transcribed spacer region (ITS) using primer pair ITS5/ITS4 (White et al. 1990), 28S large subunit nuclear ribosomal (LSU) using primer pair LR0R/LR5 (Vilgalys and Hester 1990), translation elongation factor 1-alpha gene (tef1-α) using primer pair EF1-983F/EF1-2218R (Rehner and Buckley 2005), RNA polymerase II second largest subunit (rpb2) using primer pair fRPB2-5F/fRPB2-7cR (Liu et al. 1999) and β-tubulin (tub2) using primer pair T1/T22 (O’Donnell and Cigelnik 1997). Amplification reactions were performed in a total volume of 25 μL of PCR mixtures containing 8.5 μL ddH₂O, 12.5 μL 2× PCR MasterMix (TIANGEN Co., China), 2 μL DNA template and 1 μL of each primer. The PCR thermal cycle program for LSU, ITS, tef1-α and rpb2 were set as described in Tibpromma et al. (2018). The PCR amplification condition of tub2 was set as denaturation at 94 °C for 3 minutes, followed by 35 cycles of denaturation at 94 °C for 45 seconds, annealing at 56 °C for 50 seconds and extension at 72 °C for 1 minute, with a final extension step at 72 °C for 10 minutes. PCR products were sent to the Qingke Company, Kunming City, Yunnan Province, China, for sequencing. Sequences were deposited in GenBank (Table 1).

Table 1.

GenBank accession numbers of sequences used for the phylogenetic analyses.

Organism	Strain number	GenBank accession numbers					Reference
		LSU	ITS	tub2	tef1-α	rpb2
Anteaglonium globosum	ANM 925.2T	GQ221879	NA	NA	GQ221925	NA	Mugambi and Huhndorf (2009)
A. parvulum	MFLUCC 14-0821	KU922915	NA	NA	KU922921	NA	Jayasiri et al. (2016)
Hermatomyces amphisporus	CBS 146610	LR812664	LR812664	NA	NA	NA	Delgado et al. (2020)
H. amphisporus	CBS 146611	NA	LR812663	LR812674	LR812658	LR812669	Delgado et al. (2020)
H. amphisporus	CBS 146612	NA	LR812665	LR812675	LR812659	LR812670	Delgado et al. (2020)
H. amphisporus	CBS 146613	LR812662	LR812662	LR812673	LR812657	LR812668	Delgado et al. (2020)
H. amphisporus	CBS 146614	LR812666	LR812666	LR812676	LR812660	LR812671	Delgado et al. (2020)
H. amphisporus	CBS 146615	LR812667	LR812667	LR812677	LR812661	LR812672	Delgado et al. (2020)
H. bauhiniae	MFLUCC 16-0395T	MK443378	MK443382	NA	MK443384	MK443386	Hyde et al. (2019)
H. biconisporus	KUMCC 17-0183T	MH260296	MH275063	NA	MH412771	MH412755	Tibpromma et al. (2018)
H. bifurcatus	CCF 5899	LS398262	LS398262	LS398441	LS398416	LS398343	Koukol et al. (2018)
H. bifurcatus	CCF 5900T	LS398263	LS398263	LS398442	LS398417	LS398344	Koukol et al. (2018)
H. clematidis	MFLUCC 17-2085T	MT214556	MT310603	NA	MT394735	MT394684	Phukhamsakda et al. (2020)
H. constrictus	CCF 5904T	LS398264	LS398264	LS398443	LS398418	LS398345	Koukol et al. (2018)
H. indicus	MFLUCC 14-1143T1	KU764692	KU144920	NA	KU872754	KU712488	Doilom et al. (2017)
H. indicus	MFLUCC 14-1144	KU764693	KU144921	NA	KU872755	KU712489	Doilom et al. (2017)
H. indicus	MFLUCC 14-1145	KU764694	KU144922	NA	KU872756	KU712490	Doilom et al. (2017)
H. iriomotensis	KH 361T	LC194367	LC194483	NA	LC194394	LC194449	Hashimoto et al. (2017)
H. jinghaensis	HKAS 112167T	MW989519	MW989495	NA	MZ042642	NA	This study
H. krabiensis	MFLUCC 16-0249T	KX525742	KX525750	NA	KX525758	KX525754	Tibpromma et al. (2016)
H.krabiensis (H.chiangmaiensis)	MFLUCC 16-2817 T2	KY559394	NA	NA	NA	NA	Tibpromma et al. (2017)
H. megasporus	CCF 5897	NA	LS398265	LS398444	LS398419	LS398346	Koukol et al. (2018)
H. megasporus	CCF 5898T	LS398266	LS398266	LS398445	LS398420	NA	Koukol et al. (2018)
H. nabanheensis	KUMCC 16-0149T	KY766059	KY766058	NA	KY766061	NA	Hyde et al. (2017)
H. pandanicola	MFLUCC 16-0251T	KX525743	KX525751	NA	KX525759	KX525755	Tibpromma et al. (2016)
H. reticulatus	CCF 5893	LS398267	LS398267	LS398446	LS398421	LS398347	Koukol et al. (2018)
H.reticulatus (H.subiculosus)	MFLUCC 15-0843T3	KX259523	KX259521	NA	KX259527	KX259529	Hyde et al. (2016)
H. sphaericoides	CCF 5896	NA	LS398271	LS398448	LS398425	LS398351	Koukol et al. (2018)
H. sphaericoides	CCF 5908T	LS398273	LS398273	LS398450	LS398427	LS398352	Koukol et al. (2018)
H. sphaericoides	CCF 5907	NA	LS398272	LS398449	LS398426	NA	Koukol et al. (2018)
H. sphaericoides	CCF 5895	LS398270	LS398270	LS398447	LS398424	LS398350	Koukol et al. (2018)
H. sphaericus	PMA 116080	LS398281	LS398281	LS398454	LS398431	LS398356	Koukol et al. (2018)
H. sphaericus	PMA 116081	NA	LS398283	LS398455	LS398432	LS398357	Koukol et al. (2018)
H. sphaericus	PRM 946201	NA	LS398284	LS398456	LS398433	LS398358	Koukol et al. (2018)
H. sphaericus	PRC 4116	NA	LS398275	NA	NA	NA	Koukol et al. (2018)
H. sphaericus	PRC 4105	NA	LS398286	NA	NA	NA	Koukol et al. (2018)
H. sphaericus	PRC 4104	NA	LS398278	LS398453	LS398430	LS398355	Koukol et al. (2018)
H. sphaericus	PRC 4100	NA	LS398277	LS398452	LS398429	LS398354	Koukol et al. (2018)
H. sphaericus	PRC 4106	NA	LS398279	NA	NA	NA	Koukol et al. (2018)
H. sphaericus	PMA 116085	NA	LS398280	NA	NA	NA	Koukol et al. (2018)
H. sphaericus	PMA 116082	NA	LS398285	NA	NA	NA	Koukol et al. (2018)
H. sphaericus	KZP 462	NA	LS398287	LS398457	LS398434	LS398359	Koukol et al. (2018)
H. sphaericus	PRC 4117	NA	LS398276	NA	NA	NA	Koukol et al. (2018)
H.sphaericus (H.chromolaenae)	MFLUCC 16-2818T4	KY559393	NA	NA	NA	NA	Tibpromma et al. (2017)
H.sphaericus (H.saikhuensis)	MFLUCC 16-0266T5	KX525740	KX525748	NA	KX525756	KX525752	Tibpromma et al. (2016)
H.sphaericus (H.saikhuensis)	MFLUCC 16-0267	KX525741	KX525749	NA	KX525757	KX525753	Tibpromma et al. (2016)
H.sphaericus (H.tectonae)	MFLUCC 14-1140T6	KU764695	KU144917	NA	KU872757	KU712486	Doilom et al. (2017)
H.sphaericus (H.tectonae)	MFLUCC 14-1141	KU764696	KU144918	NA	KU872758	NA	Doilom et al. (2017)
H.sphaericus (H.tectonae)	MFLUCC 14-1142	KU764697	KU144919	NA	NA	KU712487	Doilom et al. (2017)
H. sphaericus	MFLUCC 21-0036	MW989516	MW989492	MZ042643	MZ042639	MZ042636	This study
H. sphaericus	KUMCC 20-0231	MW989517	MW989493	MZ042644	MZ042640	MZ042637	This study
H. trangensis	BCC 80741T	KY790600	KY790598	NA	KY790606	KY790604	Nuankaew et al. (2019)
H. trangensis	BCC 80742	KY790601	KY790599	NA	KY790607	KY790605	Nuankaew et al. (2019)
H. tucumanensis	CCF 5912	LS398288	LS398288	LS398458	LS398435	LS398360	Koukol et al. (2018)
H. tucumanensis	CCF 5913	LS398289	LS398289	LS398459	LS398436	LS398361	Koukol et al. (2018)
H. tucumanensis	CCF 5915	LS398290	LS398290	LS398460	LS398437	LS398362	Koukol et al. (2018)
H. turbinatus	MFLUCC 21-0038T	MW989518	MW989494	MZ042645	MZ042641	MZ042638	This study
H. verrucosus	CCF 5903T	LS398292	LS398292	LS398462	LS398439	LS398364	Koukol et al. (2018)
H. verrucosus	CCF 5892	LS398291	LS398291	LS398461	LS398438	LS398363	Koukol et al. (2018)

Sequence alignment and phylogenetic analyses

Representative species used in the phylogenetic analyses were selected based on previous publications (Koukol et al. 2018; Nuankaew et al. 2019; Delgado et al. 2020; Phukhamsakda et al. 2020). Sequences were downloaded from GenBank (http://www.ncbi.nlm.nih.gov/), and their accession numbers are listed in Table 1. The newly generated sequences in this study were assembled by BioEdit 7.0.9.0 (Hall 1999). Individual gene regions were separately aligned in MAFFT v.7 web server (http://mafft.cbrc.jp/alignment/server/) (Katoh et al. 2019). The alignments of each gene were improved by manually deleting the ambiguous regions plus gaps and combined using BioEdit 7.2.3. Final alignments containing LSU, ITS, tub2, tef1-α and rpb2 were converted to NEXUS format (.nxs) using CLUSTAL X (2.0) (Thompson et al. 1997) and processed for Bayesian and maximum parsimony analysis. The FASTA format was changed into PHYLIP format via the Alignment Transformation Environment (ALTER) online program (http://www.sing-group.org/ALTER/) and used for maximum likelihood analysis (ML).

ML was carried out in CIPRES Science Gateway v.3.3 (http://www.phylo.org/portal2/; Miller et al. 2010) using RAxML-HPC2 on XSEDE (8.2.12) (Stamatakis 2014) with the GTRGAMMA substitution model and 1,000 bootstrap iterations. Maximum parsimony analysis (MP) was performed in PAUP v. 4.0b10 (Swofford 2002) with the heuristic search option and Tree-Bisection-Reconnection (TBR) of branch-swapping algorithm for 1,000 random replicates. Branches with a minimum branch length of zero were collapsed, and gaps were treated as missing data (Hillis and Bull 1993).

Bayesian analysis was executed in MrBayes v.3.2.2 (Ronquist et al. 2012). The model of evolution was estimated using MrModeltest v. 2.3 (Nylander et al. 2008) via PAUP v. 4.0b10 (Ronquist and Huelsenbeck 2003). The SYM+I+G for LSU and ITS; HKY+I for tub2; GTR+I+G for tef1-α and rpb2 were used in the final command. Markov chain Monte Carlo sampling (MCMC) in MrBayes v.3.2.2 (Ronquist et al. 2012) was used to determine posterior probabilities (PP) (Rannala and Yang 1996; Zhaxybayeva and Gogarten 2002). Bayesian analyses of six simultaneous Markov chains were run for 2,000,000 generations and trees were sampled and printed to output at every 200 generations (resulting in 10,001 total trees). The first 25% of sampled trees were discarded as part of the burn-in procedure, the remaining 7,501 trees were used to create the consensus tree and the average standard deviation of split frequencies was set as 0.01.

Phylogenetic trees were visualized in FigTree v1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/; Rambaut 2012), the tree was edited using Microsoft PowerPoint before being saved in PDF format and finally converted to JPG format using Adobe Illustrator CS6 (Adobe Systems, USA). The finalized alignments and trees were deposited in TreeBASE, submission ID: TB2:S28514 (http://purl.org/phylo/treebase/phylows/study/TB2:S28514).

Ex-type strains are indicated with superscript “T”, and newly generated sequence is shown in bold. NA represents sequences that are unavailable in GenBank. Abbreviations:

ANM A.N. Miller;

BCCBIOTEC Culture Collection, Bangkok, Thailand;

CBSCentraal Bureau voor Schimmel cultures, Utrecht, The Netherlands;

CCF Culture Collection of Fungi, Charles University, Prague, Czech Republic;

HKAS The herbarium of Cryptogams Kunming Institute of Botany Academia Sinica;

KH K. Hirayama;

KUMCC Culture Collection of Kunming Institute of Botany, Kunming, China;

KZP O. Koukol;

MFLUCC Mae Fah Luang University Culture Collection, Chiang Rai, Thailand;

PMAHerbarium of the University of Panama, Panama City, Panama;

PRCHerbarium of the Charles University, Prague, Czech Republic;

PRM Herbarium of the National Museum, Prague, Czech Republic.

T1 Type of Hermatomycesthailandicus;

T2 Type of H.chiangmaiensis;

T3 Type of H.subiculosus;

T4 Type of H.chromolaenae;

T5 Type of H.saikhuensis;

T6 Type of H.tectonae.

Results

Phylogenetic analysis

The phylogenetic analysis was conducted using 57 strains in Hermatomycetaceae, and two outgroup taxa Anteagloniumglobosum (ANM 925.2) and A.parvulum (MFLUCC 14-0821) in Pleosporales (Table 1). The aligned sequence matrix comprised five gene regions (LSU: 887 bp, ITS: 530 bp, tub2: 606 bp, tef1-α: 952 bp and rpb2: 1,028 bp) and a total of 4,003 characters (including gaps), of which 3,207 characters were constant, 174 variable characters were parsimony-uninformative and 622 characters were parsimony-informative. The Kishino-Hasegawa test shows length = 1,388 steps with CI = 0.671, RI = 0.884, RC = 0.593 and HI = 0.329. The RAxML analysis of the combined dataset yielded a best scoring tree with a final ML optimization likelihood value of -13406.555506. Estimated base frequencies were as follows: A = 0.241874, C = 0.266701, G = 0.257552, T = 0.233873; substitution rates AC = 1.188604, AG = 4.826453, AT = 1.273226, CG = 0.855218, CT = 11.409386, GT = 1.00; gamma distribution shape parameter α = 0.16102.

In the phylogenetic tree obtained from ML, MP and BI analysis (Fig. 1) the maximum likelihood analysis resulted in trees largely with similar topology and clades as in the maximum parsimony and Bayesian analyses. The new species, Hermatomycesturbinatus, is sister to H.nabanheensis (KUMCC 16-0149) with high support (94% ML, 91% MP and 1.00 BYPP, Fig. 1). Hermatomycesjinghaensis is nested between H.trangensis and H.clematidis with a strongly supported monophyletic group (98% ML, 92% MP, 1.00 PP; Fig. 1). New isolates of H.sphaericus (KUMCC 20-0231; MFLUCC 21-0036) clustered with remaining H.sphaericus strains as a monophyletic group (Fig. 1). The topology of the phylogenetic tree is in accordance with recent phylogenetic studies discussing species in Hermatomycetaceae (Nuankaew et al. 2019; Phukhamsakda et al. 2020).

Figure 1.

Phylogenetic RAxML tree based on analysis of a combined LSU, ITS, tub2, tef1-α and rpb2 and dataset. Bootstrap support values for ML and MP equal to or higher than 75% and Bayesian PP equal to or greater than 0.95 are shown at nodes. Hyphens (--) represent support values less than 75% / 0.95 BYPP. The ex-type strains are in bold and the new isolate in this study is in blue bold. The tree is rooted with Anteagloniumglobosum (ANM 925.2) and A.parvulum (MFLUCC 14-0821). The scale bar represents the expected number of nucleotide substitutions per site.

Taxonomy

Hermatomyces turbinatus

G.C. Ren & K.D. Hyde sp. nov.

479CDBD8-11CB-58C0-93ED-BED46E6A7908

558166

Facesoffungi Number No: FoF09735

Figure 2

Figure 2.

Hermatomycesturbinatus (HKAS 112724, holotype) a, b sporodochia on natural substrate c vertical section of sporodochium d conidiophores and conidiogenous cells e–h turbinate conidia i turbinate and mature lenticular conidia j–m mature lenticular conidia n germinated conidium o, p culture characters on PDA. Scale bars: 30 μm (c); 20 μm (d–n); 30 mm (o, p).

Etymology.

Referring to the turbinate shape of the conidia.

Holotype.

HKAS 112724.

Description.

Saprobic on woody litter of Dipterocarpus sp. (Dipterocarpaceae) Sexual morph Undetermined. Asexual morphColonies on natural substrate forming sporodochial conidiomata, superficial, scattered, small groups, circular or oval, sterile mycelial outer zone enclosing a black-brown velvety margin, sparse, black sporulating center, shiny, glistening, circular or oval, conidia readily liberated when agitated. Mycelium superficial, branched, septate, hyaline to pale brown, 2–3 μm wide. Conidiophores 6–8 × 2–3 μm, micronematous, straight or flexuous, smooth, short, pale brown. Conidiogenous cells 3–5 × 2–3 μm, monoblastic, integrated, terminal, determinate, often arising directly on the superficial mycelium, subsphaerical, ovoid or ampulliform, hyaline to pale brown, smooth finely verruculose. Conidia dimorphic, solitary, smooth-walled. Lenticular conidia 24–30 × 17–21 μm (x = 27 × 20 μm, n = 20), 12–15 μm thick, thick-walled, circular to oval in front view, smooth, solitary, muriform, central cells dark brown to black, peripheral cells hyaline to pale brown, forming a weakly ring, sometimes slightly constricted at septa, obovoid or oblong in lateral view, arranged in 2 rows, a row of composed of 4–6 cells, end cells pale brown to hyaline, middle cells dark brown. Turbinate conidia turbinate, pyriform, 27–36 μm in length, 19–28 µm wide in broadest part of lower cells, (x = 32 × 23 μm, n = 20), asymmetrical with the upper cells smaller than lower cells, thick-walled, smooth, septate, constricted distinct at septa, consisting of two columns with two septa composed of 2–3 rectangular to globose cells in each column, usually upper part of terminal cells dark brown, becoming hyaline towards the lower side, two cells hyaline in the lower cells swollen with oil globules.

Known host and distribution.

Dipterocarpus sp. (Thailand).

Culture characteristics.

Colonies on PDA, reaching 30–40 mm diam., after 3 weeks at 25–30 °C, circular, convex with papillate and radially furrowed at the center, rough, labate, crenate edge, fluffy, dense, gray black, in reverse darkens at the center, pale brown to gray at edge.

Material examined.

Thailand, Tak Province. Ban Na Sam Ngao District, on woody litter of Dipterocarpus sp. (Dipterocarpaceae), 22 August 2019, G. C. Ren, TSY04 (HKAS 112724, holotype), ex-type living culture, MFLUCC 21-0038.

Notes.

Hermatomycesturbinatus is introduced as a new species based on its distinct morphology, which is supported by phylogenetic analyses. In the phylogenetic analyses, H.turbinatus is distinct from extant species in this genus and formed a sister clade to H.nabanheensis with strong support (94% ML, 91% MP, 1.00 PP; Fig. 1). Hermatomycesturbinatus differs from H.nabanheensis in having turbinate conidia with two columns, while H.nabanheensis has cylindrical conidia with one or two columns. Hermatomycesturbinatus has two conidial types, and its lenticular conidia are similar to H.tectonae in shape and size. However, the turbinate conidia of H.turbinatus have 2 columns of 2–3 cells in each column, while the turbinate conidia of H.tectonae have 2 columns of 3 cells in each column. We also compared the morphological characters of H.turbinatus to other species of Hermatomyces (Table 2). Despite no molecular data being available for the three species viz. H.dimorphus, H.uniseriatus and H.truncates, H.turbinatus nonetheless differs from these species in conidial characteristics (Table 2).

Hermatomyces jinghaensis

G.C. Ren & K.D. Hyde sp. nov.

586F538B-54BB-5D95-A10A-50FE2A43AEE0

558165

Facesoffungi Number No: FoF09736

Figure 3

Figure 3.

Hermatomycesjinghaensis (HKAS 112167, holotype) a, b sporodochia on natural substrate c vertical section of sporodochium d conidiophores e, f conidiogenous cells g–l cylindrical conidia m–s mature lenticular conidia. Scale bars: 50 μm (c); 30 μm (d); 20 μm (e–r); 30 μm (s).

Etymology.

The species epithet “jinghaensis” refers to the location where the species was collected.

Holotype.

HKAS 112167.

Description.

Saprobic on unidentified woody litter. Sexual morph Undetermined. Asexual morphColonies on natural substrate forming sporodochial conidiomata, superficial, scattered, small groups, circular, sterile mycelial outer zone enclosing a black velvety margin, dense, thick, black sporulating center, shiny, glistening, circular or oval, conidia readily liberated when agitated. Mycelium superficial, branched, septate, hyaline to pale brown, 2–3 μm wide. Conidiophores 30–45 × 2–3 μm, mononematous, cylindrical, straight or flexuous, smooth, pale brown. Conidiogenous cells 4–6 × 2–3 μm, monoblastic, integrated, terminal, determinate, often arising directly on the superficial mycelium, cylindrical, ampulliform, hyaline to pale brown, smooth finely verruculose. Conidia dimorphic solitary, smooth-walled. Lenticular conidia 30–40 × 25–30 μm (x = 37 × 28 μm, n = 20), 21–25 μm thick, thick-walled, circular to oval in front view, smooth, solitary, muriform, central cells brown to dark brown, peripheral cells hyaline to subhyaline, forming a wide and distinct ring, sometimes slightly constricted at septa, obovoid or oblong in lateral view, central cells brown to dark brown, peripheral cells pale brown to brown. Cylindrical conidia 33–43 μm in length, 11–13 µm wide in broadest part of lower cells (x = 39 × 12 μm, n = 20), clavate or subcylindrical, straight or flexuous, septate, constricted distinct at the septa, with large guttules, consisting of one or two columns, each column with 6–8 cells, apical cell rectangular to globose, smooth, hyaline, smooth, basal cells acute, rectangular to cylindrical, pale brown.

Known host and distribution.

Unidentified woody litter (China)

Material examined.

China, Yunnan Province, Xishuangbanna Dai Autonomous Prefecture, Jinghong, Jingha (21°78.06'N, 101°05.61'E), on unidentified woody litter, 19 December 2019, D.N. Wanasinghe, DW57 (HKAS 112167, holotype), no living culture.

Notes.

Hermatomycesjinghaensis is introduced as a new species based on its distinct morphology and the phylogenetic results of a combined LSU, ITS, tub2, tef1-α and rpb2 dataset. Hermatomycesjinghaensis nested with H.clematidis and H.trangensis in a strongly supported monophyletic group (99% ML, 100% MP, 1.00 PP; Fig. 1). Hermatomycesjinghaensis is characterized by both lenticular and cylindrical conidia. Hermatomycesjinghaensis differs from H.clematidis in having cylindrical conidia with one or two columns, each of which has 6–8 cells with large guttules, while the latter has 5–6 cells for each column conidia. Hermatomycestrangensis differs from H.jinghaensis in having only lenticular conidia.

Hermatomyces sphaericus

(Sacc.) S. Hughes 1953.

272AC737-BC12-5F42-AA42-C0262ADA5FD2

298410

Facesoffungi Number No: FoF05259

Figure 4

Figure 4.

Hermatomycessphaericus (HKAS 112725) a, b colonies on the natural substrate c mycelia d–g young conidia h–k mature conidia (h–j surface view k thickness view) l germinated conidium m, n culture characters on PDA. Scale bars: 1000 μm (a); 200 μm (b); 20 μm (c–i, l); 30 μm (j, k); 3 cm (m, n).

Description.

Saprobic on woody litter of Dipterocarpus sp. (Dipterocarpaceae) and Ehretiaacuminata (Boraginaceae). Sexual morph Undetermined. Asexual morphColonies on natural substrate forming sporodochial conidiomata, superficial, circular or irregular, scattered or crowded, consisting of a velvety, dense, annular, gray brown, sterile mycelial outer zone and a black, glistening, abundantly sporulating granulose center, with conidia readily liberated when agitated. Mycelium 2–2.5 μm wide, superficial, composed of a tightly network of branched, septate, smooth or finely verruculose, hyaline or pale brown hyphae. Conidiophores 10–13 × 2–4 μm (x = 12 × 3 μm, n = 10) micronematous, cylindrical or forked, smooth, hyaline or pale brown, often corresponding to conidiogenous cells. Conidiogenous cells 5–8 × 3–5 μm (x = 7 × 4 μm, n = 20), monoblastic, integrated, terminal, cylindrical, hyaline to pale brown, smooth or finely verruculose. Conidia of one type, 27–29 × 26–28 μm (x = 28 × 27 μm, n = 30) μm, 19–24 μm thick, solitary, lenticular, globose, subglobose in front view, muriform, smooth, central cells brown, dark brown, outer ring of peripheral cells narrow, pale brown to brown, often constricted at septa, disk-shaped in lateral view, consisting of two rows, each row with 4–6 cells, hyaline to light brown at lower and upper cells, middle cells brown to black brown.

Known host and distribution.

Tropical and subtropical regions of Central and South America, Africa, Asia, Oceania and North America. The species were found as saprobes on Acanthaceae, Apocynaceae, Arecaceae, Asteraceae, Dipterocarpaceae, Euphorbiaceae, Fabaceae, Lamiaceae, Leguminosae, Mimosaceae, Nyctaginaceae, Oxalidaceae, Pandanaceae, Pinaceae, Rhamnaceae, and Sterculiaceae (Zhang et al 2009; Koukol et al. 2018, 2019).

Culture characteristics.

Colonies on PDA, reaching 35–40 mm diam., after 3 weeks at 25–30 °C, with circular, umbonate, fluffy, velvety, entire edge, a circular raised band, gray white, in reverse dark gray, black toward the center.

Material examined.

Thailand, Tak Province, Tha Song Yang District, on woody litter of Dipterocarpus sp. (Dipterocarpaceae), 22 August 2019, G. C. Ren, T903 (HKAS 112725), living culture, MFLUCC 21-0036; China, Yunnan Province, Xishuangbanna (21°55.19'N, 101°15.24'E), on woody litter of Ehretiaacuminata (Boraginaceae), 4 August 2020, G. C. Ren, JH39 (HKAS 112166), living culture, KUMCC 20-0231.

Notes.

The characters of our new strain of Hermatomycessphaericus (KUMCC 20-0231, MFLUCC 21-0036) are similar to the type collection (K(M)–IMI 37763) in having gray black to black sporodochia, mononematous, pale brown, smooth, monoblastic, integrated, terminal, cylindrical, hyaline to pale brown conidiogenous cells and globose to subglobose conidia (Hughes 1953). A multigene phylogeny indicates that novel strains clustered within the H.sphaericus clade (Fig. 1). We name our strain (KUMCC 20-0231, MFLUCC 21-0036) as H.sphaericus, which has been reported from different plant families and genera (Koukol et al. 2018). However, we consider this might be a species complex that need further detailed studies. Our study provides the new host records of H.sphaericus on Dipterocarpus sp. (Dipterocarpaceae) and Ehretiaacuminata (Boraginaceae), and updates sequence data for the new collections of H.sphaericus.

Discussion

This study introduces two new species of woody-based litter fungi; Hermatomycesjinghaensis from Yunnan, China and Hermatomycesturbinatus on Dipterocarpus sp. from Thailand. We also report for the first time two new records of H.sphaericus on Dipterocarpus sp. and Ehretiaacuminata in China and Thailand.

Hermatomyces (Hermatomycetaceae) is different from other similar genera in its sporodochial conidiomata and in having one to two (lenticular and cylindrical conidia) unusual conidial types (Spegazzini 1911). All species of Hermatomyces have lenticular conidia with similar characteristics, whereas some species have cylindrical and turbinate conidia, which have greater variance in shape, size, number of columns and cells. Koukol et al. (2018, 2019) have reported that multiple species may occur together on a single sample, a phenomenon we observed, which may complicate morphological identification and separation for culturing. Therefore, molecular sequence data are more reliable for the identification of Hermatomyces species (Tibpromma et al. 2016, 2017, 2018; Nuankaew et al. 2019; Phukhamsakda et al. 2020).

Hermatomycessphaericus was introduced by Hughes (1953), which may be the most widespread of species in Hermatomyces distributed across many subtropical and tropical regions worldwide (Wijayawardene et al. 2014; Doilom et al. 2017; Koukol et al. 2018, 2019; Hyde et al. 2019; Jayasiri et al. 2019; Nuankaew et al. 2019; Phukhamsakda et al. 2020). This species has been reported as saprobic on dead plant tissues of several host families (Tibpromma et al. 2016, 2017; Doilom et al. 2017; Jayasiri et al. 2019). In addition, Koukol et al. (2018) reported that H.sphaericus (ARIZ: PS0053) was isolated from seeds of Apeibamembranacea (Malvaceae), suggesting this species could be an endophyte. Previous studies have indicated that H.sphaericus is not restricted to any single host (Koukol et al. 2018, 2019; Jayasiri et al. 2019), whereas other species of Hermatomyces are saprobic on a limited number of hosts and are limited to specific regions (Rao and de Hoog 1986; Leão-Ferreira et al. 2013; Prasher and Prasher 2014; Hyde et al. 2016, 2017, 2019; Tibpromma et al. 2016, 2017, 2018; Doilom et al. 2017; Hashimoto et al. 2017; Koukol et al. 2018, 2019; Nuankaew et al. 2019; Delgado et al. 2020; Phukhamsakda et al. 2020; Table 2). In this study, our new strains of H.sphaericus had slight morphological differences in lenticular conidia size compared to the type strains and other strains of H.sphaericus (Hughes 1953, Table 2). As reported by Koukol et al. (2018), H.sphaericus is a plurivorous species, and accordingly the phenotypic variation among strains could be influenced by environmental factors and culture conditions or it could have speciated in isolated polulations (Hyde et al. 2020).

Species delineation in Hermatomyces, especially in the H.sphaericus clade, is subject to much controversy due to species inconsistency in morphological and phylogenetic status. Koukol et al. (2018) synonymized H.chromolaenae, H.saikhuensis and H.tectonae under H.sphaericus based on morphological and molecular comparisons and suspected that H.pandanicola could either be a hybrid species or incorrect sequences were used in the analysis. Koukol et al. (2019) considered that during isolation of H.biconisporus, a conidium of H.sphaericus might have been taken instead, leading to contamination when extracting DNA and the misinterpretation of its taxonomic placement. Phukhamsakda et al. (2020) further confirmed that H.biconisporus, H.pandanicola and H.sphaericus should be treated as the same species based on Genealogical Concordance Phylogenetic Species Recognition (GCPSR) analysis.

Hermatomyces had long been treated as “incertae sedis” within Ascomycota (Wijayawardene et al. 2012). Doilom et al. (2017) placed Hermatomyces in Lophiotremataceae baed on phylogenetic analyses, and consequently, Hashimoto et al. (2017) revised the family Lophiotremataceae based on morphological observations and phylogenetic analyses, and Hermatomyces was accepted in the family Hermatomycetaceae, as monophyletic. Recent studies and our study indicate Hermatomyces to be highly polyphyletic, and Hermatomyces morphology has evolved, which is mainly characterized by lenticular and cylindrical conidia (Fig. 1; Koukol et al. 2018, 2019; Hyde et al. 2019; Phukhamsakda et al. 2020). Support for a single H.sphaericus species (Fig. 1) lacks internal statistical support and includes H.biconisporus, H.chromolaenae, H.pandanicola, H.saikhuensis and H.tectonae and we suspect that this is a species complex. Tibpromma et al. (2018) also noted that H.sphaericus could be a species complex including several species and did not accept the synonymy of H.saikhuensis and H.tectonae in H.sphaericus owing to their significant base-pair differences.

In this study, we combined two non-translated loci (LSU, ITS) and three protein-coding regions (tub2, tef1-α and rpb2) to carry out phylogenetic analysis for Hermatomyces species in order to validate phylogenetic placement of the taxa within Hermatomyces. In our phylogenetic analyses, H.tectonae, H.chromolaenae, H.biconisporus, H.pandanicola and H.saikhuensis grouped together with strains of H.sphaericus (PRC 4100, PRC 4104, PMA 116081). Hermatomycessaikhuensis and H.chromolaenae are characterized by one conidium type (lenticular) similar to H.sphaericus, however, they differ in the shape, color and size of conidia (Tibpromma et al. 2016, 2017; Table 2). Hermatomycestectonae, H.biconisporus and H.pandanicola are characterized by dimorphic conidia which differ from H.sphaericus (Tibpromma et al. 2016, 2018; Doilom et al. 2017; Koukol et al. 2018; Table 2). Hermatomycessphaericus (PRC 4100, PRC 4104, PMA 116081) did not have a morphological description for inter-species comparison (Koukol et al. 2018). Further taxon sampling and more sequence data are needed to elucidate this clade.

Citation

Ren G-C, Wanasinghe DN, Monkai J, Mortimer PE, Hyde KD, Xu J-C, Pang A, Gui H (2021) Novel saprobic Hermatomyces species (Hermatomycetaceae, Pleosporales) from China (Yunnan Province) and Thailand. MycoKeys 82: 57–79. https://doi.org/10.3897/mycokeys.82.67973

Funding Statement

Open Research Fund Program of Science and Technology on Aerospace Chemical Power Laboratory (STACPL320181B04). The National Natural Science Foundation of China (NSFC21975066, NSFC21875061). The Thailand Research Fund for the grant entitled Impact of climate change on fungal diversity and biogeography in the Greater Mekong Subregion (No. RDG6130001).