Morphological and Phylogenetic Analyses Reveal Five New Species in Chaetosphaeriaceae.

Chaetosphaeriaceae is a genera-rich and highly diverse group of fungi with a worldwide distribution in terrestrial and aquatic habitats. Eight fresh collections of Chaetosphaeriaceae were obtained during investigations of hyaline-spored hyphomycetes in China and Thailand. Based on morphological characteristics and phylogenetic analysis of a combined LSU and ITS sequence dataset, Chaetosphaeria obovoidea, Codinaea aseptata, Codinaeella hyalina, Dictyochaeta guizhouensis and Paragaeumannomyces guttulatus were introduced as new species, Codinaea terminalis was reported as new host record, and Codinaea dwaya and Phialosporostilbe scutiformis were introduced as new collections. Phylogenetic analysis in this study revealed that Chaetosphaeria was polyphyletic. Detailed descriptions and illustrations of new taxa and identified species are provided, as well as an updated phylogenetic tree to confirm the placements of these eight new collections.

1. Introduction

Reblova et al. [1] validly established the Chaetosphaeriaceae to accommodate type species Chaetosphaeria Tul. and C. Tul. and their relatives, including Ascocodinaea, Melanochaeta, Melanopsammella, Porosphaerella, Porosphaerellopsis and Striatosphaeria. Subsequently, more new taxa were added to this family [2,3,4,5,6,7,8,9,10]. Reviews of Chaetosphaeriaceae were provided by Lin et al. [9] and Hyde et al. [11]. Forty-three genera were accepted in Chaetosphaeriaceae by Hyde et al. [11]. In recent studies, Zheng et al. [12] included a new genus Phialolunulospora in the family. Réblová et al. [13,14] introduced six new genera namely Arcuatospora, Achrochaeta, Ericiosphaeria, Flectospora, Phialoturbella and Tubulicolla. The lineages of “Catenularia”-like taxa were recognized as a genus Fuscocatenula by Réblová et al. [15]. Five lineages, Codinaeella, Nimesporella, Stilbochaeta, Tainosphaeriella and Xyladelphia, were proposed for “Codinaea”-like fungi by Réblová et al. [16]. Meanwhile, the key to asexual genera of Chaetosphaeriaceae was provided by Maharachchikumbura et al. [17].

Chaetosphaeria (Chaetosphaeriaceae, Chaetosphaeriales), was established by Tulasne and Tulasne [18] with the type species C. innumera Berk. and Broome ex Tul. and C. Tul. It has simple and homogeneous sexual morphs, and complex and diverse asexual morphs [3,4,5,9,10,11,19,20]. Its members have been shown to link with several asexual genera in Chaetosphaeriaceae, including Chloridium, Codinaea, Dictyochaeta and Menispora [9,21,22]. Following the “One Fungus One Name” (1F1N) principle, some Chaetosphaeria species were synonymized under respective anamorphic genera, the latter being older and prioritized, i.e., Cacumisporium, Catenularia, Chloridium, Codinaea, Dictyochaeta, Exserohilum, Gonytrichum and Menispor [4,13,14,15,23,24].

Codinaea was established by Maire [25] and typified by C. aristata Maire. Gamundi et al. (1977) synonymized Codinaea under Dictyochaeta Speg. based on a misidentification of D. fuegiana Speg., the type species of Dictyochaeta. Based on the morphological reexamination of D. fuegiana, Réblová [21] argued that Codinaea and Dictyochaeta were separate genera in Chaetosphaeriaceae. She proposed that Codinaea species have conidia with setulae, while members of Dictyochaeta do not. This proposal was accepted in later studies [11,14,26,27,28,29].

Codinaeella is established by Réblová et al. [16] for “Codinaea”-like fungi based on molecular data and morphological characters of setae and conidiophores, which is typified by Cod. minuta (Tubaki) Réblová and Hern.-Restr. Codinaeella species have a saprobic life mode on various plants and soil [16,30,31]. Réblová et al. [16] accepted eight species in Codinaeella based on molecular data and morphological evidence, and provided a synopsis of accepted species in Codinaeella [16,30,32].

Dictyochaeta was introduced by Spegazzini [32] and typified by D. fuegian. The genus has been considered polyphyletic and phylogenetically unresolved due to its long-term broad delimitation [3,8,9,29,33]. Later, Réblová et al. [14] indicated that Dictyochaeta sensu stricto including D. callimorpha, D. detriticola, D. fuegiana (type species), D. montana, D. querna and D. stratosa formed a monophyletic clade in Chaetosphaeriaceae based on combined ITS and LSU sequence analysis. Furthermore, Réblová et al. [14] reevaluated a generic concept of Dictyochaeta based on multi-loci analyses coupled with morphological comparison and culture characteristics. The keys to the accepted Dictyochaeta species were provided by Kuthubutheen and Nawawi [33] and Réblová et al. [14].

Paragaeumannomyces was typified by P. sphaerocellularis Matsush [34]. Paragaeumannomyces in Chaetosphaeriaceae was confirmed as a monophyletic clade based on the phylogenetic analyses of combined ITS and LSU sequence data [29]. The key to the 16 accepted Paragaeumannomyces species was provided by Réblová et al. [29]. Furthermore, Réblová et al. [13] introduced the new combination Paragaeumannomyces hispidus (Réblová and Seifert) Réblová and Hern.-Restr. (basionym: Chaetosphaeria hispida Réblová and Seifert) based on characteristics of a three-layered ascomatal wall. It is difficult to distinguish Paragaeumannomyces from the similar genus Ericiosphaeria morphologically, as they share semblable morphology in asci, ascospores and setae. However, the two genera can be distinguished based on a number of layers and texture of the ascomatal wall as well as molecular data [6,13,29].

The hyphomycetous genus Phialosporostilbe was verified by Sierra and Portales [35] to accommodate P. turbinate Mercado and J. Mena. The placement of Phialosporostilbe in Chaetosphaeriaceae was confirmed by Yang et al. [36], based on morphological and phylogenetic analyses of the combined ITS and LSU sequence data. The genus is characterized by synnematous conidiophores, monophialidic conidiogenous cells and hyaline, turbinate or cordiform conidia with subapical or apical appendages [36,37,38]. Phialosporostilbe can be distinguished from the similar genus Nawawia by its synnematous conidiophores [35,36,37]. Index Fungorum (May 2022) listed seven epithets under Phialosporostilbe, of which only one species has molecular data available.

In this study, eight chaetosphaeriaceous hyphomycetes were obtained from freshwater and terrestrial habitats in China and Thailand, and eventually identified five novel species (Chaetosphaeria obovoidea, Codinaea aseptata, Codinaeella hyalina, Dictyochaeta guizhouensis and Paragaeumannomyces guttulatus), a new host record Codinaea terminalis and two new collections of Codinaea dwaya and Phialosporostilbe scutiformis. These new isolated data are provided with details (Table 1). Morphological evidence and phylogenetic analysis based on a combined LSU and ITS sequence dataset confirmed the identity of these new collections, providing further evidence on the phylogenetic placement of these taxa in Chaetosphaeriaceae.

Table 1

Taxa, isolate information and GenBank accession numbers for new collections determined for this study.

Taxon	Specimen	Status	Country	Host	Habitat	GenBank Accessions
						ITS	LSU
Chaetosphaeria obovoidea	HKAS 123765	H	China	Decaying wood	F	ON502901	ON502894
Codinaea aseptata	HKAS 123758	H	China	Decaying wood	F	ON502897	ON502890
Codinaea dwaya	GZAAS 22-0071		China	Decaying wood	F	ON502896	ON502889
Codinaea terminalis	HKAS 123756		China	Dead bamboo culms	F	ON502902	ON502895
Codinaeella hyalina	HKAS 123757	H	China	Decaying wood	F	ON502898	ON502891
Dictyochaeta guizhouensis	HKAS 123753	H	China	Decaying wood	T	ON502899	ON502892
Paragaeumannomyces guttulatus	HKAS 123755	H	China	Dead bamboo culms	F	ON502900	ON502893
Phialosporostilbe scutiformis	MFLU 22-0077		Thailand	Decaying wood	F	ON678180	ON678145

Note: status: H denotes holotype; habitat: F denotes freshwater habitat and T denotes terrestrial habitat.

2. Materials and Methods

2.1. Collection, Isolation and Conservation

Samples of submerged decaying twigs and dead bamboo culms were collected from Chiang Rai Province, Thailand, and Guizhou Province, China. They were packed into plastic bags for transportation to the laboratory, and associated metadata (i.e., date, locality and host) were noted. Fungal colonies on the host surface were examined and observed using a stereomicroscope (Leica EZ4 Microsystems (Schweiz) AG, Singapore). Photomicrographs of micro-morphological characteristics were documented using a Nikon DS-Ri2 digital camera fitted to a Nikon ECLIPSE Ni compound microscope (Nikon, Japan). Measurements were made using the Tarosoft (R) Image Frame Work, and these images used for figures were processed and combined with Adobe Illustrator CS6 (Adobe Systems, San Jose, CA, USA).

Single-spore isolations were made on water agar (WA) and potato dextrose agar (PDA), and germinating conidia were transferred to fresh malt extract agar (MEA) and PDA following the method of Chomnunti et al. [39]. Dried materials were deposited in the herbaria of Mae Fah Luang University (Herb. MFLU), Chiang Rai, Thailand, Herbarium of Cryptogams, Kunming Institute of Botany, Academia Sinica (HKAS), Kunming, China, and Herbarium of Guizhou Academy of Agricultural Sciences (GZAAS), Guiyang, China. Cultures were deposited at Mae Fah Luang University Culture Collection (MFLUCC), Chiang Rai, Thailand, and Guizhou Culture Collection, China (GZCC). Faces of Fungi and Index Fungorum numbers were registered according to the guidelines in Jayasiri et al. [40] and Index Fungorum (2022).

2.2. DNA Extraction, PCR Amplification and Sequencing

Pure cultures were grown on MEA/PDA media at 25 °C for one month. Fresh fungal mycelia were scraped off from the surface of the cultures and transferred to 1.5 mL microcentrifuge tubes. Meanwhile, fungal genomic DNA was extracted with the Biospin Fungus Genomic DNA Extraction Kit (Biospin Fungus Genomic DNA Extraction Kit, BioFlux®, Shanghai, China) following the manufacturer’s instructions. LR0R and LR5 (Vilgalys and Hester 1990) and ITS5 and ITS4 (White et al. 1990) primers were used to amplify the large subunit of the ribosomal DNA (LSU) and the internal transcribed spacer (ITS) gene regions. The amplification reactions were performed in a 50 μL reaction volume, which contained 2 μL of DNA template, 2 μL of each forward and reverse primer (10 μM), 25 μL of 2× Taq PCR Master Mix with blue dye (Sangon Biotech, Shanghai, China) and 19 μL of distilled–deionized water. The following thermo-cycling parameters were used for the LSU and ITS region: initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 56 °C for 50 s, elongation at 72 °C for 1 min and a final extension period for 10 min at 72 °C. The quality of the PCR products was checked on a 1% agarose gel electrophoresis stained with ethidium bromide. Purification and sequencing of PCR products were performed at Sangon Biotech (Shanghai, China) using the same primers.

2.3. Alignments and Phylogenetic Analysis

Original sequences were verified using BioEdit v. 7.1.3.0 [41], and were assembled using SeqMan v. 7.0.0 (DNASTAR, Madison, WI, USA). Consensus sequences were submitted to the NCBI GenBank (Table 1). The new sequences were subjected to BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 1 May 2022) for preliminary determination of the possible species identification range in the GenBank database. Sequences of the ITS and LSU gene were analyzed to assess relationships among species of Chaetosphaeria, Codinaea, Codinaeella, Dictyochaeta, Paragaeumannomyces, Phialosporostilbe and relevant fungi in Chaetosphaeriaceae [6,9,10,12,14,16,20,29,36,42]. Accession numbers for sequences were retrieved from GenBank database and related publication from previous studies (Table 2 and Table S1). The alignments for sequences of each locus were performed with the online multiple alignment program MAFFT version 7 (https://mafft.cbrc.jp/alignment/server/, accessed on 3 May 2022) and then manually verified in BioEdit 7.1.3.0 [43]. The maximum likelihood (ML) and Bayesian inference (BI) analyses inferred phylogenetic relationships, based on the concatenated sequence matrix of a combined LSU and ITS dataset.

Table 2

Taxa used in this study and their GenBank accession numbers.

Taxon	Strain		GenBank Accessions
		Status	ITS	LSU
Achrochaeta talbotii	ICMP 15161		MT454480	MT454495
Adautomilanezia caesalpiniae	CC-LAMIC 102/12	T	KX821777	KU170671
Anacacumisporium appendiculatum	HMAS 245593	T	KP347129	KT001553
Arcuatospora nouae zelandiae	CBS 109474	T	MW984569	MW984552
Arcuatospora seorsa	CBS 147510	T	MW984572	MW984555
Brunneodinemasporium brasiliense	CBS 112007	T	JQ889272	JQ889288
Brunneodinemasporium jonesii	GZCC 16-0050	T	KY026058	KY026055
Calvolachnella guaviyunis	CBS 134695	T	KJ834524	KJ834525
Catenularia minor	PRM 900544	T	MW987827	MW987822
Catenularia cubensis	SMH 3258		MW987826	N/A
Chaetosphaeria acutata	CBS 101312		AF178553	AF178553
Chaetosphaeria biapiculata	SMH 3074		N/A	AF466065
Chaetosphaeria caesariata	SMH 2794		N/A	AF466060
Chaetosphaeria chalaroides	SMH 2018		N/A	AY017372
Chaetosphaeria chlorotunicata	SMH 1565	T	N/A	AF466064
Chaetosphaeria conirostris	SMH 2183		N/A	AF466066
Chaetosphaeria curvispora	CBS 125555		MH863562	MH875040
Chaetosphaeria cylindrospora	SMH 3568	T	N/A	AY017373
Chaetosphaeria decastyla	SMH 2629		N/A	AF466068
Chaetosphaeria dilabens	CBS 712 88		AF178557	AF178557
Chaetosphaeria fennica	CBS 101641		AF178562	AF178562
Chaetosphaeria fusiformis	CBS 101429		AF178554	AF178554
Chaetosphaeria hebetiseta	CBS 102340	T	AF178549	AF178549
Chaetosphaeria innumera	M R 1175		AF178551	AF178551
Chaetosphaeria jonesii	MFLUCC 15-1015	T	NR_154841	KY212761
Chaetosphaeria lateriphiala	SMH 2629		N/A	AF466070
Chaetosphaeria lentomita	MR 1265		AF178548	AF178548
Chaetosphaeria lignomollis	SMH 3015	T	EU037896	AF466073
Chaetosphaeria luquillensis	SMH 2973		N/A	AF466074
Chaetosphaeria mangrovei	MCD 069	T	MG813821	MG813820
Chaetosphaeria metallicans	PDD 92539	T	NR_119668	NG_058757
Chaetosphaeria minuta	SMH 3396		N/A	AF466075
Chaetosphaeria myriocarpa	CBS 264.76		AF178552	AF178552
Chaetosphaeria preussii	CBS 262.76		AF178561	AF178561
Chaetosphaeria pygmaea	MR 1365		AF178545	AF178545
Chaetosphaeria rivularia	CBS 127686	T	KR347356	KR347357
Chaetosphaeria sp. JEH 2019	JAUCC 2961		MN619651	MN607223
Chaetosphaeria spinosa	SMH 2754		N/A	AF466079
Chaetosphaeria sylvatica	SMH 2893		N/A	AF279419
Chaetosphaeria tropicalis	SMH 1267		N/A	AF279418
Chalarodes obpyramidata	PDD 119364		MW987828	MW987823
Chloridium caesium	CBS 102339		AF178564	AF178564
Chloridium gonytrichii	CBS 195.60		MH857954	MH869503
Chloridium virescens	CBS 152.53		MH857142	MH868678
Codinaea acaciae	OTU5		KY965397	N/A
Codinaea amazonensis	MUCL 41171		OL654076	OL654133
Codinaea assamica	CBS 242.66		MH858788	MH870426
Codinaea assamica	CBS 139907	T	OL654077	OL654134
Codinaea dwaya	CBS 261.77	T	OL654078	OL654135
Codinaea ellipsoidea	MFLUCC 18-1574	T	MK828628	MK835828
Codinaea fertilis	CBS 242.66		OL654079	OL654136
Codinaea gonytrichodes	CBS 593.93		AF178556	AF178556
Codinaea lignicola	DLUCC 0899	T	MK828630	MK835830
Codinaea pandanicola	KUMCC 16-0153	T	MH388338	MH376710
Codinaea paniculata	CBS 145098	T	MT118230	MT118201
Codinaea phasma	CBS 147516	T	OL654081	OL654138
Codinaea siamensis	CBS 194.96		OL654082	OL654139
Codinaea siamensis	MFLUCC 15-0614	T	KX609955	KX609952
Codinaea terminalis	GZCC19-0525		MW133883	N/A
Codinaea terminalis	GZCC 18-0085	T	MN104613	MN104624
Codinaeella filamentosa	CBS 147265		OL654083	OL654140
Codinaeella lambertiae	CBS 143419	T	OL654084	OL654141
Codinaeella lutea	CBS 146618	T	OL654085	OL654142
Codinaeella mimusopis	CBS 143435	T	MH107888	MH107935
Codinaeella minuta	CBS 298.61	T	OL654090	OL654147
Codinaeella minuta	CBS 966.69		AF178559	AF178559
Codinaeella minuta	417E		MZ078594	N/A
Codinaeella parvilobata	CBS 144536	T	OL654100	OL654157
Codinaeella pini	CBS 138866	T	KP004465	KP004493
Codinaeella yunnanensis	MFLUCC 17-0468		NR_168795	NG_068630
Conicomyces pseudotransvaalensis	HHUF 29956	T	LC001710	LC001708
Cryptophiale udagawae	GZCC 18-0047		MN104608	MN104619
Dendrophoma cytisporoides	CBS 144107		MT118234	MT118205
Dictyochaeta callimorpha	ICMP 15130		MT454483	MT454498
Dictyochaeta detriticola	ICMP 14948	T	MT454486	MT454501
Dictyochaeta fuegiana	ICMP 15153	T	MT454487	EF063574
Dictyochaeta montana	CBS 145342	T	MT454488	MT454502
Dictyochaeta querna	CBS 146103		MT454490	MT454504
Dictyochaeta sp	CBS 138684		MT454493	MT454507
Dictyochaeta stratosa	CBS 138739		NR 172308	MT454505
Dinemasporium americanum	CBS 127127	T	JQ889274	JQ889290
Dinemasporium pseudoindicum	CBS 127402	T	JQ889277	JQ889293
Ellisembia aurea	CBS 144403	T	MH836375	MH836376
Ellisembia folliculata	CBS 147152		OL654105	OL654162
Eucalyptostroma eucalypti	CBS 142074	T	KY173408	KY173500
Flectospora laminata	CBS 112964		MW984576	MW984558
Flectospora sp.	ICMP 23840		MW984577	MW984559
Infundibulomyces cupulatus	BCC 11929	T	EF113976	EF113979
Infundibulomyces oblongisporus	BCC 13400	T	EF113977	EF113980
Kionochaeta castaneae	GZCC 18-0025	T	MN104610	MN104621
Kionochaeta microspora	GZCC 18-0036	T	MN104607	MN104618
Leptosporella arengae	MFLUCC 15-0330	T	MG272255	MG272246
Leptosporella bambusae	MFLUCC 12-0846	T	KU940134	KU863122
Menispora ciliata	CBS 122131	T	EU488736	OL654165
Menispora tortuosa	CBS 117552		OL654110	OL654168
Menisporopsis dushanensis	GZCC 18-0084	T	MN104615	MN104626
Menisporopsis theobromae	MFLUCC 15 0055		KX609957	KX609954
Multiguttulispora dimorpha	CBS 140002		MW984582	MW984564
Multiguttulispora triseptata	IMI 353690		MW984584	MW984566
Nawawia filiformis	MFLUCC 17-2394		MH758196	MH758209
Neopseudolachnella acutispora	MAFF 244358	T	AB934065	AB934041
Neopseudolachnella magnispora	MAFF 244359	T	AB934066	AB934042
Nimesporella capillacea	IMI 358908	T	OL654114	OL654171
Paliphora intermedia	CBS 896 97		MH862682	EF204501
Paragaeumannomyces abietinus	CBS 145351	T	MT118235	MT118206
Paragaeumannomyces albidus	PDD 92537	T	EU037890	EU037898
Paragaeumannomyces bombycinus	PDD 92538	T	EU037892	N/A
Paragaeumannomyces elegans	PDD 118740	T	MT876580	N/A
Paragaeumannomyces garethjonesii	MFLUCC 15-1012	T	KY212751	KY212759
Paragaeumannomyces granulatus	ICMP 15133	T	MT876575	MT876577
Paragaeumannomyces lapazianus	SMH 2182		AY906945	MT118207
Paragaeumannomyces longisporus	ANM 1269		MT118239	MT118210
Paragaeumannomyces panamensis	SMH 3596	T	AY906948	MT118218
Paragaeumannomyces raciborskii	SMH 3119		AY906953	AY436402
Paragaeumannomyces rubicundus	SMH 3221	T	MT118242	MT118224
Paragaeumannomyces sabinianus	ILLS00121384	T	MT118243	MT118225
Paragaeumannomyces smokiensis	ILLS00121398	T	MT118240	MT118228
Phialosporostilbe scutiformis	MFLUCC 17-0227	T	MH758194	MH758207
Phialosporostilbe scutiformis	MFLUCC 18-1288		MH758199	MH758212
Phialoturbella calva	ICMP 23826	T	MW984585	MW984567
Phialoturbella lunata	MFLUCC 18-0642	T	NR 168796	MK835824
Polynema podocarpi	CBS 144415	T	MH327797	MH327833
Pseudodinemasporium fabiforme	CBS 140010	T	KR611889	KR611906
Pseudolachnea fraxini	CBS 113701	T	JQ889287	JQ889301
Pseudolachnea hispidula	MAFF 244365		AB934072	AB934048
Pseudolachnella asymmetrica	MAFF 244366		AB934073	AB934049
Pseudolachnella scolecospora	MAFF 244379		AB934086	AB934062
Pyrigemmula aurantiaca	CBS 126743	T	HM241692	HM241692
Rattania setulifer	GUFCC 15501		GU191794	HM171322
Sporoschisma longicatenatum	MFLUCC 16-0180	T	KX505871	KX358077
Sporoschisma mirabile	FMR 11247		HF677174	HF677183
Stilbochaeta aquatica	MFLUCC 15-0983	T	NR 158452	MH476569
Stilbochaeta brevisetula	ICMP 22549		OL654118	OL654175
Stilbochaeta cangshanensis	MFLUCC 17-2214	T	MK828632	MK835832
Stilbochaeta malaysiana	IMI 312436	T	OL654121	OL654178
Striatosphaeria castanea	CBS 145352	T	MT118244	MT118229
Striatosphaeria codinaeophora	SMH 1524		MT118245	AF466088
Tainosphaeria jonesii	GZCC 16-0065		KY026060	KY026057
Tainosphaeria siamensis	MFLUCC 15-0607	T	KX609956	KX609953
Tainosphaeriella aquatica	MFLUCC 17-2370		NR_173239	NG_079564
Tainosphaeriella thailandensis	MFLUCC 18-1282	T	MZ161198	MZ161196
Thozetella nivea	N/A		EU825201	EU825200
Thozetella tocklaiensis	CBS 378.58	T	MH857817	MH869349
Xyladelphia longiseta	SMH 1725		OL654131	AF279416
Zanclospora iberica	CBS 130426	T	KY853480	KY853544

Note: status: T denotes ex-type strains; “N/A” indicates no data are available in GenBank.

The website tool “ALTER” (http://www.singgroup. org/ALTER/, accessed on 3 May 2022) was used to convert the aligned fasta file for RAxML analysis [44]. Subsequently, ML analysis was performed using RAxML-HPC v.8 tool via the CIPRES Science Gateway V3.3 (https://www.phylo.org/portal2/home.action, accessed on 3 May 2022) with rapid bootstrap analysis [45,46]. Eventually, 1000 non-parametric bootstrap iterations were run with the GTRGAMMA model, and the final tree was selected amongst suboptimal trees from each run by comparing likelihood scores under the GTR-Gamma substitution model.

The aligned fasta file was converted to the nexus file format for BI analysis using AliView. BI analysis was performed using MrBayes 3.2.7a via CIPRES [45]. The best-fit model of DNA evolution was estimated using MrModeltest v. 2.2 [47]. The Bayesian Markov chain Monte Carlo (BMCMC) sampling method in MrBayes v.3.2.7a determined the posterior probabilities (PPs) [48]. Four simultaneous Markov chains were run for 1 million generations, with trees sampled every 100 generations, resulting in 10,000 trees. The first 2000 trees representing the burn-in phase of the analyses were discarded, and the remaining 8000 trees were used for calculating posterior probabilities (PPs) in the majority-rule consensus tree [49].

Phylogenetic trees were printed with FigTree v. 1.4.0 [50], and the layout was established using Adobe Illustrator CS6 (Adobe Systems, San Jose, CA, USA).

3. Results

3.1. Phylogenetic Analysis

Sequences of LSU and ITS were used to determine the phylogenetic position of the new taxa and collections. The concatenated sequence matrix comprises LSU (903 bp) and ITS (545 bp) sequence data for 151 ingroup taxa of Chaetosphaeriaceae and 2 outgroup taxa, namely Leptosporella arengae (MFLUCC 15-0330) and L. bambusae (MFLUCC 12-0846). The sequence matrix comprises 1448 characters after alignment, including the gap. The matrix had 859 distinct alignment patterns, with 14.02% undetermined characters or gaps. The estimated base frequencies were: A = 0.228939, C = 0.268473, G = 0.306331 and T = 0.196257; substitution rates AC = 1.246064, AG = 1.891246, AT = 1.625669, CG = 0.550487, CT = 6.118919, GT = 1.000000 and Tree-Length = 11.191081; distribution shape parameter α = 0.304587. Bayesian posterior probabilities (PPs) from MCMC were evaluated with a final average standard deviation of split frequencies of 0.009954. Maximum likelihood and Bayesian analyses were conducted, resulting in generally congruent topologies, and the ML analysis result with a final likelihood value of −27,871.044543 is presented in Figure 1.

Figure 1

The phylogenetic tree generated from ML analysis that is based on a concatenated LSU-ITS dataset for the Chaetosphaeriaceae family. Bootstrap support values for ML equal to or greater than 65% and Bayesian posterior probabilities (PPs) equal to or greater than 0.95 were indicated above or below the nodes as ML/PP. Leptosporella arengae (MFLUCC 15-0330) and L. bambusae (MFLUCC 12-0846) were selected as the outgroup taxa. The newly obtained sequences are indicated in blue.

This phylogenetic study confirmed that the family Chaetosphaeriaceae was a robust clade (100% ML/1.00 PP). There are eight new collections, five of which are in Chaetosphaeria, Codinaeella, Dictyochaeta, Paragaeumannomyces and Phialosporostilbe genera, respectively, while the other three belong to the genus Codinaea, under Chaetosphaeriaceae (Figure 1).

Chaetosphaeria was resolved as a polyphyletic genus in Chaetosphaeriaceae (Figure 1), consistent with many previous studies [6,9,10,13,14,15,29,51,52]. The new species, Chaetosphaeria obovoidea, and three other Chaetosphaeria species are clustered in the subclade of Chaetosphaeria III.

The phylogenetic tree shows that our three new Codinaea collections of Codinaea aseptata sp. nov., and two known species, Co. dwaya and Co. terminalis, clustered with other 16 Codinaea taxa in a monophyletic clade with highly support (90% ML/1.00 PP, Figure 1).

Eight “Codinaea”-like taxa comprised a new species of Codinaeella hyalina and eight previously identified species, representing the Codinaeella clade, which was phylogenetically well-supported by the generic placement (100% ML/1.00 PP, Figure 1). It is consistent with the studies of Réblová et al. [16].

One of the eight fresh collections was identified as a new species in Dictyochaeta species, namely D. guizhouensis. It clustered together with the other six known species and one unidentified Dictyochaeta sp. (CBS 138684) with good support (97% ML/1.00 PP, Figure 1). All species of Dictyochaeta formed into a single monophyletic clade and fit well with the narrow genetic concept of conidia without setulae [14]. Furthermore, the phylogenetic placement of the Dictyochaeta clade and the phylogenetic relationship of taxa within Dictyochaeta presented similar results to those obtained by Réblová et al. [14].

Phylogenetic placements of the Paragaeumannomyces and Phialosporostilbe genera were stable, and those genera were resolved as monophyletic lineages [29,36].

3.2. Taxonomy

Chaetosphaeria obovoidea J.Y. Zhang and Y.Z. Lu, sp. nov. (Figure 2).

Figure 2

Chaetosphaeria obovoidea (HKAS 123765, holotype). (a,b) Colonies on woody substrate; (c–f) conidiophores with pale gold or red appendant attached; (g–j) conidiogenous cells; (j–l) conidia; (l) germinating conidium; (m,n) culture on PDA from above and below. Scale bars: (c–f) 25 μm; (g–i) 20 μm; (j–l) 10 μm.

Index Fungorum number: IF559696; Facesoffungi number: FoF 11036.

Etymology: referring to obovoid conidia.

Holotype: HKAS 123765.

Saprobic on decaying wood submerged in freshwater habitats. Sexual morph: undetermined. Asexual morph: hyphomycetous. Colonies on natural substrate superficial, effuse, scattered, pale brown, with white and gold masses on the apex of conidiophores. Mycelium is composed of partly immersed, partly superficial, hyaline-to-pale brown, septate, branched hyphae. Conidiophores 93–234(–291) μm long, 3.5–5.3 μm wide at the base, macronematous, mononematous, erect, straight or slightly flexuous, occasionally branched, with intercalary conidiogenous loci, cylindrical, solitary, brown at the base, pale brown or subhyaline towards the apex, septate, smooth-walled, with pale gold or red appendants attached. Conidiogenous cells 13–51 × 3–5.5 μm ( = 33.8 × 4.6 μm, n = 20), mono- to polyphialidic, terminal, integrated, cylindrical, with funnel-shaped collarettes, brown or pale brown to subhyaline or hyaline towards the apex. Conidia 10–14.8 × 5–7.2 μm ( = 12.7 × 6 μm, n = 20), amerospores, aseptate, obovoid, pyriform to broadly clavate, rough-walled, aggregated in large and slimy mass, hyaline, round at the apex, tapering at the base and often with a small prominence.

Culture characteristics: Conidia germinating on PDA within 15 h and hyaline germ tube produced from the base of conidia. Colonies growing on PDA at 25 °C reach 17 mm in three weeks, circular, unbonate, entire, with filamentous, dense, aerial mycelium on the surface, white at the center, pale grey at the edge from above; yellowish to greyish brown to pale brown in reverse from the center to the margin of the colony, and do not produce pigmentation in culture.

Material examined: CHINA, Hainan province, Diaoluo Mountain National Nature Reserve, on decaying wood submerged in a stream, 20 August 2021, W.G. Lin, DL2 (HKAS 123765, holotype; GZAAS 22-0076, isotype), ex-type living cultures, GZCC 22-0085, GenBank accession numbers: (LSU) ON502894, (ITS) ON502901.

Notes: In a BLASTn search in GenBank, the closest match to the ITS sequence of our new isolate was Chaetosphaeria sp. (strain JEH-2019) with 97% (MN619651) similarity across 88% of the query sequence. The closest match to the LSU of the new isolate was Chaetosphaeria fusiformis (strain CBS 101429) with 99.21% (AF178554) similarity across 94% of the query sequence. The phylogenetic analysis confirmed that our new isolate of C. obovoidea formed a separate clade within the Chaetosphaeria genus and is a sister clade to Chaetosphaeria sp. (strain JEH-2019) with good support (94% ML/1.00 PP). Thus, we introduced this new isolate as a novel species in the Chaetosphaeria. Additionally, our new species formed a unique asexual morph in the natural substrate, and differs from all existing species of Chaetosphaeria in having macronematous conidiophores with pale gold or red appendants attached, hyaline, aseptate and obovoid conidia with a prominence at the base. This adds complexity and diversity to the asexual morphs of the Chaetosphaeria genus.

Codinaea aseptata J.Y. Zhang and Y.Z. Lu, sp. nov. (Figure 3).

Figure 3

Codinaea aseptata (HKAS 123758, holotype). (a) Host material; (b,c) colonies on woody substrate; (d,e) setae and conidiophores; (f–h) conidiophores and conidiogenous cells; (i) apex of seta;(j–m) conidia; (n) germinated conidium; (o) culture on PDA from above and below. Scale bars: (d,e) 100 μm; (f,k–n) 10 μm; (g–j) 20 μm.

Index Fungorum number: IF559697; Facesoffungi number: FoF 11038.

Etymology: referring to the aseptate conidia.

Holotype: HKAS 123758.

Saprobic on decaying wood submerged in freshwater habitats. Sexual morph: undetermined. Asexual morph: hyphomycetous. Colonies on the natural substrate effuse, hairy, brown. Mycelium mostly immersed, composed of branched, septate, smooth, brown hyphae. Setae 339–417 μm long, 5–6 μm wide at the base ( = 376.8 × 5.5 μm, n = 20), erect, dark brown at the base, subhyaline towards the apex, septate, unbranched, smooth, rarely fertilizable. Conidiophores 41.6–105.1 μm long, 2–3.74 μm wide at the base ( = 66 × 3 μm, n = 15), macronematous, mononematous, in groups from the mycelial knots associated with the bases of setae, shorter than setae, erect, straight or slightly flexuous, short, cylindrical, unbranched, smooth-walled, septate, pale brown at the base, subhyaline at the apex. Conidiogenous cells 5.7–20 μm ( = 12.2 μm, n = 20) long, monophialidic, integrated, determinate, terminal, cylindrical to cylindrical–lageniform, pale brown at the base, becoming subhyaline to hyaline towards the apex, with flared collarette. Conidia 13.4–16.2 × 2.1–3 μm ( = 14.2 × 2.6 μm, n = 20), acrogenous, solitary, aggregated in slimy droplets, aseptate, cylindrical or long fusiform, curved, with hair-like and 7–10 m-long appendages at both ends, hyaline, smooth-walled, with small guttules.

Culture characteristics: Conidia germinating on PDA within 15 h and germ tubes produced from the base and the upper part. Colonies growing on PDA, reaching 19 mm in diam. in 10 days at 25 °C, circular, flat, entirely to slightly filamentous, section to fan shape at the surface, taupe, becoming white towards the edge from above; yellowish-brown mycelium in the middle and pale-yellow mycelium in the outer ring in reverse, and does not produce pigmentation in culture.

Material examined: CHINA, Hainan Province, Wuzhishan City, Wuzhishan Tropical Rainforest Scenic Area, on decaying wood submerged in a freshwater stream, 15 August 2021, Zheng Luo, WZ48 (HKAS 123758, holotype; GZAAS 22-0072 isotype); ex-type living cultures, GZCC 22-0081. GenBank accession numbers: (LSU) ON502890, (ITS) ON502897.

Notes: Four morphotypes (C1–C4) of Codinaea were provided by Réblová et al. [16]. The morphological characteristics of our new isolate match well with the generic concept of Codinaea and fit well with the morphotype C1. Codinaea aseptata is most similar to Co. terminalis due to having setae in fascicles with conidiophore, phialidic and terminal conidiogenous cells and aseptate, falcate conidia with setulae at both ends [9]. However, our new isolate differs from Co. terminalis in its obviously longer and rarely fertilizable setae. BLAST results of ITS and LSU sequence data are Codinaea acacia OTU5 (96.09% similarity) and Co. paniculate MFLU 34876 (99.43 similarity), respectively. The phylogenetic tree (Figure 1) showed that our new isolate of Cod. aseptata formed an individual lineage in the Codinaea clade, but without statistical support shown in phylogenetic tree (44% ML/-). This may be due to the consideration that many close phylogenetic relatives of our new collection have not yet been discovered. Hence, Codinaea aseptata was introduced as a new species based on its distinct morphology and phylogenetic evidence.

Codinaea dwaya (Subram. and J. Bhat) Réblová and Hern.-Restr., Journal of Fungi 7 (12, no. 1097): 31 (2021) (Figure 4).

Figure 4

Codinaea dwaya (GZAAS 22-0071). (a–c) Colonies on woody substrate; (d,e) conidiophores and conidia; (f–i) conidiogenous cells; (j–l) conidia; (m) germinating conidium; (n) culture on PDA. Scale bars: (d,e) 100 μm; (f–m) 20 μm.

Index Fungorum number: IF 842192; Facesoffungi number: FoF 11037.

Holotype: MUBL 2351.

Saprobic on decaying wood submerged in freshwater habitats. Sexual morph: undetermined. Asexual morph: hyphomycetous. Colonies on natural substrate superficial, effuse, scattered, brown, with glistening masses of conidia on the apex of conidiophores. Mycelium 1.3–2.4 μm wide, mostly immersed, composed of pale brown, septate hyphae. Conidiophores (360–)440–620 × 7.6–14 μm ( = 527.7 × 9.9 μm, n = 20), macronematous, mononematous, erect, straight or slightly flexuous, occasionally branched, cylindrical, solitary, septate, smooth-walled, dark brown, becoming paler brown towards the apex. Conidiogenous cells (17–)35–62.5 × 5.6–8 μm ( = 50.2 × 6.6 μm, n = 20), monophialidic, terminal, integrated, cylindrical, guttulata, with an inconspicuous apical collarette. Conidia in succession by percurrent proliferation from a single fertile locus, spherical, 13.4–17 μm diam. ( = 15.5 μm), amerospores, aseptate, acrogenous, in groups, accumulating in a slimy mass at the tip of the phialide, rough-walled, hyaline, with 7–13 hair-like and 6.4–9.9 μm-long appendages at both ends.

Culture characteristics: Conidia germinating on PDA within 16 h and germ tube produced from conidia. Colonies growing on PDA, reaching 25 mm in diam. in 10 days at 25 °C, circular. Mycelium slightly raised, taupe dark brown with a white protuberance in the center, entirely to slightly filamentous, becoming taupe to dark brown towards the edge in reverse and not producing pigmentation in culture.

Material examined: CHINA, Hainan province, Diaoluo Mountain National Nature Reserve, on decaying wood submerged in a freshwater stream, 20 August 2021, Wei-Guo Lin, DL1-1 (GZAAS 22-0071), living cultures, GZCC 22-0080, GenBank accession numbers: (LSU) ON502889, (ITS) ON502896.

Notes: Following BLASTn searches of NCBI GenBank, the closest matches of the LSU and ITS sequences of our new isolate is Codinaea dwaya (strain CBS 261.77; LSU, OL654135, 99.88% shared identity; ITS, OL654078, 97.79% shared identity). Our new collection fits well with the description of species of Co. dwaya in mononematous conidiophores, integrated, terminal, cylindrical, phialidic conidiogenous cells and hyaline, spherical, aseptate conidia with setulae [16,53,54,55]. Phylogenetically, our isolate grouped together with Co. dwaya (CBS 261.77) with high support value (100% ML/1.00 PP, Figure 1). Thus, we identified this isolate as a new collection of Co. dwaya from a freshwater habitat.

Codinaea terminalis (C.G. Lin and K.D. Hyde) Réblová and Hern.-Restr., Journal of Fungi 7(12, no. 1097): 44 (2021). (Figure 5).

Figure 5

Codinaea terminalis (HKAS 123756). (a) Host material; (b,c) colonies on host surface; (d,e) conidiophores with conidiogenous cell; (f–h) conidiogenous cells; (i–m) conidia; (n) germinating condium; (o,p) culture on PDA from above and below. Scale bars: (b) 200 μm; (c) 100 μm; (d,e) 20 μm; (f–o) 10 μm.

Index Fungorum number: IF 56706; Facesoffungi number: FoF 06287.

Holotype: MFLU 19-0214.

Saprobic on dead bamboo culms in freshwater habitats. Sexual morph: undetermined. Asexual morph: hyphomycetous. Colonies on natural substrate superficial, effuse, gregarious, white, shining, globose aggregated in a large mass. Mycelium mostly immersed, composed of septate, smooth, branched and pale brown hyphae. Setae 99–277 µm long, 3.1–4.7 µm wide at the base ( = 153 × 3.8 µm, n = 25), fertile, straight or slightly flexuous, dark brown at the base, paler towards the apex, septate, smooth-walled, unbranched, cylindrical. Conidiophores 34–62 × 3–4 µm ( = 48.3 × 3.4 µm, n = 20), macronematous, mononematous, erect, in groups from the mycelial knots from the bases of setae, cylindrical, slightly swollen at the base, straight or slightly flexuous, brown at the base becoming pale brown or subhyaline towards the apex, 2–4-septate, unbranched, smooth, guttules. Conidiogenous cells 14.5–36 µm long ( = 24 µm, n = 20), monophialidic, terminal, integrated, cylindrical, brown at the base, pale brown to subhyaline towards the apex, narrowing below the flared and conspicuous collarette. Conidia 13.5–16 × 2.5–3.3 µm ( = 14.5 × 2.9 µm, n = 35), aggregated in a glistening mass of conidia at the tip of conidiophores, amerospores, acrogenous, fusiform, mostly curved, guttules, rough-walled, hyaline, with hair-like and 9.6–11.5 µm-long appendages at both ends.

Culture characteristics: Conidia germinating on PDA within 15 h and germ tubes produced from conidia. Colonies growing on PDA, reaching 19 mm in diam. in 10 days at 25 °C, circular, flat with a protuberance in the center, white, entire margin with thin mycelia; pale yellow to white from center to edge in reverse and not producing pigmentation in culture.

Material examined: CHINA, Guizhou province, Zunyi city, Chishui county, Hushi town, Chishui Alsophila Natural Reserve (28°29′43″ N, 106°0′24″ E), on dead bamboo culms from a stream, 22 September 2019, Jing-Yi Zhang, Y124 (HKAS 123756 = GZAAS 22-0070); living culture, GZCC 22-0086. GenBank accession numbers: (LSU) ON502895, (ITS) ON502902.

Notes: Codinaea terminalis was introduced by Lin et al. [9], isolated from decaying leaves in China. The morphology of our isolate shares similar characters with the ex-type strain Codinaea terminalis (GZCC 18–0085). However, there are some differences in the size of the microstructure such as obviously longer setulae (9.6–11.5 µm vs. 4–9.5 μm). Our new isolate clustered among two Dictyochaeta terminalis strains (GZCC 18–0085 and GZCC19-0525) with strong statistical support (99% ML/1.00 PP, Figure 1). Thus, we identified this isolate as a new host record of Co. terminalis on dead bamboo culms in China.

Codinaeella hyalina J.Y. Zhang and Y.Z. Lu, sp. nov. (Figure 6).

Figure 6

Codinaeella hyalina (HKAS 123757, holotype). (a,b) Colonies on woody substrate; (c,d) conidiophores and conidiogenous cells; (e–g) conidiogenous cells (arrows indicate collarettes); (h–l) conidia; (m) germinating conidium; (n,o) culture on PDA from above and below. Scale bars: (c–e) 20 μm; (f–m) 10 μm.

Index Fungorum number: IF559701; Facesoffungi number: FoF 11039.

Etymology: referring to its hyaline conidia.

Holotype: HKAS 123757.

Saprobic on decaying wood submerged in freshwater habitats. Sexual morph: undetermined. Asexual morph: hyphomycetous. Colonies on natural substrate superficial, effuse, mostly single, or arise in groups of 2–3 from knots of hyphal cells, dark brown, with glistening mass of conidia at the apex of conidiophores. Mycelium mostly immersed, composed of pale brown, septate hyphae. Conidiophores 93–143 × 3.4–5.1 μm ( = 121 × 4.4 μm), macronematous, mononematous, single or in small groups, erect, straight or slightly flexuous, smooth, dark brown at the base, becoming paler to subhyaline towards the apex, 2–7-septate, smooth, guttulate. Conidiogenous cells 23.6–68 × 3.2–5.5 μm ( = 50.3 × 4.3 μm), mono- or polyphialidic, with discrete, lateral phialides, integrated, terminal, with lateral openings formed by successive sympodial elongation, cylindrical to cylindrical–lageniform, with funnel-shaped collarettes, brown at the base and becoming subhyaline to hyaline towards the apex, smooth-walled. Conidia 15.9–21.6 × 5.8–7 μm ( = 18.6 × 6.3 μm, n = 20), amerospores, aseptate, often unilateral ventricose, reniform, aggregated in large and slimy mass, rough-walled, hyaline, with hair-like and 4–7 μm-long appendages at both ends.

Culture characteristics: Conidia germinating on PDA within 12 h. and germ tubes produced from conidia. Colonies growing on PDA, slow growth, reaching 20 mm in diam. in 20 days at 25 °C, circular, flat, with dense, white mycelium on the surface with undulate margin, from below olivaceous brown at the center, yellow at the edge and does not produce pigmentation in culture.

Material examined: CHINA, Guizhou province, Zunyi city, Xishui county, Taolin town, Tianlong, on submerged decaying wood in a freshwater stream, 13 February 2021, Jian Ma, TL24 (HKAS 123757, holotype; GZAAS22-0073 isotype), ex-type living culture, GZCC 22-0082. GenBank accession numbers: (LSU) ON502891, (ITS) ON502898.

Notes: Following BLASTn searches, the closest matches of our new isolate Codinaeella hyalina of ITS and LSU sequence data were Codinaeella minuta 417E (MZ078594, 96.57% similarity) and Cod. minuta ATCC 20960 (OL654146, 99.08% similarity), respectively. In the phylogeny (Figure 1), our new isolate formed a separate clade, and is basal to other species of Codinaeella with a high bootstrap support value (100% ML/1.00 PP). Morphologically, our new isolate shares similar characters to Cod. mimusopis in having macronematous, mononematous conidiophores, conidiogenous cell with lateral phialides and hyaline, aseptate conidia with filiform appendages at both ends [16,30]. However, our new species Cod. hyalina differs from Cod. mimusopis by having wider conidiogenous cells (3.2–5.5 μm vs. 3(–3.5) μm) and wider reniform conidia (15.9–21.6 × 5.8–7 μm vs. 16–18(–20) × 2.5–3(–3.5) μm). Additionally, Cod. hyalina differs from existing species of Codinaeella in having obviously wider conidia. Therefore, we introduce Cod. hyalina as a new species based on its distinct morphology and phylogenetic evidence.

Dictyochaeta guizhouensis J.Y. Zhang and Y.Z. Lu, sp. nov. (Figure 7).

Figure 7

Dictyochaeta guizhouensis (HKAS 123753, holotype). (a,b) Colonies on dead wood; (c–e) conidiophores; (f,g) conidiogenous cells; (h–j) conidia; (k,l) culture on PDA from above and below. Scale bars: (c–e) 50 μm; (f,g) 20 μm; (h–j) 10 μm.

Index Fungorum number: IF559699; Facesoffungi number: FoF 11040.

Etymology: referring to the province “Guizhou”, where this species was collected.

Holotype: HKAS 123753.

Saprobic on dead wood in land. Sexual morph: undetermined. Asexual morph: hyphomycetous. Colonies on natural substrate superficial, effuse, gregarious, hairy, brown. Conidiophores 133–240 × 4–6.8 µm ( = 170.7 × 4.8 µm, n = 10), macronematous, mononematous, erect, straight, unbranched, multiseptate, smooth, dark brown at the base, becoming pale brown towards the apex. Conidiogenous cells 8.6–20 × 2.9–4.8 µm ( = 14.8 × 3.8 µm, n = 10), polyphialidic, terminal, integrated, with flared collarette, cylindrical, pale brown to subhyaline or hyaline towards the apex. Conidia 13–16 × 1.5–2.2 µm ( = 15 × 1.8 µm, n = 20), aggregated in a glistening and small mass of conidia at the tip of conidiophores, amerospores, acrogenous, asymmetrical, falcate, straight or slightly curved, often rounded at the apex, tapering toward the basal end, rough-walled, hyaline.

Cultural characteristics: Conidia germinating on PDA within 15 h and germ tubes produced from conidia. Colonies grew on PDA medium at 25 °C, circular, with an entire margin, umbonate, with dense, white mycelium in the middle and yellowish-brown mycelium in the outer ring on the surface; in contrast, they are cream yellow in the middle, while yellowish-brown in the center and the outer ring.

Material examined: CHINA, Guizhou province, Xishui county, Xishui Reserve, 7 September 2020, Yong-Zhong Lu, XSBHq(011) (HKAS 123753, holotype; GZAAS 22-0074, isotype), ex-type living culture, GZCC 22-0083. GenBank accession numbers: (LSU) ON502892, (ITS) ON502899.

Notes: In a BLASTn search in GenBank, the closest match to the LSU and ITS sequences of our new isolate (GZCC 22-0083) exhibited 100% similarity across 100% of the query sequence and 98.49% similarity across 90% of the query sequence to Dictyochaeta sp. (strain CBS 138684), respectively. The phylogenetic tree depicted that our new collection is closely related to unidentified taxon Dictyochaeta sp. (CBS 138684) with 97% ML/1.00 PP support (Figure 1). Dictyochaeta sp. (CBS 138684) was described by Réblová et al. [14] with its sexual and asexual morphs. However, there was insufficient evidence of morphological characteristics due to a sample ageing; therefore, Dictyochaeta sp. (CBS 138684) was treated as an unidentified species [14]. Our new isolate formed an asexual morph in the natural substrate and is characterized by a lack of setae, microconidia and polyphialidic conidiohenous cells with conspicuous collarette, which can be differentiated from Dictyochaeta sp. (CBS 138684). Given the remarkable differences in morphology between our new collection (HKAS 123753) and Dictyochaeta sp. (CBS 138684), we maintain Dictyochaeta sp. as an unidentified species and introduce our new collection as a novel species in Dictyochaeta.

Paragaeumannomyces guttulatus J.Y. Zhang and Y.Z. Lu, sp. nov. (Figure 8).

Figure 8

Paragaeumannomyces guttulatus (HKAS 123755, holotype). (a) Colonies (arrow) on host surface; (b) seta; (c–e) conidiogenous cell with conidia; (f–k) conidia; (l) germinating conidium; (m,n) colonies on PDA from above and below. Scale bars: (a) 100 μm; (b) 20 μm; (c–l) 10 μm.

Index Fungorum number: IF559700; Facesoffungi number: FoF 11041.

Etymology: referring to its conidia with big guttules.

Holotype: HKAS 123755.

Saprobic on dead bamboo culms in freshwater habitats. Sexual morph: undetermined. Asexual morph: hyphomycetous. Colonies on natural substrate superficial, effuse, gregarious. Mycelium composed of partly immersed, partly superficial, septate, pale brown hyphae. Setae scattered over entire colonies, aseptate, dark brown, stiff, pointed, 65–110.5 μm long ( = 85 μm, n = 13). Conidiophores often reduced to conidiogenous cells, semi-macronematous to micronematous, mononematous, flexuous, unbranched, pale to moderately brown, thin-walled. Conidiogenous cells 6.4–10.3 × 5.4–8 μm ( = 8 × 6.5 µm, n = 20), monophialidic, flask-shaped, cup-shaped collarette, hyaline to light brown, guttulate. Conidia 9.4–11.5 μm diam. ( = 10.5 μm, n = 30), enteroblastic, acrogenous, aseptate, hyaline, globose to subglobose or ellipsoid, guttulate, with 2–6 hair-like and 8.8–13.5 µm-long appendages.

Culture characteristics: conidia germinating on PDA within 15 h and the hyaline germ tubes germinates from a point of the conidia. Colonies on MEA at 26 °C, slow growth, reach 5 mm in diam. in 30 days, circular, flat, with entire margin, taupe brown from above, while dark brown in reverse, and do not produce pigmentation in culture.

Material examined: CHINA, Guizhou province, Zunyi city, Chishui county, Hushi town, Chishui Alsophila Natural Reserve (28°29′43″ N, 106°0′24″ E), on dead bamboo culms from a freshwater stream, 22 September 2019, Jing-Yi Zhang, Y132 (HKAS 123755, holotype; GZAAS 22-0076, isotype); ex-type living culture, GZCC 22-0084. GenBank accession numbers: (LSU) ON502893, (ITS) ON502900.

Notes: Following BLASTn searches, the closest match to our new collection Paragaeumannomyces guttulatus is Chaetosphaeria panamensis (LSU, MT118218, 98.46% shared identity; ITS, KY212752, 92.68%). The phylogenetic results confirm that our new isolate P. guttulatus formed a separate clade within Paragaeumannomyces and shared a sister relationship with P. panamensis (S.M.H. 3596) with 94% ML/1.00 PP support (Figure 1). Chaetosphaeria panamensis was introduced by Huhndorf and Fernández [4] with sexual and asexual morphs. Later, Chaetosphaeria panamensis was transferred to the Paragaeumannomyces (P. panamensis) by Réblová et al. [29] based on generic concept and phylogenetic analysis. Morphologically, our new species P. guttulatus formed an asexual morph in the natural substrate and is similar to P. panamensis in having a “Craspedodidymum”-like asexual morph. However, Paragaeumannomyces guttulatus differs from P. panamensis in having bigger conidiogenous cells and smaller and rounder conidia with longer appendages [4,6]. Thus, we introduce P. guttulatus as a new species based on morphological and phylogenetic analyses.

Phialosporostilbe scutiformis N.G. Liu, J. Yang and K.D. Hyde (2019). (Figure 9).

Figure 9

Phialosporostilbe scutiformis (MFLU 22-0077). (a,b) Colonies on woody substrate; (c) conidiophores; (d,e) conidiogenous cells with conidia; (f,g) conidia; (h) germinated conidium; (i,j) culture on PDA from above and below. Scale bars: (a,b) 200 μm; (c) 100 μm; (d,e) 20 μm; (f–h) 10 μm.

Index Fungorum number: IF555325; Facesoffungi number: FoF 04869.

Holotype: MFLU 18-1502.

Saprobic on decaying wood submerged in freshwater habitats. Sexual morph: undetermined. Asexual morph: hyphomycetous. Colonies on natural substrate superficial, effuse, scattered or gregarious, white, with glistening mass of conidia surround the conidiophores. Mycelium 2.1–3.2 μm ( = 2.7 μm) wide, composed of partly immersed, partly superficial, septate, brown hyphae. Setae 247–358 μm ( = 309 μm), rarely fertilizable, straight or slightly flexuous, dark brown at the base, paler towards the apex, mutiseptate, smooth-walled, unbranched, cylindrical; the lower part is surrounded and compacted by conidiophores. Conidiophores macronematous, synnematous, 11–13.7 μm ( = 12.5 μm) wide at the synnemata, always spread laterally along the upper half of the synnemata, erect, flexuous, unbranched, septate, smooth-walled, dark brown at the base, becoming pale brown and slightly tapering towards the apex, cylindrical. Conidiogenous cells 26–54 × 3.4–5.1 µm ( = 36 × 4.4 μm), monophialidic, terminal, integrated, clavate, thick-walled, rough, brown to subhyaline towards the apex, with an inconspicuous apical collarette. Conidia 6–7.9 μm ( = 7.1 μm, n = 20) long at each above edge, 9–14.4 μm ( = 10.8 μm, n = 20) long at the side, amerospores, acrogenous, round–tetrahedral, triangular from above, rough-walled, hyaline, with (6.5–)9.4–14 µm ( = 10.8 μm, n = 20)-long appendages at each corner.

Cultural characteristics: Conidia germinating on PDA within 15 h and germ tubes produced from the corner of conidia. Colonies on PDA medium, circular, reaching 13 mm in diam. in 10 days at 25 °C, entirely to slightly filamentous, with dense, yellowish-brown mycelium in the middle and thin, slightly yellow mycelium in the edge; yellowish in reverse and not producing pigmentation in culture.

Material examined: THAILAND, Chiang Rai province, on submerged decaying wood in a freshwater stream, 6 March 2021, Jing-Yi Zhang, Y269-2 (MFLU 22-0077); living culture, MFLUCC 22-0053.

GenBank accession numbers: (LSU) ON678145, (ITS) ON678180.

Notes: Phialosporostilbe scutiformis was introduced by Yang et al. [36] based on two specimens collected from freshwater habitats in China and Thailand. A BLASTn search of the ITS and LSU sequence data indicated that our new isolate is closely related to P. scutiformis (MFLUCC 17-0227) with 100% similarity. The morphology of our isolate and the paratype specimen of P. scutiformis (HKAS 102205) are indistinguishable, except that the big guttule in conidia was not observed in our collection. This may be due to the different observation periods. Phylogenetic analysis confirmed that our isolate MFLUCC 22-0053 grouped with Phialosporostilbe scutiformis strains (MFLUCC 17-0227 and MFLUCC 18-1288) with strong bootstrap support (99% ML/1.00 PP, Figure 1). Therefore, we identified this isolate as a new collection of P. scutiformis based on identical morphology and phylogenetic analysis.

4. Discussion

Most Chaetosphaeriaceous species are saprobic on wood or decaying plants from terrestrial and freshwater habitats, sometimes occurring on soil, while some are fungicolous taxa [1,2,6,11,12,14,20,29,56,57,58]. Chaetosphaeria mangrovei was only one species reported from the marine habitat [20]. In this study, five new species among eight newly collections, namely Chaetosphaeria obovoidea, Codinaea aseptata, Codinaeella hyalina, Dictyochaeta guizhouensis and Paragaeumannomyces guttulatus, were documented from China. This increased the number of Chaetosphaeriaceous species and exhibited high diversity, and undiscovered in China. Additionally, three previously known species including a new host record and two new collections were described and identified based on morphological comparison and phylogenetic analysis of a combined LSU and ITS sequence dataset.

The tree topologies resulting from phylogenetic reconstruction revealed Chaetosphaeriaceae are a well-resolved family (Figure 1). However, phylogenetic relationships of some genera in Chaetosphaeriaceae are problematic and uncertain [6,9,13,14,15,16,36]. The type genus Chaetosphaeria (Chaetosphaeriaceae, Chaetosphaeriales) is speciose and phylogenetically unresolved [1,3,4,5,6,9,10,22,29]. Its sexual morph is characterized by black, papillate ascomata, persistent paraphyses, clavate to cylindrical and unitunicate asci with a shallow, refractive, J-apical ring and hyaline, allantoid or ellipsoid, fusiform to filiform curved, septate ascospores with guttules [6,11,17,19,21,57,59]. The asexual morph is described as hyphomycetes with diversity, and is considered to be able to distinguish Chaetosphaeria species [3,10,11,20,23,51,59]. However, the high variability of asexual morphology complicates the phylogenetic placement of Chaetosphaeria [9,10,20,51]. Therefore, further studies on a complicated teleomorph–anamorph relationship are warranted, alongside molecular support to confirm the phylogenetic status of Chaetosphaeria.