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Research Article
Taxonomy and phylogeny of the novel rhytidhysteron-like collections in the Greater Mekong Subregion
expand article infoGuang-Cong Ren‡§, Dhanushka N. Wanasinghe|, Rajesh Jeewon#, Jutamart Monkai‡, Peter E. Mortimer|, Kevin D. Hyde‡, Jian-Chu Xu|, Heng Gui|
‡ Mae Fah Luang University, Chiang Rai, Thailand
§ Guiyang Nursing Vocational College, Guiyang, China
| Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
¶ World Agroforestry Centre, Kunming, China
# University of Mauritius, Moka, Mauritius

Corresponding author: Dhanushka N. Wanasinghe ( dnadeeshan@gmail.com )

Corresponding author: Heng Gui ( guiheng@mail.kib.ac.cn )

Academic editor: Thorsten Lumbsch
© 2022 Guang-Cong Ren, Dhanushka N. Wanasinghe, Rajesh Jeewon, Jutamart Monkai, Peter E. Mortimer, Kevin D. Hyde, Jian-Chu Xu, Heng Gui.
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.
Citation: Ren G-C, Wanasinghe DN, Jeewon R, Monkai J, Mortimer PE, Hyde KD, Xu J-C, Gui H (2022) Taxonomy and phylogeny of the novel rhytidhysteron-like collections in the Greater Mekong Subregion. MycoKeys 86: 65-85. https://doi.org/10.3897/mycokeys.86.70668
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Abstract

During our survey into the diversity of woody litter fungi across the Greater Mekong Subregion, three rhytidhysteron-like taxa were collected from dead woody twigs in China and Thailand. These were further investigated based on morphological observations and multi-gene phylogenetic analyses of a combined DNA data matrix containing SSU, LSU, ITS, and tef1-α sequence data. A new species of Rhytidhysteron, R. xiaokongense sp. nov. is introduced with its asexual morph, and it is characterized by semi-immersed, subglobose to ampulliform conidiomata, dark brown, oblong to ellipsoidal, 1-septate, conidia, which are granular in appearance when mature. In addition to the new species, two new records from Thailand are reported viz. Rhytidhysteron tectonae on woody litter of Betula sp. (Betulaceae) and Fabaceae sp. and Rhytidhysteron neorufulum on woody litter of Tectona grandis (Lamiaceae). Morphological descriptions, illustrations, taxonomic notes and phylogenetic analyses are provided for all entries.

Keywords

Ascomycota, one new taxon, phylogeny, saprobic, taxonomy, Yunnan

Introduction

Hysteriaceae was introduced by Chevallier (1826) with Hysterium as the type genus, which was characterized by hysterothecial or apothecioidal, carbonaceous ascomata with a pronounced, longitudinal slit running the length of the long axis, 8-spored, clavate to cylindric asci with an ocular chamber as well as obovoid, clavate, ellipsoid or fusoid, hyaline to light- or dark brown, one to multi-septate or muriform, smooth-walled ascospores with or without a sheath (Boehm et al. 2009b; Hongsanan et al. 2020; Hyde et al. 2020a). In recent outlines of Dothideomycetes (Hongsanan et al. 2020; Pem et al. 2020; Wijayawardene et al. 2020), 14 genera have been accepted in Hysteriaceae.

Rhytidhysteron was introduced by Spegazzini (1881) to accommodate two species: Rhytidhysteron brasiliense (type species) and R. viride in Patellariaceae (Clements and Shear 1931; Kutorga and Hawksworth 1997). Boehm et al. (2009a, b) transferred Rhytidhysteron from Patellariaceae to Hysteriaceae based on molecular data. Subsequent studies introduced more taxa and records in Rhytidhysteron with both morphological and molecular evidence (Thambugala et al. 2016; Doilom et al. 2017; Cobos-Villagrán et al. 2020; Dayarathne et al. 2020; de Silva et al. 2020; Hyde et al. 2020a, b; Mapook et al. 2020; Wanasinghe et al. 2021). Currently, 24 species are recognized in Rhytidhysteron (Species Fungorum 2021; Wanasinghe et al. 2021).

Rhytidhysteron species have been documented from a wide range of hosts in various countries such as Australia, Bermuda, Bolivia, Brazil, China, Colombia, Cuba, France, Hawaii, India, New Zealand, Thailand, Ukraine, USA, and Venezuela (Kutorga and Hawksworth 1997; de Silva et al. 2020). Most Rhytidhysteron species are identified as saprobes on woody-based substrates in terrestrial habitats as well as from mangrove wood in marine habitats (Thambugala et al. 2016; Kumar et al. 2019; Hyde et al. 2020a, b; Wanasinghe et al. 2021). However, they have also been reported as endophytes or weak pathogens on woody plants and seldom as human pathogens (Soto and Lucking 2017; de Silva et al. 2020). From a biotechnological perspective, Rhytidhysteron species have great potential for their commercial applications and in industry. In particular, interest in secondary metabolites has rekindled in recent years, for instance with the discovery of palmarumycins. The latter is a potential inhibitor of thioredoxin–thioredoxin reductase cellular redox systems, with potential antimicrobial and antifungal properties (Murillo et al. 2009). Other Rhytidhysteron species discovered from the Southeast Asian region, such as R. bruguierae (MFLUCC 17-1515) and R. chromolaenae (MFLUCC 17-1516) also showed antimicrobial activity against Mucor plumbeus (Mapook et al. 2020) and hence this demonstrates a potential biotechnological application.

The Greater Mekong Subregion (GMS) is regarded as a global biodiversity hotspot due to its widely varying environmental conditions. Accordingly, the GMS harbors a diverse array of numerous florae, fauna and microorganisms (Li et al. 2018). Woody litter microfungi is an overlooked group of fungi in GMS and based on previous fungal estimates, there is undoubtedly a large number of new species yet to be described from this region. Our ongoing studies into the diversity of microfungi of the GMS are actively contributing towards filling in the knowledge gap in fungal taxonomy, phylogeny, host association and ecological distribution of Rhytidhysteron species in this region (Luo et al. 2018; Bao et al. 2019; Dong et al. 2020; Hyde et al. 2020b; Monkai et al. 2020, 2021; Wanasinghe et al. 2020, 2021; Yasanthika et al. 2020). Our specific objectives of this study are as follows: 1) to describe a novel species of Rhytidhysteron with evidence from morphology and DNA sequence data; 2) to characterize (based on morphology and phylogeny) additional new records of Rhytidhysteron; 3) to investigate the phylogenetic relationships of our Rhytidhysteron samples based on DNA sequence analyses from rDNA and protein coding genes and update the taxonomy of Rhytidhysteron.

Materials and methods

Samples collection and morphological analyses

Woody litter samples were collected from China (Kunming, Yunnan Province) during the wet season (August 2019) and during the dry season (December 2019) collections were done in Thailand (Chiang Rai and Tak Provinces). Samples were brought to the laboratory in 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 one week in the dark. Digital images of the fruiting structures were captured with a Canon (EOS 600D) digital camera fitted to a Nikon ECLIPSE Ni compound microscope. Squash mount preparations were prepared to determine micro-morphology and free hand sections of sporocarps made to observe the shapes of ascomata/conidiomata and peridium structures. Measurements of morphological structures were taken from the widest part of each structure. When possible, more than 30 measurements were made. Measurements were taken using the Tarosoft (R) Image Frame Work program. Figures were processed using Adobe Photoshop CS6. Field data are presented in ‘Material examined’. Other details pertaining to good practices of morphological examinations were done following guidelines by Senanayake et al. (2020). New species are established based on recommendations proposed by Jeewon and Hyde (2016). Type specimens were deposited in the herbarium of the 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

Genomic DNA was extracted from the mycelium grown on PDA at 25–30 °C for one week using a Biospin Fungus Genomic DNA Extraction Kit (BioFlux Hangzhou, P. R. China). Three partial rDNA genes and a protein coding gene were processed in our study, including the small ribosomal subunit RNA (SSU) using the primer pair NS1/NS4 (White et al. 1990), internal transcribed spacer region (ITS) using the primer pair ITS5/ITS4 (White et al. 1990), large nuclear ribosomal subunit (LSU) using primer pair LR0R/LR5 (Vilgalys and Hester 1990), translation elongation factor 1-alpha gene (tef1-α) using primer pair 983F/2218R (Rehner and Buckley 2005). Amplification reactions were performed in a total volume of 25 μL of PCR mixtures containing 8.5 μL ddH2O, 12.5 μL 2X PCR MasterMix (TIANGEN Co., China), 2 μL DNA template and 1 μL of each primer. PCR thermal cycle program for SSU, LSU, ITS, and tef1-α were set as described in Wanasinghe et al. (2020). The PCR products were sent to the Qingke Company, Kunming City, Yunnan Province, China, for sequencing. Sequences were deposited in GenBank (Table 1).

Table 1.
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GenBank accession numbers of sequences used for the phylogenetic analyses.

Taxon Strain number GenBank accession numbers Reference
SSU LSU ITS tef1-α
Gloniopsis calami MFLUCC 15-0739 KX669034 NG_059715 KX669036 KX671965 Hyde et al. (2016)
Gloniopsis praelonga CBS 112415 FJ161134 FJ161173 NA FJ161090 Boehm et al. (2009a)
Rhytidhysteron bruguierae MFLUCC 18-0398T MN017901 MN017833 NA MN077056 Dayarathne et al. (2020)
Rhytidhysteron bruguierae MFLUCC 17-1515 MN632463 MN632452 MN632457 MN635661 Mapook et al. (2020)
Rhytidhysteron bruguierae MFLUCC 17 1511 MN632465 MN632454 MN632459 NA Mapook et al. (2020)
Rhytidhysteron bruguierae MFLUCC 17-1502 MN632464 MN632453 MN632458 MN635662 Mapook et al. (2020)
Rhytidhysteron bruguierae MFLUCC 17-1509 MN632466 MN632455 MN632460 NA Mapook et al. (2020)
Rhytidhysteron camporesii HKAS 104277T NA MN429072 MN429069 MN442087 Hyde et al. (2020a)
Rhytidhysteron chromolaenae MFLUCC 17-1516T MN632467 MN632456 MN632461 MN635663 Mapook et al. (2020)
Rhytidhysteron erioi MFLU 16-0584T NA MN429071 MN429068 MN442086 Hyde et al. (2020a)
Rhytidhysteron hongheense KUMCC 20-0222T MW264224 MW264194 MW264215 MW256816 Wanasinghe et al. (2021)
Rhytidhysteron hongheense HKAS112348 MW541831 MW541820 MW54182 MW556132 Wanasinghe et al. (2021)
Rhytidhysteron hongheense HKAS112349 MW541832 MW541821 MW541825 MW556133 Wanasinghe et al. (2021)
Rhytidhysteron hysterinum EB 0351 NA GU397350 NA GU397340 Boehm et al. (2009b)
Rhytidhysteron magnoliae MFLUCC 18-0719T MN989382 MN989384 MN989383 MN997309 de Silva et al. (2020)
Rhytidhysteron mangrovei MFLUCC 18-1113T NA MK357777 MK425188 MK450030 Kumar et al. (2019)
Rhytidhysteron neorufulum MFLUCC 13-0216T KU377571 KU377566 KU377561 KU510400 Thambugala et al. (2016)
Rhytidhysteron neorufulum GKM 361A GU296192 GQ221893 NA NA Thambugala et al. (2016)
Rhytidhysteron neorufulum HUEFS 192194 NA KF914915 NA NA Thambugala et al. (2016)
Rhytidhysteron neorufulum MFLUCC 12-0528 KJ418119 KJ418117 KJ418118 NA Thambugala et al. (2016)
Rhytidhysteron neorufulum CBS 306.38 AF164375 FJ469672 NA GU349031 Thambugala et al. (2016)
Rhytidhysteron neorufulum MFLUCC 12-0011 KJ418110 KJ418109 KJ206287 NA Thambugala et al. (2016)
Rhytidhysteron neorufulum MFLUCC 12-0567 KJ546129 KJ526126 KJ546124 NA Thambugala et al. (2016)
Rhytidhysteron neorufulum MFLUCC 12-0569 KJ546131 KJ526128 KJ546126 NA Thambugala et al. (2016)
Rhytidhysteron neorufulum EB 0381 GU397366 GU397351 NA NA Thambugala et al. (2016)
Rhytidhysteron neorufulum MFLUCC 21-0035 MZ346025 MZ346015 MZ346020 MZ356249 This study
Rhytidhysteron opuntiae GKM 1190 NA GQ221892 NA GU397341 Mugambi et al. (2009)
Rhytidhysteron rufulum MFLUCC 14-0577T KU377570 KU377565 KU377560 KU510399 Thambugala et al. (2016)
Rhytidhysteron rufulum EB 0384 GU397368 GU397354 NA NA Boehm et al. (2009b)
Rhytidhysteron rufulum EB 0382 NA GU397352 NA NA Boehm et al. (2009b)
Rhytidhysteron rufulum EB 0383 GU397367 GU397353 NA NA Boehm et al. (2009b)
Rhytidhysteron rufulum MFLUCC 12-0013 KJ418113 KJ418111 KJ418112 NA de Silva et al. (2020)
Rhytidhysteron tectonae MFLUCC 13-0710T KU712457 KU764698 KU144936 KU872760 Doilom et al. (2017)
Rhytidhysteron tectonae MFLUCC 21-0037 MZ346023 MZ346013 MZ346018 MZ356247 This study
Rhytidhysteron tectonae MFLUCC 21-0034 MZ346024 MZ346014 MZ346019 MZ356248 This study
Rhytidhysteron thailandicum MFLUCC 14-0503T KU377569 KU377564 KU377559 KU497490 Thambugala et al. (2016)
Rhytidhysteron thailandicum MFLUCC 12-0530 KJ546128 KJ526125 KJ546123 NA Thambugala et al. (2016)
Rhytidhysteron thailandicum MFLU17-0788 MT093495 MT093472 MT093733 NA de Silva et al. (2020)
Rhytidhysteron xiaokongense KUMCC 20-0158 MZ346021 MZ346011 MZ346016 MZ356245 This study
Rhytidhysteron xiaokongense KUMCC 20-0160T MZ346022 MZ346012 MZ346017 MZ356246 This study

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

Phylogenetic analyses

Representative species used in the phylogenetic analyses were selected based on previous publications (Thambugala et al. 2016; Mapook et al. 2020; Wanasinghe et al. 2021). 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 and gaps, and then combined using BioEdit 7.2.3. Final alignments containing SSU, LSU, ITS, and tef1-α were converted to NEXUS format (.nxs) using CLUSTAL X (2.0) and PAUP v. 4.0b10 (Thompson et al. 1997; Swofford 2002) 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). ML and MP bootstrap values (ML) ≥ 75% are given above each node of the phylogenetic tree (Fig. 1).

Figure 1. 

RAxML tree based on a combined dataset of partial SSU, LSU, ITS, and tef1-α sequence analyses. 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 the 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. The tree is rooted with Gloniopsis calami (MFLUCC 15-0739) and G. praelonga (CBS 112415).

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 HKY+I for SSU; GTR+I+G for ITS, LSU and tef1-α 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 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. Branches with Bayesian posterior probabilities (BYPP) ≥ 0.95 are indicated above each node of the phylogenetic tree (Fig. 1). 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, then 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:S28620 (http://purl.org/phylo/treebase/phylows/study/TB2:S28620).

Results

Phylogenetic analysis

The phylogenetic analysis was conducted using 38 strains in Rhytidhysteron, and two outgroup taxa viz. Gloniopsis calami (MFLUCC 15-0739) and G. praelonga (CBS 112415) in Pleosporales (Table 1). The aligned sequence matrix comprised four gene regions (SSU: 1018 bp, LSU: 891 bp, ITS: 742 bp and tef1-α: 953 bp) and a total of 3,604 characters (including gaps), of which 3,095 characters were constant, 161 variable characters were parsimony-uninformative and 348 characters were parsimony-informative. The Kishino-Hasegawa test shows length = 928 steps with CI = 0.696, RI = 0.846, RC = 0.589 and HI = 0.304. The RAxML analysis of the combined dataset yielded a best-scoring tree with a final ML optimization likelihood value of -10181.226009. The matrix had 723 distinct alignment patterns, with 26.6% of undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.242390, C = 0.244261, G = 0.276352, T = 0.236997; substitution rates AC = 1.457846, AG = 2.708684, AT = 1.298658, CG = 0.909442, CT = 6.323746, GT = 1.00; gamma distribution shape parameter α = 0.02.

Topologies of the phylogenetic trees under ML, MP and BI criteria recovered for each gene dataset were visually compared, and the overall tree topology was similar to those obtained from the combined dataset (Figure 1). Our analyzed molecular data generated phylogeny of Rhytidhysteron species was consistent with those of Wanasinghe et al. (2021). The maximum likelihood tree generated based on sequence analysis of the combined (ribosomal DNA: SSU, LSU and ITS; and protein coding gene: tef1-α) dataset recovered three major monophyletic clades within Rhytidhysteron (A-C, Figure 1) and two basal lineages viz. R. hysterinum (EB 0351) and R. opuntiae (GKM 1190). Clade A comprises Rhytidhysteron magnoliae, R. neorufulum, R. rufulum and R. tectonae with 96% ML, 98% MP and 1.00 BYPP support values.

One of our new isolates, MFLUCC 21-0035 grouped with another nine Rhytidhysteron neorufulum strains (CBS 306.38, EB 0381, GKM 361A, HUEFS 192194, MFLUCC 12-0011, MFLUCC 12-0528, MFLUCC 12-0567, MFLUCC 12-0569, MFLUCC 13-0216, MFLUCC 21-0035). However, this relationship is not statistically supported in Bayesian analysis, retrieving 79% and 77% support values in ML and MP, respectively (sub clade A1, Figure 1). Rhytidhysteron magnoliae (MFLUCC 18-0719) constitutes an independent lineage and is a sister taxon to others in sub clade A1, and this was not statistically supported.

Two newly generated sequences MFLUCC 21-0034 and MFLUCC 21-0037 grouped with the type strain of Rhytidhysteron tectonae (MFLUCC 13-0710) as a monophyletic clade within Clade A (subclade A2, Figure 1). This association was supported by 85% ML, 92% MP and 1.00 BYPP bootstrap values (subclade A2, Figure 1). Five strains of Rhytidhysteron rufulum (EB 0382, EB 0383, EB 0384, MFLUCC 12-0013, MFLUCC 14-0577) constitute another strongly monophyletic group basal to Clade A.

Two of our newly generated sequences, Rhytidhysteron xiaokongense (KUMCC 20-0158, KUMCC 20-0160), grouped with R. bruguierae (MFLUCC 17-1511, MFLUCC 17-1502, MFLUCC 17-1509, MFLUCC 17-1515, MFLUCC 18-0398), R. erioi (MFLU 16-0584), R. mangrovei (MFLUCC 18-1113) and R. thailandicum (MFLU 17-0788, MFLUCC 12-0530, MFLUCC 14-0503). These taxa form a monophyletic clade (Clade B) in Rhytidhysteron with 93% ML, 91% MP and 1.00 BYPP bootstrap values. Within this clade (Clade B), Rhytidhysteron xiaokongense (KUMCC 20-0158 and KUMCC 20-0160) clusters together (subclade B1) with high bootstrap values (100% ML, 100% MP and 1.00 BYPP) and is sister to Rhytidhysteron thailandicum. However, the latter relationship was only supported by BI analysis with 0.96 BYPP.

Rhytidhysteron camporesii (HKAS104277), R. chromolaenae (MFLUCC 17-1516) and R. hongheense (HKAS112348, HKAS112349, KUMCC 20-0222) grouped as a monophyletic clade. This relationship is statistically supported with 100% ML, 99% MP and 1.00 BYPP values (Figure 1). Rhytidhysteron hysterinum (EB 0351) and R. opuntiae (GKM 1190) nested as basal lineages in Rhytidhysteron (Figure 1).

Taxonomy

Rhytidhysteron xiaokongense G.C. Ren & K.D. Hyde, sp. nov.

MycoBank No: 558453
Facesoffungi Number No: FoF09903
Figure 2

Etymology

The species epithet reflects the location where the species was collected.

Holotype

HKAS 112728.

Diagnosis

Similar to R. hysterinum and R. rufulum, but differs in some conidial features.

Description

Saprobic on woody litter of Prunus sp. Sexual morph Undetermined. Asexual morph Conidiomata 448–464 × 324–422 µm (x̄ = 454 × 378 μm, n = 5), solitary, scattered, semi-immersed in the host, black, unilocular, subglobose to ampulliform. Ostioles 178–227 × 166–234 µm (x̄ = 205 × 198 μm, n = 6), central, short papillate. Conidiomata wall 30–40 μm thick, 4–6 layers, reddish-brown to dark brown cells of textura angularis. Conidiogenous cells 5–8 × 3–6 µm (x̄ = 6.8 × 4.5 μm, n = 10), subglobose or ellipsoidal, hyaline, smooth, forming in a single layer over the entire inner surface of the wall, discrete, producing a single conidium at the apex. Conidia 20–25 × 8–10 μm (x̄ = 22 × 9 μm, n = 20), hyaline to yellowish-brown when immature, becoming brown to dark brown at maturity, oblong to ellipsoidal, with rounded ends, straight to slightly curved, aseptate when immature, becoming 1-septate when mature, with granular appearance, slightly constricted at septa.

Habitat and distribution

Known to inhabit woody litter of Prunus sp. (Yunnan, China) (this study).

Material examined

China, Yunnan Province, Kunming city, Xiaokong Mountain (25.171311°N, 102.703690°E), on dead wood of Prunus sp. (Rosaceae), 21-Dec-2019, G.C. Ren, KM18 (HKAS 112728, holotype), ex-type living culture KUMCC 20-0160; KM17 (HKAS 112727, paratype), ex-paratype living culture KUMCC 20-0158.

Notes

Rhytidhysteron xiaokongense is similar to R. hysterinum and R. rufulum in having black, unilocular, subglobose conidiomata and dark brown, 1-septate conidia. However, some of the conidia features in these species are different: R. xiaokongense has oblong to ellipsoidal conidia with rounded ends, whereas the conidia of R. rufulum and R. hysterinum have a truncated base with a pore in the middle of the septum (Samuels and Müller 1979). In the phylogenetic analyses, R. xiaokongense is distinct from R. rufulum and R. hysterinum and is more closely related to R. thailandicum. Rhytidhysteron xiaokongense has 1-septate, dark brown, oblong to ellipsoidal conidia, while R. thailandicum has globose to subglobose, hyaline conidia (Thambugala et al. 2016). The sequence data from both mycelium and fruiting bodies confirms that single spore isolation was successfully performed.

Figure 2. 

Rhytidhysteron xiaokongense (HKAS 112728, holotype) a, b conidiomata on natural wood surface c sections through conidioma d ostiolar neck e conidioma wall f–h conidiogenous cells and developing conidia i–m conidia n germinated conidium o, p culture characters on PDA (o = above, p = reverse). Scale bars: 100 μm (c, d); 50 μm (e); 15 μm (f–h); 10 μm (i–m); 20 μm (n); 25 mm (o, p).

Rhytidhysteron tectonae Doilom & K.D. Hyde, Fungal Diversity. 82: 107–182 (2017)

MycoBank No: 551964
Facesoffungi number No: FoF01849
Figure 3

Description

Saprobic on decaying wood. Sexual morph Hysterothecia 550–950 µm long, 450–600 µm high, 400–500 diam. (x̄ = 800 × 500 × 450 µm, n = 5), semi-immersed to superficial, scattered, apothecial, erumpent from the substrate, dark brown to black, coriaceous, elongate with a longitudinal slit. Exciple 70–110 µm (x̄ = 90 µm, n = 15), thick-walled, composed of brown to dark brown cells of textura globulosa to angularis. Hamathecium comprising 1–2 μm wide, numerous, septate, branched, pseudoparaphyses. Asci 170–200 × 10–12 μm (x̄ = 190 × 11, n = 15), 8-spored, bitunicate, cylindrical, with short pedicel, rounded at the apex, with an ocular chamber. Ascospores 25–29 × 8–10 µm (x̄ = 27 × 9 µm, n = 20), uniseriate, hyaline to brown, 1–3-septate, smooth-walled, ellipsoidal to fusoid, straight or curved, rounded to slightly pointed at both ends, guttulate. Asexual morph Undetermined.

Figure 3. 

Rhytidhysteron tectonae (HKAS 115533) a, b Hysterothecium on wood c vertical section through hysterothecia d exciple e pseudoparaphyses f–i immature and mature asci j ocular chamber. k–r immature and mature ascospores s Germinating ascospore t, u culture characters on PDA (t = above view, u = reverse view). Scale bars: 300 μm (c); 50 μm (d); 30 μm (e); 50 μm (f–i); 10 μm (j–r); 15 μm (s); 25 mm (t, u).

Habitat and distribution

Known to inhabit dead branches of Tectona grandis, Betula sp. (Betulaceae) and Fabaceae sp (Thailand) (Doilom et al. 2017; this study).

Material examined

Thailand, Chiang Rai Province, Mae Yao District, on dead woody twigs of Betula sp. (Betulaceae), 23-Sep-2019, G.C. Ren, MY09 (HKAS 115533), living culture MFLUCC 21-0037; Thailand, Chiang Rai Province, Mae Fah Luang University, on dead woody twigs of Fabaceae, 5-Jul-2019, G.C. Ren, RMFLU19001 (HKAS 115532), living culture MFLUCC 21-0034.

Notes

Rhytidhysteron tectonae was introduced by Doilom et al. (2017) based on morphological and phylogenetic analyses from dead branches of Tectona grandis in Thailand. Based on our phylogenetic analysis of the combined SSU, LSU, ITS, and tef1-α sequence data, our collections (MFLUCC 21-0034 and MFLUCC 21-0037) cluster with the strain of R. tectonae (MFLUCC 13-0710) with 85% ML, 92% MP, 1.00 PP bootstrap support (Figure 1). Our collection shares similar morphological features with R. tectonae (MFLU 14-0607). However, our new collection has smaller hysterothecia (800 × 500 × 450 μm vs 2175 × 585 × 523 μm) and longer asci (190 μm vs 155 μm) in comparison to the type. Based on morphological characteristics and phylogenetic analysis, we introduce MFLUCC 21-0034 and MFLUCC 21-0037 as new host records of R. tectonae from decaying wood of Betula sp. and Fabaceae sp. in Thailand.

Rhytidhysteron neorufulum Thambug. & K.D. Hyde, Cryptog. Mycol. 37(1): 110 (2016)

MycoBank No: 551865
Facesoffungi number No: FoF01840
Figure 4

Description

Saprobic on decaying wood of Tectona grandis. Sexual morph Hysterothecia 1400–2100 μm long, 350–500 μm high, 600–1000 μm diam. (x̄ = 1780 × 400 × 700 μm, n = 5), superficial, black, solitary to aggregated, coriaceous, smooth, elliptical or irregular in shape, elongated with a longitudinal slit. Exciple 75–115μm (x̄ = 90, n = 20) wide, composed of several layers of brown to dark brown, thick-walled cells of textura angularis. Hamathecium 2–3.5 μm wide, dense, septate pseudoparaphyses, constricted at the septum, filiform, pale-yellow pigmented, forming epithecium above the asci and enclosed in a gelatinous matrix. Asci 190–260 × 13–18 μm (x̄ = 230 × 16 μm, n = 10), 8-spored, bitunicate, clavate to cylindrical, with a short furcate pedicel, apically rounded, without a distinct ocular chamber. Ascospores 36–44 × 11–17 μm (x̄ = 41 × 13 μm, n = 30), uni-seriate, yellowish to brown, with 1–3-septa, ellipsoidal to fusiform, slightly rounded or pointed at both ends, constricted at the central septum, with granular appearance. Asexual morph Undetermined.

Figure 4. 

Rhytidhysteron neorufulum (HKAS 115534) a, b Hysterothecium on wood c vertical section through hysterothecia d exciple e pseudoparaphyses f–h immature asci and mature asci i–m immature ascospores and mature ascospores n germinating ascospore o, p culture characters on PDA (o = above view, p = reverse view). Scale bars: 1000 μm (a, b); 200 μm (c); 15 μm (d); 20 μm (e); 50 μm (f–h); 10 μm (i–m); 20 μm (n); 20 mm (o, p).

Habitat and distribution

Bursera sp (Mexico), Hevea brasiliensis and Tectona grandis (Thailand) (Thambugala et al. 2016; Cobos-Villagran et al. 2020; this study).

Material examined

Thailand, Tak Province, Mogro District, Amphoe Umphang, on dead woods of Tectona grandis (Lamiaceae), 20-Aug-2019, G.C. Ren, T203 (HKAS 115534), living culture MFLUCC 21-0035.

Notes

Rhytidhysteron neorufulum was introduced by Thambugala et al. (2016) based on both morphological and phylogenetic analyses of a combined dataset of LSU, SSU and tef1-α sequence data. Thambugala et al. (2016) accounted R. neorufulum (MFLUCC 13-0216) from decaying woody stems and twigs in Thailand. Our new collection shares similar morphology to that of the type description of Rhytidhysteron neorufulum (MFLUCC 13-0216) in having superficial, coriaceous, elliptical or irregular, elongated hysterothecia with a longitudinal slit, bitunicate, cylindrical, short furcate pedicel asci and yellowish to brown, ellipsoidal to fusiform ascospores with 1–3-septa (Thambugala et al. 2016). However, our new collection has larger asci (190–260 × 13–18 μm vs 185–220 × 9.5–13 μm) and ascospores (36–44 × 11–17 μm vs 19–31 × 8–13 μm) in comparison to the type of Rhytidhysteron neorufulum (MFLUCC 13-0216). The multi-gene phylogenetic analysis based on combined SSU, LSU, ITS, and tef1-α sequence data showed that our collection is related to Rhytidhysteron neorufulum (Figure 1).

Key to asexual morphs of Rhytidhysteron species

1 Asexual morph has two types of conidia 2
– Asexual morph has only one type of conidia 3
2 Comprising paraphyses R. hysterinum
– Paraphyses are absent R. rufulum
3 Diplodia-like conidia R. xiaokongense
– Aposphaeria-like conidia R. thailandicum

Discussion

Rhytidhysteron is one of the first genera that trainee mycologists working on microfungi find in nature, as the hysterothecia are conspicuous (Hyde et al. 2020a). Species also easily germinate in culture and can easily be sequenced (Hyde et al. 2020a). Thus, it is even more remarkable that we found a new species in this study, indicating we are far from finding all species in this genus, and that more collections need be done on other continents (Hyde et al. 2020c). Most of Rhytidhysteron species are saprobes, which are essential for ecosystems functioning in terrestrial habitats and are commonly recognized as key biotic agents of wood decomposition, playing a vital role in carbon and nitrogen cycling in arid ecosystems, soil stability, plant biomass decomposition, and endophytic interactions with plants (Lustenhouwer et al. 2020; Dossa et al. 2021). Furthermore, Rhytidhysteron species have numerous antimicrobial and antifungal applications (Murillo et al. 2009; Mapook et al. 2020), and the discovery of new species provides new resources for future applied research in the field of biotechnology and industry.

Since the genus was established in 1881, a total of 24 species have been found to date, and the most commonly encountered species are Rhytidhysteron neorufulum and R. rufulum, so it might be difficult for mycologists to find new species within Rhytidhysteron. Rhytidhysteron is mainly identified via its sexual morph (Dayarathne et al. 2020; de Silva et al. 2020; Hyde et al. 2020a, b; Mapook et al. 2020; Wanasinghe et al. 2021). The asexual morphs of Rhytidhysteron have been reported as aposphaeria-like or diplodia-like, including R. hysterinum and R. rufulum (Samuels and Müller 1979). Thambugala et al. (2016) confirmed the asexual-sexual morph connection for R. thailandicum by aposphaeria-like asexual morphs forming in culture on PDA. Herein, we found a diplodia-like asexual morph of Rhytidhysteron from woody litter of Prunus sp. in China. In comparison to the occurrence of the sexual morph of Rhytidhysteron, asexual morphs seldom form under natural conditions. The discovery of this new species provides an important reference for the study of the asexual morphs of Rhytidhysteron. Moreover, findings from this study further enrich GMS Rhytidhysteron species diversity.

In our phylogenetic analyses, the new species, Rhytidhysteron xiaokongense was basal to R. thailandicum (Fig. 1). Although species in Rhytidhysteron are morphologically similar, our new species is an asexual form of the species found in nature, so it is easy to distinguish from other speices excluding the asexual forms of R. hysterinum, R. rufulum and R. thailandicum. Rhytidhysteron xiaokongense shares similar morphological characters to R. hysterinum and R. rufulum in having black, unilocular, subglobose conidiomata and dark brown, 1-septate conidia but conidial features differ (Samuels and Müller 1979). Rhytidhysteron thailandicum can be differentiated from R. xiaokongense with respects to its globose to subglobose, hyaline conidia (Thambugala et al. 2016). To further support the establishment of the new taxon as proposed by Jeewon and Hyde (2016), we examined the nucleotide differences within the ITS regions (ITS1-5.8S-ITS2) gene region. Comparison of the 507 nucleotides across the ITS regions reveals 39 bp (7.7%) differences between Rhytidhysteron thailandicum and R. xiaokongense.

Rhytidhysteron species are widely distributed throughout the globe (de Silva et al. 2020); however, they appear to be particularly abundant in Asia, where they are well studied. There is an abundance of species and collections in the Greater Mekong Subregion (China and Thailand), such as R. brasiliense, R. camporesii, R. chromolaenae, R. erioi, R. hongheense, R. hysterinum, R. magnoliae, R. mangrovei, R. neorufulum, R. tectonae and R. thailandicum (Thambugala et al. 2016; Doilom et al. 2017; Soto-Medina et al. 2017; Kumar et al. 2019; Cobos-Villagran et al. 2020; Dayarathne et al. 2020; de Silva et al. 2020; Hyde et al. 2020a; Mapook et al. 2020; Wanasinghe et al. 2021). We provide morphological and phylogenetic data for three species of Rhytidhysteron collected from the Greater Mekong Subregion: one new species, Rhytidhysteron xiaokongense, as a geographical record from China, two new host records of R. tectonae from woody litter of Betula sp and Fabaceae sp, and one new host record of R. neorufulum from woody litter of Tectona grandis. Based on our current work and that of past studies (de Silva et al. 2020; Hyde et al. 2020a, b; Mapook et al. 2020; Wanasinghe et al. 2021), it is clear that species within Rhytidhysteron are likely cosmopolitan and not host-specific, with evidence of the same species being found on a number of different hosts. Importantly, the morphology of a single species sometimes shows slight variations under different environmental conditions, geographical regions, hosts and different life modes (Senanayake et al. 2020). It is therefore crucial to collect more species of Rhytidhysteron across different geographic regions and hosts, obtain more cultures and sequence data, and describe their morphology to improve knowledge of taxonomy and phylogeny.

Acknowledgements

This work was supported by the Strategic Priority Research Program of Chinese Academy of Sciences (Grant No. XDA2602020). We thank the support from the National Natural Science Foundation of China (NSFC32001296). 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 the 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, High-End Foreign Experts” in the High-Level Talent Recruitment Plan of Yunnan Province (2021) and Chinese Academy of Sciences (grant no. 41761144055) for financial support. Austin G. Smith at World Agroforestry (ICRAF), Kunming Institute of Botany, China, is thanked for English editing.

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