Taxonomic and Phylogenetic Insights into Novel Ascomycota from Forest Woody Litter.

While surveying the mycobiomes of dead woody litter in Yunnan Province, China, numerous isolates with affinity to Pleosporales (Dothideomycetes, Ascomycota) were recovered. The present work characterizes two species associated with dead woody twigs found in terrestrial habitats in the Kunming area of Yunnan. The novel taxa were recognized based on a polyphasic approach, including morphological examination and multiple gene phylogenetic analyses (non-translated loci and protein-coding regions). Neokalmusia jonahhulmei sp. nov. is introduced in Didymosphaeriaceae (Pleosporales) as a woody-based saprobic ascomycete that possesses multiloculate ascostromata immersed under a black clypeus-like structure, and three-septate, brown, fusiform, guttulate ascospores. Thyridaria jonahhulmei (Thyridariaceae) is introduced with teleomorphic and anamorphic (coelomycetous) characteristics. The teleomorph has the following characteristics: globose to subglobose ascomata with an ostiolum, a pruinose layer of yellow to reddish- or orange-brown material appearing around the top of the ostiolar necks, and brown, ellipsoid to fusoid, two-to-three-septate, euseptate, rough-walled ascospores; the anamorph features pycnidial conidiomata, phialidic, ampulliform to doliiform, conidiogenous cells, and brown, guttulate, ellipsoidal, aseptate conidia.

1. Introduction

Forest ecosystems produce a large quantity of litter in various forms, such as leaves, branches, twigs, inflorescence, and other debris. Plant litter maintains pathways involved in nutrient cycling that are crucial to forest productivity [1,2,3,4,5,6]. A great proportion of total forest carbon (C) is contained in these woody debris, providing habitats for thousands of organisms, including fungi. Fungi play a critical role in the decomposition of woody litter as they are capable of actively decomposing lignin and other recalcitrant components found in these materials. However, comprehensive studies of the fungal taxonomic systematics of woody litter are scarce.

Most of the described woody-based fungal species lack biological and ecological information and reliable taxonomic status. Researchers have paid more attention to economic species, such as those producing edible mushrooms, and less attention to the narrowly distributed microfungal species responsible for the decomposition and recycling of woody material. However, in recent years, there have been numerous studies into woody-based microfungal occurrences in Yunnan Province, leading to reports of higher microfungal diversity, especially in Dothideomycetes [7,8,9,10,11,12,13,14,15,16]. At the Centre for Mountain Futures (Kunming Institute of Botany), researchers are investigating microfungal diversity across several substrates in southwest China, including leaf and woody litter, in order to clarify their taxonomy using morphology in conjunction with multigene phylogeny. Accordingly, we have isolated ascomycetes (Pleosporales) from woody litter, collected in a Pinus yunnanensis forest in Yunnan Province (Kunming), China (Figure 1). This study assesses the systematic categorization of two taxonomic novelties in Neokalmusia and Thyridaria, assesses morphological characteristics on natural substrates and in cultures, and conducts phylogenetic analyses.

Figure 1

Habitat. (a–d) Mixed forest (predominantly Fagaceae and Pinus spp., with Pinus yunnanensis being the most abundant tree species); (e) collector.

2. Materials and Methods

2.1. Isolates and Specimens

During fieldwork in the Kunming region, Yunnan, China, characteristic black ascomata/conidiomata on dead woody twigs were collected during the dry season. The local environment features mixed forests (predominantly Fagaceae and Pinus spp., with Pinus yunnanensis being the most abundant tree species) (Figure 1) and a humid temperate climate at elevation 2080 masl. Specimens were stored in Ziploc plastic bags and taken to the laboratory. Isolations were made from single ascospores, according to the methods of Wanasinghe et al. [11]. Voucher specimens were preserved in the herbarium of the Kunming Institute of Botany (KUN-HKAS), and the living cultures were placed in the Culture Collection of Kunming Institute of Botany (KUMCC), Kunming, China. Nomenclatural novelties were deposited in MycoBank as outlined in http://www.MycoBank.org (accessed on 17 March 2022).

2.2. Morphological Observations

Examination of external structures of the fungal specimens were made using a Motic SMZ 168 Series stereo-microscope (Motic Asia, Kowloon, Hong Kong). Micro-morphological characters were examined and evaluated following the protocols provided by Wanasinghe et al. [10]. Macroscopic images of colonies were documented using an iPhone XS Max (Apple Inc., Cupertino, CA, USA) with daylight. Photoplates and images were processed using Adobe Photoshop CS6 (Adobe Systems, San Jose, CA, USA).

2.3. DNA Extraction, PCR Amplifications and Sequencing

The genomic DNA of each isolate was extracted from scraped mycelia following Wanasinghe et al. [10] using the Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux, Shanghai, China). Primers for PCR amplification used were ITS (internal transcribed spacers) = ITS5/ITS4 [17], LSU (partial 28S large subunit rDNA) = LR0R/LR5 [18,19], SSU (partial 18S small subunit rDNA) = NS1/NS4 [17], tef1 (translation elongation factor 1-α) = EF1-983F/EF1-2218R [20,21], and rpb2 (RNA polymerase II second largest subunit) = fRPB2-5f/fRPB2-7cR [22]. The PCR protocols were programmed as described in The PCR protocols of SSU, LSU, ITS, and tef1 and were programmed as described in Wanasinghe et al. [11]. The PCR amplification condition of rpb2 was set as denaturation at 98 °C for 2 min, followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 52 °C for 10 s, and extension at 72 °C for 20 s, with a final extension step at 72 °C for 5 min. DNA sequencing were performed at a private company for sequencing (BGI, Ltd., Shenzhen, China).

2.4. Molecular Phylogenetic Analyses

2.4.1. Sequencing and Sequence Alignment

Sequences generated from SSU, LSU, ITS, tef1, and rpb2 were first used for BLASTn analyses. Based on BLAST similarities and relevant publications [23,24,25], closely related sequences were downloaded from GenBank (Table 1 and Table 2). Loci were aligned using MAFFT v. 7 [26,27]) under default conditions. Final improvements were made when necessary using BioEdit v.7.0.5.2 software [28].

Table 1

Taxa used in the phylogenetic analyses of Didymosphaeriaceae and their corresponding GenBank numbers of partial ITS, LSU, SSU, tef1, and rpb2 sequences. Isolates/sequences in bold were isolated/sequenced in the present study.

Species	Strain	GenBank Accession Numbers
		ITS	LSU	SSU	tef1	rpb2
Alloconiothyrium camelliae	NTUCC 17-032-1	MT112294	MT071221	MT071270	MT232967	-
Alloconiothyrium camelliae	NTUCC 17-032-2	MT112295	MT071222	MT071271	MT232965	-
Austropleospora keteleeriae	MFLUCC 18-1551	NR_163349	MK347910	NG_070075	MK360045	MK434909
Austropleospora ochracea	KUMCC 20-0020	MT799859	MT808321	MT799860	MT872714	-
Austropleospora osteospermi	MFLUCC 17-2429	MK347757	MK347863	MK347974	MK360044	MK434884
Bambusistroma didymosporum	MFLU 15-0057	KP761733	KP761737	KP761730	KP761727	KP761720
Bambusistroma didymosporum	MFLU 15-0058	KP761734	KP761738	KP761731	KP761728	KP761721
Bimuria novae-zelandiae	CBS 107.79	MH861181	AY016338	AY016356	DQ471087	DQ470917
Bimuria omanensis	SQUCC 15280	NR_173301	-	NG_071257	MT279046	-
Chromolaenicola lampangensis	MFLUCC 17-1462	MN325016	MN325010	MN325004	MN335649	MN335654
Chromolaenicola nanensis	MFLUCC 17-1477	MN325014	MN325008	MN325002	MN335647	MN335653
Chromolaenicola thailandensis	MFLUCC 17-1475	MN325019	MN325013	MN325007	MN335652	MN335656
Cylindroaseptospora leucaenae	MFLUCC 17-2424	NR_163333	MK347856	NG_066310	MK360047	-
Cylindroaseptospora siamensis	MFLUCC 17-2527	NR_163337	MK347866	NG_066311	MK360048	-
Deniquelata barringtoniae	MFLUCC 16-0271	MH275059	-	MH260291	MH412766	MH412753
Deniquelata hypolithi	CBS 146988	MZ064429	-	NG_076735	MZ078250	MZ078201
Deniquelata vittalii	NFCCI4249	MF406218	MF622059	MF182395	MF182398	MF168942
Didymocrea sadasivanii	CBS 438.65	MH858658	DQ384066	DQ384103	-	-
Didymosphaeria rubi ulmifolii	CBS 100299	MH862698	AY642523	JX496124	-	-
Didymosphaeria rubi ulmifolii	MFLUCC 140023	KJ436586	NG_063557	KJ436586	-	-
Kalmusia ebuli	CBS 123120	KF796674	JN851818	JN644073	-	-
Kalmusia erioi	MFLU 18-0832	MN473058	MN473046	MN473052	MN481599	-
Kalmusia italica	MFLUCC 14 0560	KP325440	KP325442	KP325441	-	-
Kalmusia variisporum	CBS 121517	NR_145165	-	JX496143	-	-
Kalmusibambusa triseptata	MFLUCC 13-0232	KY682697	KY682696	KY682695	-	-
Karstenula rhodostoma	CBS 690.94	-	GU296154	GU301821	GU349067	GU371788
Karstenula rhodostoma	CBS 691.94	LC014559	AB797241	AB807531	AB808506	-
Laburnicola hawksworthii	MFLUCC 13 0602	KU743194	KU743196	KU743195	-	-
Laburnicola muriformis	MFLUCC 16 0290	KU743197	KU743199	KU743198	KU743213	-
Laburnicola muriformis	MFLUCC 14 0921	KU743200	KU743202	KU743201	-	-
Laburnicola rhizohalophila	CGMCC 8756	KJ125522	-	KJ125523	KJ125525	KJ125524
Letendraea cordylinicola	MFLUCC 11 0150	KM213996	KM214002	KM213999	-	-
Letendraea cordylinicola	MFLUCC 11 0148	NR_154118	KM214001	NG_059530	-	-
Letendraea helminthicola	CBS 884.85	MK404145	AY016345	AY016362	MK404174	MK404164
Letendraea padouk	CBS 485.70	-	GU296162	AY849951	-	-
Montagnula bellevaliae	MFLUCC 14 0924	NR_155377	KT443904	KT443902	KX949743	-
Montagnula chromolaenicola	MFLUCC 17-1469	NR_168866	NG_070157	NG_070948	MT235773	MT235809
Montagnula cirsii	MFLUCC 13 0680	KX274242	KX274255	KX274249	KX284707	-
Montagnula krabiensis	MFLUCC 16-0250	MH275070	MH260343	MH260303	MH412776	-
Montagnula thailandica	MFLUCC 17-1508	MT214352	NG_070158	NG_070949	MT235774	MT235810
Neokalmusia arundinis	MFLU 17-0754	MT649882	MT649880	MT649878	MT663766	-
Neokalmusia arundinis	MFLUCC 15-0463	NR_165852	NG_068372	NG_068237	KY244024	-
Neokalmusia arundinis	MFLUCC 14-0222	KX965731	KX986344	KX954400	KY271091	-
Neokalmusia brevispora	KT 2313	LC014574	AB524460	AB524601	AB539113	-
Neokalmusia brevispora	KT 1466	LC014573	AB524459	AB524600	AB539112	-
Neokalmusia didymospora	MFLUCC 11-0613	-	KP091435	KP091434	-	-
Neokalmusia jonahhulmei	KUMCC 21-0818	ON007043	ON007039	ON007048	ON009133	ON009137
Neokalmusia jonahhulmei	KUMCC 21-0819	ON007044	ON007040	ON007049	ON009134	ON009138
Neokalmusia kunmingensis	KUMCC 18-0120	MK079886	MK079887	MK079889	MK070172	-
Neokalmusia scabrispora	KT 1023	LC014575	AB524452	AB524593	AB539106	-
Neokalmusia scabrispora	KT 2202	LC014576	AB524453	AB524594	AB539107	-
Neokalmusia thailandica	MFLUCC 16-0405	NR_154255	KY706137	NG_059792	KY706145	KY706148
Neokalmusia thailandica	MFLUCC 16-0399	KY706141	KY706136	KY706131	-	-
Neptunomyces aureus	CMG12	MK912121	-	-	MK948000	-
Neptunomyces aureus	CMG13	MK912122	-	-	MK948001	-
Neptunomyces aureus	CMG14	MK912123	-	-	MK948002	-
Paracamarosporium fagi	CPC 24890	NR_154318	-	NG_070630	-	-
Paracamarosporium fagi	CPC 24892	KR611887	-	KR611905	-	-
Paracamarosporium hawaiiense	CBS 120025	JX496027	EU295655	JX496140	-	-
Paraconiothyrium cyclothyrioides	CBS 972.95	JX496119	AY642524	JX496232	-	-
Paraconiothyrium estuarinum	CBS 109850	JX496016	AY642522	JX496129	-	-
Paramassariosphaeria anthostomoides	CBS 615.86	MH862005	GU205246	GU205223	-	-
Paramassariosphaeria clematidicola	MFLU 16-0172	KU743206	KU743208	KU743207	-	-
Paraphaeosphaeria rosae	MFLUCC 17-2547	MG828935	MG829150	MG829044	MG829222	-
Paraphaeosphaeria rosae	MFLUCC 17-2549	MG828937	MG829152	MG829046	MG829223	-
Paraphaeosphaeria rosicola	MFLUCC 15-0042	NR_157528	MG829153	MG829047	-	-
Phaeodothis winteri	CBS 182.58	-	GU296183	GU301857	-	-
Pseudocamarosporium propinquum	MFLUCC 13 0544	KJ747049	KJ819949	KJ813280	-	-
Pseudocamarosporium pteleae	MFLUCC 17-0724	NR_157536	MG829166	MG829061	MG829233	-
Pseudocamarosporium ulmi-minoris	MFLUCC 17-0671	NR_157537	MG829167	MG829062	-	-
Pseudodidymocyrtis lobariellae	KRAM Flakus 25130	NR_169714	NG_070349	NG_068933	-	-
Pseudopithomyces entadae	MFLUCC 17-0917		MK347835	NG_066305	MK360083	MK434899
Pseudopithomyces kunmingnensis	MFLUCC 17-0314	MF173607	MF173606	MF173605	-	-
Pseudopithomyces rosae	MFLUCC 15-0035	MG828953	MG829168	MG829064	-	-
Spegazzinia deightonii	yone 212		AB797292	AB807582	AB808558	-
Spegazzinia radermacherae	MFLUCC 17-2285	MK347740	MK347848	MK347957	MK360088	MK434893
Spegazzinia tessarthra	SH 287	JQ673429	AB797294	AB807584	AB808560	-
Tremateia arundicola	MFLU 16 1275	KX274241	KX274254	KX274248	KX284706	-
Tremateia chromolaenae	MFLUCC 17-1425	NR_168868	NG_070160	NG_068710	MT235778	MT235816
Tremateia guiyangensis	GZAAS01	KX274240	KX274253	KX274247	KX284705	-
Tremateia murispora	GZCC 18-2787	NR_165916	MK972750	MK972751	MK986482	-
Tremateia thailandensis	MFLUCC 17-1430	NR_168869	NG_070161	NG_068711	MT235781	MT235819
Verrucoconiothyrium nitidae	CBS 119209	EU552112	-	EU552112	-	-
Vicosamyces venturisporus	CDA1494	MF802825	-	MF802828	-	-
Vicosamyces venturisporus	CDA1495	MF802826	-	MF802829	-	-
Vicosamyces venturisporus	CDA495	MF802827	-	MF802830	-	-
Xenocamarosporium acaciae	CPC 24755	NR_137982	-	NG_058163	-	-
Xenocamarosporium acaciae	MFLUCC 17-2432	MK347766	MK347873	MK347983	MK360093	-

Table 2

Taxa used in the phylogenetic analyses of Thyridariaceae and their corresponding GenBank numbers of partial ITS, LSU, SSU, tef1, and rpb2 sequences. Isolates/sequences in bold were isolated/sequenced in the present study.

Species	Strain	GenBank Accession Numbers
		ITS	LSU	SSU	tef1	rpb2
Arthopyrenia salicis	CBS 368.94	KF443410	AY538339	AY538333	KF443404	KF443397
Chromolaenomyces appendiculatus	MFLUCC 17-1455	NR_168862	NG_068705	MT214394	MT235770	MT235806
Cycasicola goaensis	MFLUCC 17-0754	MG828885	MG829001	MG829112	MG829198	-
Cycasicola leucaenae	MFLUCC17-0914	NR_163322	MK347942	MK347833	MK360046	MK434900
Liua muriformis	KUMCC 18-0177	MK433599	MK433598	MK433595	MK426798	MK426799
Neoroussoella alishanense	AKW 11 FU31018	MK503818	MK503824	MK503830	MK336182	MN037757
Neoroussoella alishanense	AKW 03 FU31016	MK503816	MK503822	MK503828	MK336181	MN037756
Neoroussoella entadae	MFLUCC 18-0243	MK347786	MK348004	MK347893	MK360065	MK434866
Neoroussoella leucaenae	MFLUCC 18-1544	MK347767	MK347984	MK347874	MK360067	MK434876
Occultibambusa bambusae	MFLUCC 11-0394	KU940124	KU863113	-	KU940194	KU940171
Occultibambusa bambusae	MFLUCC 13-0855	KU940123	KU863112	KU872116	KU940193	KU940170
Ohleria modesta	MGC	KX650562	KX650562	-	KX650533	KX650582
Ohleria modesta	OM	KX650563	KX650563	KX650513	KX650534	KX650583
Parathyridaria clematidis	MFLUCC 17-2154	MT310645	MT214601	MT226712	MT394657	MT394712
Parathyridaria clematidis	MFLUCC 17-2157	MT310644	MT214600	MT226711	MT394656	MT394711
Parathyridaria clematidis	MFLUCC 17-2160	MT310643	MT214599	MT226710	MT394655	MT394710
Parathyridaria clematidis	MFLUCC 17-2185	MT310642	MT214598	NG_070668	MT394654	MT394709
Parathyridaria ellipsoidea	KNU-JJ-1829	LC552950	LC552952	-	-	-
Parathyridaria flabelliae	MUT 4886	KR014358	KP671720	KT587317	MN605910	MN605930
Parathyridaria flabelliae	MUT 4859	KR014355	KP671716	KT587315	MN605909	MN605929
Parathyridaria percutanea	CBS 128203	KF322117	KF366448	KF366450	KF407988	KF366453
Parathyridaria percutanea	CBS 868.95	KF322118	KF366449	KF366451	KF407987	KF366452
Parathyridaria philadelphi	CBS 143432	MH107905	NG_063958	-	MH108023	-
Parathyridaria ramulicola	MUT 4397	KC339235	KF636775	MN556311	MN605913	MN605933
Parathyridaria ramulicola	CBS 141479	NR_147657	KX650565	KX650514	KX650536	KX650584
Parathyridaria robiniae	MUT 2452	MG813183	MG816491	MN556312	MN605903	MN605923
Parathyridaria robiniae	MUT 4893	KM355998	MN556328	KM355993	MN605904	MN605924
Parathyridaria robiniae	MFLUCC 14-1119	KY511142	KY511141	-	KY549682	-
Parathyridaria rosae	MFLU 17-0623	NR_157530	NG_059873	-	-	-
Parathyridaria serratifoliae	MFLUCC 17-2210	MT310646	MT214602	NG_070669	MT394658	MT394713
Parathyridaria tyrrhenica	MUT 4966	KR014366	KP671740	KT587309	MN605911	MN605931
Parathyridaria tyrrhenica	MUT 5371	KU314951	MN556329	KU314952	MN605912	MN605932
Parathyridaria virginianae	MFLUCC 17-2163	MT310647	NG_073853	NG_070670	MT394659	MT394714
Parathyridariella dematiacea	MUT 4419	KC339245	KF636786	MN556313	MN605905	MN605925
Parathyridariella dematiacea	MUT 5310	KU255057	MN556330	MN556314	MN605907	MN605927
Parathyridariella dematiacea	MUT 5381	KU314959	MN556331	KU314960	MN605908	MN605928
Parathyridariella dematiacea	MUT 4884	MN556317	KP671726	KT587329	MN605906	MN605926
Pseudothyridariella chromolaenae	MFLUCC 17-1472	NR_168863	NG_068706	MT214395	MT235771	MT235807
Pseudothyridariella mahakoshae	NFCCI 4215	MG020435	MG020438	MG020441	MG023140	MG020446
Roussoella hysterioides	CBS 546.94	KF443405	KF443381	AY642528	KF443399	KF443392
Roussoella intermedia	CBS 170.96	KF443407	KF443382	KF443390	KF443398	KF443394
Roussoella japanensis	MAFF 239636	KJ474829	AB524621	AB524480	AB539114	AB539101
Roussoella margidorensis	MUT 5329	KU314944	MN556322	MN556309	MN605897	MN605917
Roussoella mediterranea	MUT 5306	KU255054	MN556323	MN556310	MN605898	MN605918
Roussoella padinae	MUT 5341	KU158153	MN556325	KU158176	MN605900	MN605920
Roussoella pustulans	KT 1709	KJ474830	AB524623	AB524482	AB539116	AB539103
Roussoella siamensis	MFLUCC 11-0149	KJ474837	KJ474845	KU872125	KJ474854	KJ474861
Thyridaria acaciae	CBS 138873	KP004469	KP004497	-	-	-
Thyridaria aureobrunnea	MFLUCC 21-0090	MZ538528	MZ538562	-	-	-
Thyridaria broussonetiae	TB	KX650567	KX650567	-	KX650538	KX650585
Thyridaria broussonetiae	TB1a	KX650569	KX650569	-	-	-
Thyridaria broussonetiae	TB2	KX650570	KX650570	-	KX650540	KX650587
Thyridaria broussonetiae	TB1	KX650568	KX650568	KX650515	KX650539	KX650586
Thyridaria jonahhulmei	KUMCC 21-0816	ON007041	ON007037	ON007046	ON009131	ON009135
Thyridaria jonahhulmei	KUMCC 21-0817	ON007042	ON007038	ON007047	ON009132	ON009136
Thyridariella mangrovei	NFCCI 4214	MG020436	MG020439	MG020442	MG020444	MG020447
Thyridariella mangrovei	NFCCI 4213	MG020434	MG020437	MG020440	MG020443	MG020445
Torula herbarum	CBS 111855	KF443409	KF443386	KF443391	KF443403	KF443396
Torula herbarum	CBS 595.96	KF443408	KF443385	KF443387	KF443402	KF443395

2.4.2. Phylogenetic Analyses

The single-gene data sets were examined for topological incongruence among loci for members of the analyses. The conflict-free single alignments for each locus were combined into a multi-locus dataset. Concatenated alignment was used to construct maximum-likelihood (ML) and Bayesian (BI) phylogenetic analyses. MrModeltest v. 2.3 [29] was used under the Akaike Information Criterion (AIC) implemented in PAUP v. 4.0b10 to determine the evolutionary models for Bayesian and maximum-likelihood analyses.

The CIPRES webportal [30] was used to execute RAxML [31] and Bayesian analyses [32]. RAxML-HPC2 on XSEDE v. 8.2.10 [30] was used with default parameters and 1000 bootstrap repetitions to construct the ML analysis.

MrBayes analyses were performed setting GTR+I+G as the evolutionary model, with 2 M generations, sampling every 1000 generations, ending the run automatically when standard deviations of split frequencies dropped below 0.01, and with a burnin fraction of 0.25. ML bootstrap values equal or greater than 70% and BYPP greater than 0.95 are given above each node of every tree.

FigTree v1.4.0 program [33] was used to visualize the phylogenetic trees and reorganized in Microsoft Power Point (2007). The finalized datasets and trees were submitted to TreeBASE, submission ID: 29569 (http://purl.org/phylo/treebase/phylows/study/TB2:S29569 (accessed on 17 March 2022)).

3. Results

3.1. Phylogenetic Analyses

Two analyses were performed in this study. The first is a phylogenetic overview of the genera treated in Didymosphaeriaceae (Figure 2), while the remaining alignment represents the genera in Thyridariaceae (Figure 3). Other details related to both ML and BI analyses from Didymosphaeriaceae and Thyridariaceae datasets are presented in Table 3.

Figure 2

RAxML phylogram generated from combined dataset of partial SSU, LSU, ITS, tef1, and rpb2 DNA sequence analyses for Didymosphaeriaceae. The tree is rooted to Bambusistroma didymosporum (MFLU 15-0057, MFLU 15-0058). Bootstrap supports ML (MLB) ≥ 70% and Bayesian posterior probabilities (BYPP) ≥ 0.95 are given as MLB/BYPP above the branches. Branches with an asterisk (*) indicate MLB = 100% and BYPP = 1.00. The newly generated isolates are in blue.

Figure 3

Phylogram generated from RAxML based on a concatenated sequence dataset of partial SSU, LSU, ITS, tef1, and rpb2 DNA for Thyridariaceae. Bootstrap values equal to or greater than 70% for ML (MLB) and Bayesian posterior probabilities (BYPP) ≥ 0.95 are shown at each node (as MLB/BYPP). An asterisk (*) represents branches with MLB = 100% and BYPP = 1.00. The new isolates are show in in blue.

Table 3

Maximum-likelihood (ML) and Bayesian (BI) analyses results for each sequenced dataset.

Analyses		Didymosphaeriaceae	Thyridariaceae
Number of taxa		88	59
Gene regions		SSU, LSU, ITS, tef1, and rpb2	SSU, LSU, ITS, tef1, and rpb2
Number of character positions (including gaps)		5016	4529
ML optimization likelihood value		−35,672.743881	−30,606.10565
Distinct alignment patterns in the matrix		2249	1796
Number of undetermined characters or gaps (%)		41.88%	19.56%
Estimated base frequencies	A	0.240418	0.249274
	C	0.253351	0.25578
	G	0.270784	0.267476
	T	0.235446	0.227469
Substitution rates	AC	1.561664	1.486771
	AG	3.248718	3.744601
	AT	1.433496	1.706836
	CG	1.323566	1.014483
	CT	7.428045	7.933665
	GT	1.0	1.0
Proportion of invariable sites (I)		0.396829	0.505108
Gamma distribution shape parameter (α)		0.454368	0.442817
Number of generated trees in BI		11,301	2501
Number of trees sampled in BI after 25% were discarded as burn-in		8476	1876
Final split frequency		0.009959	0.009966
The total of unique site patterns		2252	1798

3.2. Taxonomy

Luttr. ex M.E. Barr, Prodromus to class Loculoascomycetes: 67 (1987)

Munk, Dansk botanisk Arkiv 15 (2): 128 (1953)

Ariyaw. & K.D. Hyde, Fungal Diversity 68: 92 (2014)

Wanas. & Mortimer sp. nov. (Figure 4)

Figure 4

Neokalmusia jonahhulmei (HKAS122910, holotype). (a,b) Ascostromata on the bamboo culms; (c) horizontal section of ascostromata; (d,e) vertical sections of ascomata; (f) vertical section through two locules; (g) close-up of ostiole; (h) peridium; (i) pseudoparaphyses; (j–l) asci; (m–r) ascospores (r germinated ascospore); (s,t) colonies on PDA after 21 days. Scale bars, (d–f) 100 µm; (g) 50 µm; (h,m–r) 10 µm; (i) 5 µm; (j–l) 20 µm.

MycoBank: MB843400

Etymology: The epithet is derived from Jonah Hulme Mortimer, who is the collector of this fungus.

Holotype: HKAS122910

Saprobic on dead bamboo culms. Teleomorph: Ascomata 200–300 μm high × 1200–1500 µm diam. (M = 180 × 1400 µm, n = 5), scattered or in groups, immersed under a black clypeus-like structure, composed of host epidermis and fungal mycelium, hemispherical, dark brown to black, multi-loculate. Locules 100–170 μm high, 150–280 μm diam. (M = 137.6 × 217.8 µm, n = 5), immersed within ascostromata, dark brown to black, subglobose to ampulliform, ostiolate. Peridium 10–12 μm wide at the base, 15–30 μm wide at the sides, comprising several layers; outer layers dark brown to brown, with compressed cells of textura angularis; inner layers hyaline, with compressed pseudoparenchymatous cells, arranged in textura angularis. Hamathecium contains 2–3 μm wide, branched, septate, cellular pseudoparaphyses. Asci 65–85 × 10–15 μm (M = 72.8 × 11.8 μm, n = 15), eight-spored, bitunicate, cylindrical to clavate, curved, short pedicel with slightly furcate ends, apically rounded. Ascospores 15–17 × 6–7 μm (M = 15.5 × 6.4 µm, n = 30), bi-seriate, overlapping and are initially hyaline, turning brown at maturity, fusiform, three-transversely septate, slightly curved, constricted at the septa, conically rounded at the ends, and smooth-walled, guttulated, without a distinct mucilaginous sheath. Anamorph: undetermined.

Culture characteristics: colonies reaching 4 cm diameter on PDA after 2 weeks at 20 °C. Mycelium dense, circular, slightly raised, smooth on surface and undulated floccose edge. Colony grey at the centre and coffee brown near margin from the top and dark brown at the bottom. Hyphae septate, branched, hyaline, thin, and smooth-walled.

Known distribution: Yunnan, China, on dead woody litter.

Material examined: China, Yunnan, Kunming, Wuhua, 25.131198 N, 102.590770 E, 2080 m, on dead bamboo culms (Phyllostachys sp.), 20 March 2021, Jonah Hulme Mortimer, PEM03-6-2-1 (HKAS122910, holotype), ex-holotype culture, KUMCC 21-0818. ibid. 25.131178 N, 102.590749 E, PEM03-6-2-4 (HKAS122911), living culture, KUMCC 21-0819.

Notes: The new fungus was collected from dead bamboo culms in Kunming. It is characterized as a typical Neokalmusia taxon based on its immersed, hemispherical multi-loculate ascomata under a black clypeus-like structure, bitunicate, clavate, apically rounded asci and bi-seriate, fusiform, brown ascospores with transverse septa [34]. It has a close phylogenetic affinity to Neokalmusia brevispora (KT 2313, KT 1466) and N. kunmingensis (KUMCC 18-0120), with 100 MLB and 1.00 BYPP support values (Figure 2). Neokalmusia brevispora and N. kunmingensis are reported as saprobes from Japan on the Sasa sp. and on dead bamboo culms in China, respectively [34,35,36]. These three species are morphologically similar with few dimensional differences in asci and ascospores. Neokalmusia brevispora has comparatively larger asci and ascospores (80–118 × 10.5–15 μm; 18–26.5 × 4–7 μm [34]) than N. kunmingensis (63–77 × 9.6–11.4 μm; 13–15 × 4–5 μm [36]) and our new collection (65–85 × 10–15 μm; 15–17 × 6–7 μm, this study). Further comparison of the ITS regions reveals Neokalmusia brevispora and N. kunmingensis feature nucleotide differences from N. jonahhulmei of 66/695 (9.5%) and 55/560 (9.8%), respectively. Comparison of the tef1 nucleotides of Neokalmusia brevispora and N. kunmingensis with our new strains revealed nucleotide differences of 31/917 (3.4%) and 26/904 (2.9%), respectively. The rpb2 region is not available for Neokalmusia brevispora and N. kunmingensis for comparison with our new species. This is the first time rpb2 sequence data for a Neokalmusia species have been provided (Table 1).

Ariyawansa et al. [34] established Neokalmusia to accommodate N. brevispora and N. scabrispora, which are characterized by immersed and subglobose to oblong ascomata with multiple perithecia, thin-walled clypeus-like structure, and verrucose ascospores. These two were found on dead culms of Sasa and Phyllostachys species in Japan, respectively [34,35]. Subsequent studies added four other species viz. N. arundinis (Italy) [37], N. didymospora (Thailand) [38], N kunmingensis (China) [36], and N. thailandica (Thailand) [37]. In this study, we introduce the seventh species in the genus from Phyllostachys species in China. All specimens of these species were obtained from dead culms of Bambusoideae (Poaceae) in China, Italy, Japan, and Thailand; therefore, Neokalmusia appear to be saprobic on bambusicolous hosts in temperate to tropical environments.

Q. Tian & K.D. Hyde, Fungal Diversity 63 (1): 254 (2013)

Sacc., Grevillea 4 (29): 21 (1875)

Wanas. & Mortimer sp. nov. (Figure 5 and Figure 6)

Figure 5

Sexual morph of Thyridaria jonahhulmei (HKAS122912, holotype). (a,b) Ascomata on the dead woody twigs; (c,d) cross section of ascomata; (e) close-up of ostiole; (f) peridium; (g) pseudoparaphyses; (h–j) asci; (k–p) ascospores. Scale bars, (d) 200 µm; (e) 50 µm; (f,h–j) 20 µm; (f,k–p) 10 µm.

Figure 6

Asexual morph of Thyridaria jonahhulmei (KUMCC 21-081, ex-type culture). (a) Germinated ascospore (b–d) colony on PDA (c from the bottom); (e–g) immersed pycnidia in PDA; (h) squashed conidiomata; (i,j) conidiogenous cells; (k) conidia. Scale bars, (h) 100 µm; (i,j) 5 µm; (k) 10 µm.

MycoBank: MB843401

Etymology: The epithet is derived from Jonah Hulme Mortimer, who is the collector of this fungus.

Holotype: HKAS122912

Saprobic on dead twigs of Fagaceae sp. Teleomorph: Ascomata 550–750 μm high, 200–300 μm diam. (M = 679.3 × 256.4 µm, n = 5), solitary or gregarious, immersed, coriaceous, heavily pigmented, globose to subglobose, ostiolate, yellow to reddish- or orange-brown pruinose layer forming around the apices of the ostiolar necks. Ostiole 100–150 μm long, 40–70 μm diam. (M = 121.1 × 57.8 µm, n = 5), central papillate, comprising hyaline periphyses. Peridium 15–25 μm wide, wider at the apex (60–70 μm), composed with two layers, with outer stratum comprising pale brown to brown, compressed, thick-walled cells of textura angularis, fused with the host tissues, and inner stratum multi-layered and composed with lightly pigmented to hyaline cells of textura angularis. Hamathecium comprised of 2–2.5 μm wide, branched, septate, cellular pseudoparaphyses, situated between and above the asci, embedded in a gelatinous matrix. Asci 150–200 × 18–22 μm (M = 170.2 × 20.1 μm, n = 25), eight-spored, bitunicate, fissitunicate, cylindrical to cylindric-clavate, long pedicellate (30–60 μm), and apex rounded with an ocular chamber. Ascospores 25–35 × 8–12.5 μm (M = 29.3 × 9.7 µm, n = 30), one-to-two-seriate, overlapping, and pale or yellowish brown when young, turning yellowish brown to brown at maturity, narrowly ellipsoid to fusoid, ends narrowly rounded (sometimes pointed), straight or curved, two-to-three-transversely septate, with median euseptum, slightly constricted at the septa, containing several guttules, with surface finely punctate to verruculose. Anamorph: Coelomycetous. Conidiomata (1–1.5 mm diam.), pycnidial, scattered or grouped, immersed and heavily pigmented. Pycnidial wall comprised with several strata, with brown-walled pseudoparenchymatous cells at outer margin becoming hyaline and thin-walled towards the inner conidiogenous cell-layer. Conidiogenous cells phoma-like, phialidic, ampulliform to doliiform, hyaline, flexuous, and smooth, with a short collarette. Conidia 5.5–8 × 2.5–3.5 μm (M = 6.9 × 3.1 μm, n = 50), first hyaline, turning pale brown, one-celled, straight or curved, ellipsoidal, rounded at both ends, thin and smooth-walled, comprising numerous guttules.

Culture characteristics: Colonies spreading on PDA up-to 4 cm diameter after 2 weeks at 20 °C, circular, whitish at the beginning, and becoming slightly raised and greenish-grey after 4 weeks, reverse dark brown. Hyphae septate, branched, hyaline, thin, smooth-walled, producing conidia after six weeks.

Known distribution: Yunnan, China, on dead woody litter.

Material examined: Material examined: China, Yunnan, Kunming, Wuhua, 25.131178 N, 102.590726 E, 2080 m, on dead twigs of woody litter of Fagaceae sp., 30 January 2021, Jonah Hulme Mortimer, PM03-2-1 (HKAS122912, holotype), ex-holotype culture, KUMCC 21-0816. ibid. 25.131157 N, 102.590708 E, 20 March 2021, PM03-2-3 (HKAS122913), living culture, KUMCC 21-0817.

Notes: Based on multi-gene sequence analyses (SSU, LSU, ITS, tef1 and rpb2), isolates KUMCC 21-0816 and KUMCC 21-0817 cluster with Thyridaria acaciae (CBS 138873), T. aureobrunnea (MFLUCC 21-0090) and T. broussonetiae (TB, TB1a, TB1, TB2) with 100 MLB and 1.00 BYPP bootstrap support values (Figure 3). Thyridaria acaciae is reported from Tanzania (on Acacia tortilis) [39], T. aureobrunnea from decaying wood in Thailand [40], and T. broussonetiae from Croatia (Hippocrepis emerus), Hungary (Amorpha fruticosa), and Italy (Broussonetia papyrifera) [41], whereas our new isolate is from dead woody twigs in Yunnan, China. Morphologically Thyridaria jonahhulmei has a close affinity to T. aureobrunnea and T. broussonetiae in its ascomata, asci, and ascospore characteristics. The asci dimensions of Thyridaria jonahhulmei (150–200 × 18–22 μm) are comparatively larger than T. aureobrunnea (45–61.5 × 7–8.5 µm) and are not significantly different from T. broussonetiae (109–183 × 12–19 µm). Comparison of the 546 ITS (+5.8S) nucleotides reveals 43 (7.9%) nucleotide differences between T. aureobrunnea and T. jonahhulmei. Thyridaria aureobrunnea lacks tef1 and rpb2 gene regions for comparison. Comparison of the 514 ITS (+5.8S) nucleotides of Thyridaria broussonetiae and T. jonahhulmei reveals 25 (4.86%) nucleotide differences, and in tef1 and rpb2 there are 8/712 (1.12%) and 18/1025 (1.75%) nucleotide differences, respectively. Thyridaria acaciae is known only from its asexual morph, and therefore it is not possible to compare their sexual morphologies. However, the asexual morph of Thyridaria jonahhulmei is similar to Thyridaria acacia. They both have ampulliform to doliiform conidiogenous cells and pale brown, aseptate conidia. Thyridaria acacia has subcylindrical conidia that lack guttules, whereas Thyridaria jonahhulmei has ellipsoidal conidia with large guttules. Comparison of the 469 ITS (+5.8S) nucleotides of these two strains reveals 23 (4.9%) nucleotide differences while tef1 and rpb2 gene regions were unavailable for comparison. Therefore, we recognize these isolates belong to two distinct species [42].

Excluding Thyridaria acaciae, T. aureobrunnea and T. broussonetiae, our new species resembles T. eminens (30 × 12 µm [43]), T. koae (14–21 × 6–9 µm [44]), T. minor (15–18 × 5–6.5 µm [45]), T. sambucina (12–15.5 µm [46]), T. subrufa (12–15 × 4–5 µm [46]), and T. triseptata (15–18 µm [47]) with its three-septate pigmented ascospores. Among them, Thyridaria eminens (collected from dead Streblus asper in Philippines [43]) is morphologically closely affiliated with T. jonahhulmei by ascospore dimensions. However, all of these species lack molecular data for further phylogenetic characterization.

4. Discussion

In this study, we describe and illustrate two new species of microfungi on dead woody litter, Neokalmusia jonahhulmei (Didymosphaeriaceae) and Thyridaria jonahhulmei (Thyridariaceae), from Kunming, Yunnan, based on morphological and molecular analyses (Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5). Neokalmusia jonahhulmei is introduced with only its sexual characteristics, whereas Thyridaria jonahhulmei is accounted for with both asexual and sexual morphological features.

Didymosphaeriaceae was introduced by Munk [48], and, given that the family is composed of 33 genera, it is considered one of the most specious families in the order Pleosporales [23,25]. Members of Didymosphaeriaceae are known to form numerous different types of life modes, including saprobes, pathogens, or endophytes, and can be found both on land or in water [23,49]. We have presented representative sequence data of all currently available genera listed in Hongsanan et al. [23] and Samarakoon et al. [25] for the phylogenetic analyses (except Barria, Curreya, Julella, and Lineostroma, for which no DNA-based sequence data were available). Additionally, we included the sequences of Pseudodidymocyrtis lobariellae that were introduced by Flakus et al. [50] as a lichenicolous fungus from Bolivia on Lobariella pallida. Multi-gene phylogenetic analyses (Figure 2) revealed that Pseudodidymocyrtis clusters with Kalmusia species, and from the morphological perspective, these two genera appear to have a close resemblance. Therefore, generic delimitation needs defining among these two genera, possibly by using more fresh collections with additional morpho–phylo data.

Even though Thyridaria is one of the oldest genera (introduced in 1875) in Ascomycota, no exact family was available to accommodate this genus [51]. More than 30 species are included in Thyridaria [52]; however, for many of these species, data are lacking illustrations, descriptions, or DNA-based molecular data, leading to ambiguous taxonomic relationships. Therefore, Thyridaria species have suffered from uncertain family placement and have been assigned to different families in Dothideomycetes at various times [41,53,54,55,56,57,58,59,60,61]. Hyde et al. [51] considered its unique morphology and the phylogenetic placement of Thyridaria rubronotata in the Dothideomycetes backbone tree and introduced Thyridariaceae to accommodate this genus. Recently, Jaklitsch and Voglmayr [41] studied several thyridaria-like genera and constructed a multi-gene analysis to clarify intergeneric taxonomic affinities of Thyridaria in the Pleosporales. They found that thyridaria-like genera are distributed among at least nine clades of the Pleosporales. Barr [61] thoroughly reviewed various descriptions of Thyridaria and suggested that a key characteristic of the genus is an ample subiculum surrounding fruiting bodies that are produced under the ascomata wall or in host tissues. Similarly, the intensive study of Jaklitsch and Voglmayr [41] pointed out that this feature is exclusive to delineate Thyridaria species from its phylogenetically closely related affiliates. We also observed that our new Thyridaria species was characterized with a pruinose layer of yellow to reddish- or orange-brown material forming around the apices of the ostiolar necks. The subiculum of Thyridaria species could be an adaptation for its terrestrial-based habitat, and their potential advantages should be further clarified with extensive sampling from both aquatic and non-aquatic habitats.

By contrast, wood-decaying Basidiomycota in tropical China are well studied, which has facilitated a better understanding of global Basidiomycota species diversity and systematics [62,63,64,65,66,67]. Nonetheless, the study of habitat properties and potential lifestyles of woody-based microfungal species is especially important to understanding their functional aspects. The trophic mode of many species can be highly variable, with the ability to switch between mutualistic, pathogenic, and saprotrophic strategies. Pathogenic taxa that cause leaf spots may begin as endophytes, but environmental stressors may necessitate becoming pathogenic or eventually saprotrophic after plant tissue [68,69]. Another example is Scedosporiosis, the human disease caused by Pseudallescheria/Scedosporium complex species (PSC), which can grow saprobically in wood [70,71]. Therefore, owing to their importance in all ecosystems, saprotrophic fungi simply cannot be overlooked in any region.

This study provides some insights into the saprotrophic taxa and contributes knowledge of microfungi associated with woody litter in Yunnan, China by introducing two novel species. Furthermore, these species were discovered by a 5-year-old boy, unassisted by any mycologists at the time of collection, thus highlighting how young enthusiasts can be involved in field studies and make a significant impact.