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Novel saprobic Hermatomyces species (Hermatomycetaceae, Pleosporales) from China (Yunnan Province) and Thailand
expand article infoGuang-Cong Ren§, Dhanushka N. Wanasinghe|, Jutamart Monkai§, Peter E. Mortimer, Kevin D. Hyde§, Jian-Chu Xu, Aimin Pang#, Heng Gui
‡ Guiyang Nursing Vocational College, Guiyang, China
§ Mae Fah Luang University, Chiang Rai, Thailand
| World Agroforestry, Kunming, China
¶ Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
# Hubei Institute of Aerospace Chemotechnology, Xiangyang, China
Open Access

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 Ehretia acuminata (Boraginaceae). Both morphology and multigene analyses revealed two taxa as new species (Hermatomyces turbinatus and H. jinghaensis) and the remaining collections as new records of H. sphaericus. Hermatomyces turbinatus 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. Hermatomyces jinghaensis 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.

Keywords

2 new species, hyphomycetes, phylogeny, taxonomy, woody litter fungi

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).

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;

BCC BIOTEC Culture Collection, Bangkok, Thailand;

CBS Centraal 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;

PMA Herbarium of the University of Panama, Panama City, Panama;

PRC Herbarium of the Charles University, Prague, Czech Republic;

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

T1 Type of Hermatomyces thailandicus;

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 Anteaglonium globosum (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, Hermatomyces turbinatus, is sister to H. nabanheensis (KUMCC 16-0149) with high support (94% ML, 91% MP and 1.00 BYPP, Fig. 1). Hermatomyces jinghaensis 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 Anteaglonium globosum (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.

MycoBank No: 558166
Figure 2

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 morph Colonies 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.

Figure 2. 

Hermatomyces turbinatus (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).

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

Hermatomyces turbinatus 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). Hermatomyces turbinatus differs from H. nabanheensis in having turbinate conidia with two columns, while H. nabanheensis has cylindrical conidia with one or two columns. Hermatomyces turbinatus 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).

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., Sabal minor 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., Ehretia acuminata 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)

Hermatomyces jinghaensis G.C. Ren & K.D. Hyde, sp. nov.

MycoBank No: 558165
Figure 3

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 morph Colonies 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.

Figure 3. 

Hermatomyces jinghaensis (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).

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

Hermatomyces jinghaensis 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. Hermatomyces jinghaensis nested with H. clematidis and H. trangensis in a strongly supported monophyletic group (99% ML, 100% MP, 1.00 PP; Fig. 1). Hermatomyces jinghaensis is characterized by both lenticular and cylindrical conidia. Hermatomyces jinghaensis 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. Hermatomyces trangensis differs from H. jinghaensis in having only lenticular conidia.

Hermatomyces sphaericus (Sacc.) S. Hughes 1953.

MycoBank No: 298410
Figure 4

Description

Saprobic on woody litter of Dipterocarpus sp. (Dipterocarpaceae) and Ehretia acuminata (Boraginaceae). Sexual morph Undetermined. Asexual morph Colonies 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.

Figure 4. 

Hermatomyces sphaericus (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).

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 Ehretia acuminata (Boraginaceae), 4 August 2020, G. C. Ren, JH39 (HKAS 112166), living culture, KUMCC 20-0231.

Notes

The characters of our new strain of Hermatomyces sphaericus (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 Ehretia acuminata (Boraginaceae), and updates sequence data for the new collections of H. sphaericus.

Discussion

This study introduces two new species of woody-based litter fungi; Hermatomyces jinghaensis from Yunnan, China and Hermatomyces turbinatus on Dipterocarpus sp. from Thailand. We also report for the first time two new records of H. sphaericus on Dipterocarpus sp. and Ehretia acuminata 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).

Hermatomyces sphaericus 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 Apeiba membranacea (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). Hermatomyces saikhuensis 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). Hermatomyces tectonae, 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). Hermatomyces sphaericus (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.

Acknowledgements

This work was supported by Open Research Fund Program of Science and Technology on Aerospace Chemical Power Laboratory (STACPL320181B04). We thank the support from the National Natural Science Foundation of China (NSFC21975066, NSFC21875061). We also would like to thank the Thailand Research Fund for the grant entitled Impact of climate change on fungal diversity and biogeography in the Greater Mekong Subregion (No. RDG6130001). Dhanushka Wanasinghe thanks CAS President’s International Fellowship Initiative (PIFI) for funding his postdoctoral research (number 2021FYB0005), the Postdoctoral Fund from Human Resources and Social Security Bureau of Yunnan Province and the National Science Foundation of China and Chinese Academy of Sciences (grant no. 41761144055) for financial support.

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