Lignicolous Freshwater Fungi from Plateau Lakes in China (I): Morphological and Phylogenetic Analyses Reveal Eight Species of Lentitheciaceae, Including New Genus, New Species and New Records

Abstract

During the investigation of lignicolous freshwater fungi in plateau lakes in Yunnan Province, China, eight Lentitheciaceae species were collected from five lakes viz. Luguhu, Qiluhu, Xingyunhu, Cibihu, and Xihu lake. Based on morphological characters and phylogenetic analysis of combined ITS, LSU, SSU, and tef 1-α sequence data, a new genus Paralentithecium, two new species (Paralentithecium suae, and Setoseptoria suae), three new records (Halobyssothecium phragmitis, H. unicellulare, and Lentithecium yunnanensis) and three known species viz. Halobyssothecium aquifusiforme, Lentithecium pseudoclioninum, and Setoseptoria bambusae are reported.

1. Introduction

Lignicolous freshwater fungi are those fungi that grow on submerged woody debris in freshwater habitats, including lentic (e.g., lakes, ponds, swamps, and pools), lotic (e.g., rivers, streams, creeks, brooks), and other habitats (e.g., cooling tower, tree holes) [1,2,3]. They play an important role in the material and energy cycle of freshwater ecosystems [4,5,6,7,8]. Lignicolous freshwater fungi are a highly diverse group, with the majority belonging to Dothideomycetes and Sordariomycetes (Ascomycota), and a few species in Eurotiomycetes and Orbiliomycetes [3,9,10,11,12]. Lignicolous freshwater fungi have been investigated worldwide, but mainly in lotic habitats of tropical, subtropical, and temperate regions [10,13,14,15,16], with a few from lentic habitats [17]. Those fungi in lentic habitats are poorly studied. This study collects submerged decaying wood from plateau lakes in Yunnan, China, to investigate the species diversity of lignicolous freshwater fungi in the lakes.

Yunnan Province is in the southwest of China, it is a low-latitude, high-altitude inland province, and is one of the biodiversity hotspots in the Yunnan–Guizhou Plateau [18]. Yunnan has three climatic zones, tropical (southwest, south, and southeast borders), subtropical (west, middle, and east), and temperate regions (high-elevation area in the northwest) [19]. The special geographical location and climatic conditions endow Yunnan with abundant natural resources. There are plateau cold-resistant biomes in the west and tropical biomes in the south and southwest. Plateau lakes are an important part of terrestrial lakes and an important part of regional water cycling. They are distributed at higher altitudes, have a large number, and have a wide basin area. They are sensitive to climate change and have made outstanding contributions to coping with global climate change and shaping regional biodiversity patterns [20,21]. There have been several biological studies conducted on plateau lakes in Yunnan, such as waterbirds [22,23], invasive fish [24], water plants [25,26,27], and microorganisms [2,17,28,29,30,31,32]. Yunnan has abundant lignicolous freshwater fungi resources, and from 1986 to 2021, a total of 281 lignicolous freshwater fungi taxa have been reported. These species were mainly reported in lotic habitats (rivers, streams), with only 53 (19%) species from plateau lakes [12].

Species of Lentitheciaceae from freshwater habitats are mainly in Halobyssothecium, Lentithecium, Setoseptoria, and Tingoldiago [14,15,33,34,35,36,37]. The family was introduced by Zhang et al. [35] to accommodate those lentitheciaceous taxa that have narrow peridia, fusiform to broadly cylindrical pseudoparaphyses, hyaline ascospores with 1–3-transverse septa and containing refractive globules, surrounded by a mucilaginous sheath or extended appendage-like sheaths and asexual morphs are stagonospora-like or dendrophoma-like [14,38,39,40]. Currently, more than 100 species are reported in Lentitheciaceae. The last treatment of Lentitheciaceae was provided by Wijayawardene et al. [41] with acceptance of 18 genera: Crassoascoma [42], Darksidea [43], Groenewaldia [44], Halobyssothecium [45], Katumotoa [46], Keissleriella [47], Lentithecium [35], Murilentithecium [40], Neolentithecia [48], Neoophiosphaerella [34], Phragmocamarosporium [49], Pleurophoma [50,51], Poaceascoma [52], Pseudokeissleriella [53], Pseudomurilentithecium [54], Setoseptoria [37], Tingoldiago [55], and Towyspora [56].

We are investigating the diversity of lignicolous freshwater fungi from plateau lakes in Yunnan Province, and 13 collections of lentitheciaceous-like taxa were obtained. Based on morphological and multigene phylogenetic analysis, a new genus Paralentithecium is introduced to accommodate P. aquaticum, and a new taxon P. suae, Setoseptoria suae sp. nov. and new records Halobyssothecium and Lentithecium are described and illustrated. The sexual morph of Halobyssothecium phragmitis is also introduced.

2. Materials and Methods

2.1. Samples Collection

The fresh samples were submerged in lake water with a diameter of less than 2 cm and a length of more than 20 cm, including tree trunks, branches, twigs, and rotten branches of grasses. The specimens in this study were collected from Dali City (Cibihu, Xihu, and Erhai Lakes), Lijiang City (Luguhu Lake), and Yuxi City (Xingyunhu and Qiluhu Lakes) in Yunnan. The collected samples were placed in plastic ziplock bags and were taken back to the laboratory for processing.

2.2. Sample Processing and Cultivation

The samples were brought to the laboratory in ziplock bags to avoid moisture loss and then trimmed to 15 cm in length with pruning scissors. Each sample with a label number that is attached to the end of the sample with a thumbtack (Figure 1a). In addition, plastic boxes with the size of 24 cm × 16 cm × 6 cm were prepared. First, rinse the inside of the plastic box with sterile water, then wipe the entire plastic box with 75% alcohol. After drying, put two layers of sterilized tissues on the bottom of the box, lay three sterilized straws on the tissues to prevent the sample from directly touching the sterilized tissues, and add an appropriate amount of sterile water (the water soaks sterile tissues, but accumulates at the bottom), and then arrange the processed samples horizontally on the straw, ten samples in each plastic box, and label the boxes in obvious places (Figure 1b,c). The samples were placed on a culture rack and incubated at room temperature for one week (Figure 1d).

2.3. Morphological Studies and Isolation

Macromorphological characters of samples were observed using Optec SZ 760 compound stereomicroscope (Chongqing Optec Instrument Co., Ltd., Chongqing, China). The temporarily prepared microscope slide was placed under a Nikon ECLIPSE Ni-U compound stereomicroscope (Nikon, Tokyo, Japan) for observation and microscopic morphological photography. The morphology of colonies on native substrates was photographed with a Nikon SMZ1000 stereo-zoom microscope. Indian ink was used to reveal the presence of a gelatinous sheath around the ascospores or conidia. The measurements of photomicrographs were obtained using Tarosoft (R) Image Frame Work version 0.9.7. Images were edited with Adobe Photoshop CS5 Extended version 12.0.0.0 software (Adobe Systems, San Jose, CA, USA).

Single spore isolations were performed as follows: the tip of a sterile toothpick dipped in sterile water was used to capture the conidia of the target colony directly from the specimen; the conidia were then streaked on the surface of water agar (WA, Composition: Agar 20 g/L, Chloramphenicol 0.1 g/L) or potato dextrose agar (PDA, CM123, Composition: Potato infusion 5.0 g/L, Dextrose 20 g/L, Agar 20 g/L, Chloramphenicol 0.1 g/L, from Beijing Bridge Technology Co., Ltd., Beijing, China) and incubated at room temperature overnight. The single germinated conidia were transferred to fresh PDA medium and incubated at room temperature. A few of the remaining germinated spores in the media plate were separated along with agar using a needle and transferred onto water-mounted glass slides for photographs to capture the germination position of the germ tubes.

After finalizing the observation and isolation, the specimens were dried under natural light, wrapped in absorbent paper, and placed in a ziplock bag with mothballs. Specimens were deposited in the herbarium of Kunming Institute of Botany, Academia Sinica (KUN-HKAS). The living cultures were deposited in the China General Microbiological Culture Collection Center (CGMCC) and Kunming Institute of Botany Culture Collection (KUNCC). MycoBank numbers are registered in the MycoBank database (https://www.mycobank.org/Registration%20home (accessed on 4 August 2023)). Entries will be added to the Greater Mekong Subregion database [57].

2.4. DNA Extraction, PCR Amplification and Sequencing

DNA extraction, PCR amplification, sequencing, and phylogenetic analysis were done following the methods of Dissanayake et al. [58]. Mycelia for DNA extraction from each isolate was grown on PDA for 3–4 weeks at room temperature. Total genomic DNA was extracted from 100 to 300 mg axenic mycelium via scraping from the edges of the growing culture using a sterile scalpel and transferred to a 1.5 mL microcentrifuge tube using sterilized inoculum needles. The mycelium was ground to a fine powder with liquid nitrogen or quartz sand to break the cells for DNA extraction. When the cultures could not be maintained with some of the collected samples, fruiting structures (20–50 mg) were removed from the natural substrate using a sterile scalpel placed on sterile paper and then transferred to a 1.5 mL microcentrifuge tube. DNA was extracted with the TreliefTM Plant Genomic DNA Kit (TSP101) following the manufacturer’s guidelines.

Four gene regions, ITS, LSU, SSU, and tef 1-α were amplified using ITS5/ITS4 [59], LR0R/LR5 [60], NS1/NS4 [59], and EF1-983F/EF1-2218R [61] primer pairs, respectively. The PCR mixture contained 12.5 µL of 2× Power Taq PCR MasterMix (a premix and ready-to-use solution, including 0.1 Units/µL Taq DNA Polymerase, 500 µm dNTP Mixture each (dATP, dCTP, dGTP, dTTP), 20 mm Tris–HCl pH 8.3, 100 Mm KCl, 3 mM MgCl₂, stabilizer, and enhancer), 1 µL of each primer including forwarding primer and reverse primer (10 µm), 1 µL template DNA extract and 9.5 µL deionized water. The PCR thermal cycling conditions of ITS and SSU were as follows: 94 °C for s min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 50 s, elongation at 72 °C for 1 min, and a final extension at 72 °C for 10 min; LSU and tef 1-α were as follows: 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 50 s, elongation at 72 °C for 1 min, and a final extension at 72 °C for 10 min. PCR products were then purified using minicolumns, purification resin, and buffer according to the manufacturer’s protocols (Amersham product code: 27-9602-01). The sequences were carried out at Beijing Tsingke Biological Engineering Technology and Services Co., Ltd. (Beijing, China).

2.5. Phylogenetic Analyses

ITS, LSU, SSU, and tef 1-α sequence data used for phylogenetic analysis are selected based on the preliminary identification results and the related publications [14,15]. The sequences were aligned using MAFFT online service: multiple alignment program MAFFT v.7 (http://mafft.cbrc.jp/alignment/server/index.html (accessed on 30 August 2023)) [62], and sequence trimming was performed with trimAl v1.2 for Windows, and all parameters were set by default (http://trimal.cgenomics.org for specific operation steps (accessed on 30 August 2023)) [63]. The sequence dataset was combined using SquenceMatrix v.1.7.8 [64]. FASTA alignment formats were changed to PHYLIP and NEXUS formats by the website: ALignment Transformation EnviRonment (ALTER) (http://sing.ei.uvigo.es/ALTER/ (accessed on 30 August 2023)) [65]. The alignments and phylogenetic trees were deposited in TreeBASE (http://www.treebase.org/ (accessed on 31 August 2023), accession number: 30729-30733).

The single-gene phylogenetic tree was obtained based on maximum likelihood (ML) only, and the multigene phylogenetic tree was obtained based on maximum likelihood (ML) and Bayesian criterion (BI). ML tree and BI tree were run on the CIPRES Science Gateway portal [66,67,68,69]. MrModeltest v. 2.3 [70] was run under the AIC (Akaike Information Criterion) implemented in PAUP v. 4.0b10. to evaluate the best-fit model in both ML and BI analyses. ML analyses for the datasets were performed with RAxML-HPC2 on XSEDE v. 8.2.10 [66] using the determined best-fit substitution model with 1000 bootstrap iterations. The BI analysis was computed with MrBayes v. 3.2.6 [69]. Six simultaneous Markov chains were run with a suitable number of generations, and trees were sampled every 100th generation, ending the run automatically when the standard deviation of split frequencies dropped below 0.01. Alignment gaps were treated as missing characters in the analysis of the combined data set, where they will occur in relatively conserved regions. Trees were inferred using the heuristic search option with 1000 random sequence additions, with maxtrees set at 1000. Phylogenetic trees were visualized using FigTree v1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 31 August 2023)), editing and typesetting using Adobe Illustrator (AI) (Adobe Systems Inc., San Jose, CA, USA). The new sequences were submitted in GenBank, and the strain information used in this paper is provided in

Table 1. Taxa used in the phylogenetic analyses and their corresponding GenBank accession numbers.

Species	Strain/Voucher Number	GenBank Accession Number
		LSU	SSU	ITS	tef 1-α
Bambusicola bambusae	MFLUCC 11–0614 T	JX442035	JX442039	NR_121546	KP761722
Bambusicola irregulispora	MFLUCC 11–0437 T	JX442036	JX442040	NR_121547	KP761723
Bambusicola massarinia	MFLUCC 11–0389 T	JX442037	JX442041	JX442033	KP761725
Bambusicola splendida	MFLUCC 11–0439 T	JX442038	JX442042	NR121549	KP761726
Crassoascoma potentillae	UESTCC 21.0010	OK161254	OK161233	OK161237	OK181165
Crassoascoma potentillae	UESTCC 21.0011	OK161255	OK161234	OK161238	OK181166
Crassoascoma potentillae	UESTCC 21.0012	OK161256	OK161235	OK161239	OK181167
Crassoascoma potentillae	CGMCC 3.20483 T	OK161257	OK161236	OK161240	OK181168
Darksidea alpha	CBS 135650 T	KP184019	KP184049	NR_137619	KP184166
Darksidea beta	CBS 135637 T	KP184023	KP184074	NR_137957	KP184189
Darksidea delta	CBS 135638 T	KP184024	KP184069	NR_137075	KP184184
Darksidea epsilon	CBS 135658 T	KP184029	KP184070	NR_137959	KP184186
Darksidea gamma	CBS 135634 T	KP184031	KP184073	NR_137587	KP184188
Darksidea zeta	CBS 135640 T	KP184013	KP184071	NR_137958	KP184191
Halobyssothecium aquifusiforme	GZCC 20–0481 T	OP377925	OP378010	OP377825	OP473005
Halobyssothecium aquifusiforme	MFLUCC 19–0305	OP377929	OP378014	OP377829	OP473008
Halobyssothecium aquifusiforme	KUNCC 22–12665	OR335346	OR335329	OR335289	OR367662
Halobyssothecium bambusicola	MFLUCC 20–0226 T	MT068489	MT068494	MN833419	MT477868
Halobyssothecium cangshanense	DLUCC 0143 T	KU991149	KU991150	–	–
Halobyssothecium caohaiense	GZCC 19–0482 T	MW133831	MW134611	OP377841	OP473019
Halobyssothecium carbonneanum	CBS 144076 T	MH069699	–	MH062991	–
Halobyssothecium estuariae	MFLUCC 19–0386 T	MN598871	MN598868	MN598890	MN597050
Halobyssothecium estuariae	MFLUCC 19–0387 T	MN598872	MN598869	MN598891	MN597051
Halobyssothecium kunmingense	KUMCC 19–0101 T	MN913732	MT864313	MT627715	MT954408
Halobyssothecium obiones	20AV2566	–	–	KX263862	–
Halobyssothecium obiones	27AV2385	–	–	KX263864	–
Halobyssothecium obiones	MFLUCC 15–0381 T	MH376744	MH376745	MH377060	MH376746
Halobyssothecium phragmitis	MFLUCC 20–0223 T	MT068486	MT068491	MT232435	MT477865
Halobyssothecium phragmitis	MFLUCC 20–0225	MT068488	MT068493	MT232437	MT477867
Halobyssothecium phragmitis	HKAS 127181	OR506189	OR506192	OR506177	OR513794
Halobyssothecium thailandica	MFLUCC 21–0062 T	MZ433248	MZ429435	MZ429434	–
Halobyssothecium unicellulare	MD129	KX505375	KX505373	–	–
Halobyssothecium unicellulare	KUNCC 22–12413	OR335347	OR335330	OR335290
Halobyssothecium unicellulare	MD6004 T	KX505376	KX505374	–	–
Halobyssothecium versicolor	MFLUCC 20–0222 T	MT068485	MW346047	MT232434	MT477864
Halobyssothecium voraginesporum	CBS H-22560 T	KX499520	KX499519	–	–
Kalmusia scabrispora	KT2202	AB524594	AB524453	LC014576	AB539107
Karstenula rhodostoma	CBS 690.94	GU301821	GU296154	–	GU349067
Katumotoa bambusicola	KT 1517a T	AB524595	AB524454	LC014560	AB539108
Keissleriella bambusicola	KUMCC 18–0122 T	MK995880	MK995878	MK995881	MN213156
Keissleriella breviasca	KT 581	AB807587	AB797297	AB811454	AB808566
Keissleriella breviasca	KT 649 T	AB807588	AB797298	AB811455	AB808567
Keissleriella camporesiana	MFLUCC 15–0029 T	MN401741	MN401743	MN401745	MN397907
Keissleriella camporesii	MFLUCC 15–0117 T	MN252886	MN252907	MN252879	–
Keissleriella caraganae	KUMCC 18–0164 T	MK359439	MK359444	MK359434	MK359073
Keissleriella cirsii	MFLUCC 16–0454 T	KY497780	KY497782	KY497783	KY497786
Keissleriella cladophila	CBS 104.55 T	GU301822	GU296155	MH857391	GU349043
Keissleriella culmifida	KT2308	AB807591	AB797301	LC014561	AB808570
Keissleriella culmifida	KT2642	AB807592	AB797302	LC014562	AB808571
Keissleriella dactylidicola	MFLUCC 13–0866 T	KT315506	KT315505	–	KT315507
Keissleriella dactylidis	MFLUCC 13–0751 T	KP197668	KP197666	KP197667	KP197669
Keissleriella genistae	CBS 113798	GU205222	GU205242	–	–
Keissleriella gloeospora	KT829	AB807589	AB797299	LC014563	AB808568
Keissleriella linearis	IFRD2008	FJ795435	FJ795478	–	–
Keissleriella linearis	MFLUCC 19–0410	MN598873	MN598870	MN598892	MN607978
Keissleriella linearis	MFLUCC 20–0224	MT068487	MT068492	MT232436	MT477866
Keissleriella phragmiticola	CPC 33249	MT223903	–	MT223808	MT223715
Keissleriella phragmiticola	MFLUCC 17–0779 T	MG829014	–	MG828904	–
Keissleriella poagena	CBS 136767	KJ869170	–	KJ869112	–
Keissleriella quadriseptata	KT2292 T	AB807593	AB797303	AB811456	AB808572
Keissleriella rara	CBS 118429	GU479791	GU479757	–	–
Keissleriella rosacearum	MFLUCC 15–0045 T	MG829015	MG829123	–	–
Keissleriella rosae	MFLUCC 15–0180 T	MG829016	MG922549	–	–
Keissleriella rosarum	MFLUCC 15–0089 T	MG829017	MG829124	MG828905	–
Keissleriella sparticola	MFLUCC 14–0196 T	KP639571	–	–	–
Keissleriella tamaricicola	MFLUCC 14–0168 T	KU900300	–	KU900328	–
Keissleriella taminensis	KT571	AB807595	AB797305	LC014564	AB808574
Keissleriella taminensis	KT594	AB807596	AB797306	–	–
Keissleriella taminensis	KT678	AB807597	AB797307	LC014565	AB808575
Keissleriella trichophoricola	CBS 136770 T	KJ869171	–	KJ869113	–
Keissleriella yonaguniensis	HHUF 30138 T	AB807594	AB797304	AB811457	AB808573
Keissleriella sp.	KT895	AB807590	AB797300	–	AB808569
Latorua caligans	CBS 576.65 T	MH870362	–	MH858723	–
Latorua grootfonteinensis	CBS 369.72 T	MH877741	–	–	–
Lentithecium clioninum	KT1149A T	AB807540	AB797250	LC014566	AB808515
Lentithecium clioninum	KT1220	AB807541	AB797251	LC014567	AB808516
Lentithecium fluviatile	CBS 122367	FJ795451	FJ795493	–	GU349074
Lentithecium fluviatile	CBS 123090	FJ795450	FJ795492	–	–
Lentithecium pseudoclioninum	KT1113 T	AB807544	AB797254	AB809632	AB808520
Lentithecium pseudoclioninum	GZCC 19–0483	MW133832	MW134612	OM692194	–
Lentithecium pseudoclioninum	KUNCC 22–12414	OR335348	OR335331	OR335291	–
Lentithecium pseudoclioninum	KUNCC 22–12415	OR335349	OR335331	OR335291	–
Lentithecium yunnanensis	KUNCC 22–10776 T	ON227127	ON227123	ON227126	ON228074
Lentithecium yunnanensis	KUNCC 22–10777	ON227124	ON227122	ON227125	ON228075
Lentithecium yunnanensis	KUNCC 22–12420	OR335350	OR335333	OR335293	OR367664
Lentithecium yunnanensis	KUNCC 22–12421	OR335351	OR335334	OR335294	OR367665
Lentithecium yunnanensis	KUNCC 22–12422	OR335352	OR335335	OR335295	OR367666
Longipedicellata aptrootii	MFLUCC 10–0297 T	KU238894	KU238895	KU238893	KU238892
Longipedicellata aptrootii	MFLUCC 18–0988	MN913744	–	MT627733	–
Macrodiplodiopsis desmazieri	CBS 140062 T	KR873272	–	KR873240	–
Massarina cisti	CBS 266.62	FJ795447	FJ795490	LC014568	AB808514
Massarina eburnea	CBS 139697	AB521735	AB521718	LC014569	AB808517
Massarina eburnea	CBS 473.64	GU301840	GU296170	AF383959	GU349040
Multiseptospora thailandica	MFLUCC 11–0183 T	KP744490	KP753955	KP744447	KU705657
Murilentithecium clematidis	MFLUCC 14–0561	KM408758	KM408760	KM408756	KM454444
Murilentithecium clematidis	MFLUCC 14–0562 T	KM408759	KM408761	KM408757	KM454445
Murilentithecium lonicerae	MFLUCC 18–0675 T	MK214373	MK214376	MK214370	MK214379
Murilentithecium rosae	MFLUCC 15–0044 T	MG829030	MG829137	MG828920	–
Neolentithecia changchunensis	CCMJ10012 T	MZ518790	MZ518820	MZ519071	–
Neoophiosphaerella sasicola	KT1706 T	AB524599	AB524458	LC014577	AB539111
Parabambusicola thysanolaenae	KUMCC 18–0147 T	MK098199	MK098205	MK098190	MK098209
Parabambusicola thysanolaenae	KUMCC 18–0148	MK098198	MK098202	MK098193	MK098211
Paraconiothyrium brasiliense	CBS 100299 T	JX496124	AY642523	JX496011	–
Paraphaeosphaeria michotii	MFLUCC 13–0349 T	KJ939282	KJ939285	KJ939279	–
Paraphaeosphaeria minitans	CBS 122788	EU754173	EU754074	–	GU349083
Phragmocamarosporium hederae	MFLUCC 13–0552 T	KP842915	KP842918	–	–
Phragmocamarosporium platani	MFLUCC 14–1191 T	KP842916	KP842919	–	–
Phragmocamarosporium rosae	MFLUCC 17–0797 T	MG829051	MG829156	–	MG829225
Pleomonodictys descalsii	CBS 142298 T	KY853522	–	KY853461	–
Pleomonodictys capensis	CBS 968.97 T	KY853521	–	KY853460	–
Pleurophoma ossicola	CBS139905 T	KR476769	–	KR476736	–
Pleurophoma ossicola	CPC24985	KR476770	–	KR476737	–
Pleurophoma pleurospora	CBS130329 T	JF740327	–	–	–
Poaceascoma aquaticum	MFLUCC 14–0048 T	KT324690	KT324691	–	–
Poaceascoma halophila	MFLUCC 15–0949 T	MF615399	MF615400	–	–
Poaceascoma helicoides	MFLUCC 11–0136 T	KP998462	KP998463	KP998459	KP998461
Poaceascoma taiwanense	MFLUCC 18–0083 T	MG831567	MG831568	MG831569	–
Paralentithecium aquaticum	CBS 123099 T	GU301823	GU296156	NR_160229	GU349068
Paralentithecium suae	CGMCC 3.24265 T	OQ732683	OQ875040	OQ874972	OR367672
Pseudokeissleriella bambusicola	CGMCC 3.20950 T	ON614138	ON614096	ON614135	ON639623
Pseudokeissleriella bambusicola	UESTCC 22.0028	ON614137	ON614095	ON614134	ON639622
Setoseptoria arundelensis	MFLUCC 17–0759 T	MG829073	MG829173	MG828962	–
Setoseptoria arundinacea	CBS 123131	GU456320	GU456298	–	GU456281
Setoseptoria arundinacea	CBS 619.86	GU301824	GU296157	–	–
Setoseptoria arundinacea	MAFF 239460	AB807574	AB797284	LC014594	AB808550
Setoseptoria arundinacea	MAFF 243842 T	AB807575	AB797285	LC014595	AB808551
Setoseptoria bambusae	GZCC 17–0044	OP377919	OP378004	OP377820	OP472999
Setoseptoria bambusae	KUNCC 22–12416	OR335353	OR335336	OR335296	OR367667
Setoseptoria bambusae	KUNCC 22–12417	OR335354	OR335337	OR335297	OR367668
Setoseptoria bambusae	KUNCC 22–12418	OR335355	OR335338	OR335298	OR367669
Setoseptoria englandensis	MFLUCC 17–0778 T	MG829074	MG829174	MG828963	–
Setoseptoria lulworthcovensis	MFLU 18–0110 T	MG829075	MG829175	–	–
Setoseptoria magniarundinacea	KT1174	AB807576	AB797286	LC014596	AB808552
Setoseptoria phragmitis	CBS 114802 T	KF251752	–	KF251249	KF253199
Setoseptoria phragmitis	CBS 114966	KF251753	–	KF251250	KF253200
Setoseptoria scirpi	MFLUCC 14–0811 T	KY770982	KY770980	MF939637	KY770981
Setoseptoria suae	CGMCC 3.24266 T	OQ874972	OQ875041	OQ874972	OR367673
Splanchnonema platani	CBS 221.37	MH867404	–	MH855894	DQ677908
Splanchnonema platani	CBS 222.37	KR909316	KR909318	KR909311	KR909319
Tingoldiago clavata	MFLUCC 19–0495	MN857180	MN857188	MN857184	–
Tingoldiago clavata	MFLUCC 19–0496 T	MN857178	MN857186	MN857182	–
Tingoldiago clavata	MFLUCC 19–0498	MN857179	MN857187	MN857183	–
Tingoldiago graminicola	KH155	AB521745	AB521728	LC014599	AB808562
Tingoldiago graminicola	KH68 T	AB521743	AB521726	LC014598	AB808561
Tingoldiago graminicola	KT891	AB521744	AB521727	LC014600	AB808563
Tingoldiago hydei	MFLUCC 19–0499 T	MN857177	–	MN857181	–
Towyspora aestuari	MFLUCC 15–1274 T	KU248852	KU248853	NR_148095	–

Notes: The ex-type cultures are indicated using “T” after strain numbers; newly generated sequences are indicated in bold. “–” stands for no sequence data in GenBank.

.

3. Results

3.1. Phylogenetic Analysis

The combined ITS, LSU, SSU, and tef 1-α dataset comprises 147 taxa, including nine genera of Lentitheciaceae, with Pleomonodictys capensis (CBS 968.97) and P. descalsii (CBS 142298) as outgroup taxa (Figure 2). The dataset comprised 3777 characters (LSU: 1285 bp; SSU: 1021 bp; ITS: 539 bp; tef 1-α: 932 bp, including gaps). Maximum likelihood (ML) analysis and Bayesian analysis produced similar topologies that were consistent across the major clades. The likelihood of the final tree is evaluated and optimized under GAMMA. The best RAxML tree with a final likelihood value of −31,318.755060 is presented (Figure 2). The matrix had 1636 distinct alignment patterns, with 27.52% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.239720, C = 0.248808, G = 0.272414, T = 0.239058; substitution rates AC = 1.212047, AG = 2.534776, AT = 1.388124, CG = 1.249521, CT = 7.002685, GT = 1.000000, α = 0.226056, Tree-Length: 3.286461. Bayesian analyses generated 4412 trees (average standard deviation of split frequencies: 0.009960) from which 3310 were sampled after 25% of the trees were discarded as burn-in. The alignment contained a total of 1441 unique site patterns. Bootstrap support values with an ML greater than 75%, and Bayesian posterior probabilities (PP) greater than 0.97 are given above the nodes.

The multigene phylogenetic analyses showed that the 13 fresh collections clustered within Lentitheciaceae. Five known species, Halobyssothecium aquifusiforme (KUNCC 22-12665), H. phragmitis (KUN-HKAS 127181), H. unicellulare (KUNCC 22-12413), Lentithecium pseudoclioninum (KUNCC 22-12414 and KUNCC 22-12415), L. yunnanensis (KUNCC 22-124201, KUNCC 22-12421 and KUNCC 22-12422) and Setoseptoria bambusae (KUNCC 22-12416, KUNCC 22-12417 and KUNCC 22-12418) clustered with their ex-type strains, respectively. Paralentithecium suae (KUNCC 22-12412) clustered sister to P. aquaticum (CBS 123099) in an independent clade within Lentitheciaceae. Setoseptoria suae (KUNCC 22-12419) was placed sister to S. phragmitis (CBS 114804 and CBS 114966). Single-gene phylogenies are shown as Supplemental Materials (Figures S1–S4) because they resulted in being less informative and resolutive than those based on the four-loci concatenated tree.

3.2. Taxonomy

Halobyssothecium aquifusiforme J. Yang, Jian K. Liu & K.D. Hyde, Fungal Diversity 119: 39 (2023). Figure 3.

Index Fungorum number: IF559450; Facesoffungi number: FoF12783.

Saprobic on submerged decaying wood in a freshwater lake. Asexual morph: Undetermined. Sexual morph: Ascomata 354–382 µm high, 328–366 µm wide, immersed, clustered, sometimes solitary, scattered, subglobose or ellipsoidal, dark brown to black, carbonaceous, uniloculate, ostiolate. Ostiolar neck central, 86–114 µm long, 138–168 µm wide, papillate, rounded, short, dark brown, composed of several layers of pseudoparenchymatous cells. Peridium 22–35 µm thick, composed of several layers of pseudoparenchymatous cells, an outer layer composed of black cells, arranged in a textura angularis, inner layer composed of hyaline, flattened cells, arranged in a textura angularis. Pseudoparaphyses about 2 µm wide, branched, septate, hyaline, filamentous, anastomosing above the asci. Asci 97–129 × 13–16 µm ($\overline{x}$ = 113 × 14 µm, n = 20) µm, 8-spored, clavate to subcylindrical, bitunicate, fissitunicate, apex rounded, short pedicellate, with an ocular chamber. Ascospores (20–)24–27 × 7–8 µm ($\overline{x}$ = 25 × 8 µm, n = 40), overlapping, uniseriate to biseriate, central cells are brown to dark brown, 1-septate when young, 3-septate when mature, constricted at the septa, slightly curved, fusiform, guttulate, conical and narrowly rounded at the ends, one cell on the central septum side is swollen, lacking gelatinous sheaths or appendages.

Culture characteristics: Ascospore germinating on PDA within 12 h. Colonies on PDA reaching 3 cm diameter in 6 weeks at room temperature. Mycelium superficial, initially white, later becoming brown to black, with pale brown dense aerial mycelium on the surface, mastoid, marginal mycelium smooth, sparse, brown to black; from below, light brown at the center, dark brown at the margin.

Material examined: China, Yunnan Province, Dali City, Eryuan County, Cibihu Lake, 26°09′59″ N, 99°55′27″ E (2050 m), on unknown submerged decaying wood, 21 July 2021, S.P. Huang and L.L. Li, L788 (KUN-HKAS 124599), living cultures (KUNCC 22-12665).

Known host and distribution: China, Guizhou Province, Anshun City, Gaodang village, 26.071° N, 105.698° E, Suoluo River, on decaying wood submerged in a freshwater stream HKAS 112638, (holotype), HKAS 112641 (paratype) [15].

Notes: The phylogenetic analysis showed that our new strain, KUNCC 22-12665 clustered sister to Halobyssothecium aquifusiforme (GZCC 20-0481 and MFLUCC 19-0305) with 99% ML/1.00 PP supports (Figure 2). Our species is similar to H. aquifusiforme in having immersed, subglobose ascomata, and fusiform, guttulate, septate ascospores which are constricted at the septum [15]. We, therefore, identified our new collection as H. aquifusiforme and provided detailed descriptions and illustrations for it. Halobyssothecium aquifusiforme is an aquatic species that was collected on submerged decaying wood in a freshwater stream in Guizhou, China. Our two new collections were collected from a plateau lake in Yunnan.

Halobyssothecium phragmitis M.S. Calabon, E.B.G. Jones, S. Tibell & K.D. Hyde, Mycological Progress 20: 711 (2021). Figure 4.

Index Fungorum number: IF558090; Facesoffungi number: FoF 09431.

Saprobic on submerged decaying wood in a freshwater lake. Sexual morph: Ascomata 529–566 µm high, 545–691 µm wide, immersed or semi-immersed, solitary to gregarious, scattered, subglobose or ellipsoidal, dark brown, subcarbonaceous or coriaceous, uniloculate, with indistinct ostiolate. Ostiolar neck 172–265 µm high, 184–320 µm wide, central, papillate, rounded, short, dark brown, composed of several layers of pseudoparenchymatous cells. Peridium 26–77 µm thick, composed of several layers of pseudoparenchymatous cells, an outer layer composed of brown cells, arranged in a textura angularis and textura globulosa, and an inner layer composed of hyaline, flattened cells, arranged in a textura angularis. Pseudoparaphyses 2–3 µm wide, septate, hyaline, filiform, branched, anastomosing above the asci. Asci (102–)111–130(–137) × 10–12 µm ($\overline{x}$ = 121 × 11 µm, n = 30), 8-spored, clavate to subcylindrical, bitunicate, fissitunicate, short pedicellate with an ocular chamber. Ascospores 22–27 × 5–6 µm ($\overline{x}$ = 25 × 6 µm, n = 40), overlapping, uniseriate to biseriate, fusiform with narrow ends, cells swollen nearly central septum and gradually narrow toward ends, slightly curved, pale brown to dark brown and lightening from central cells to the end cells, 1-septate when young, 5-septate when mature, and constricted at the septa, lacking gelatinous sheaths or appendages. Asexual morph: Coelomycetes [14].

Material examined: China, Yunnan Province, Dali City, Eryuan County, Xihu Lake, 26°00′33″ N, 100°03′35″ E (1970 m), on unknown submerged decaying wood, 8 May 2021, S.P. Huang and L.L Li, L783 (KUN-HKAS 127181).

Known host and distribution: SWEDEN, Gotland, Kappelshamnsviken, on dead Phragmites culm (Poaceae), MFLU 20–0550 (holotype); ibid., Sudersand, on dead Phragmites (Poaceae) stem, MFLU 20–0552 (paratype) [14].

Notes: Halobyssothecium phragmitis was introduced by Calabon et al. [14], and only the asexual morph is known. This species was collected on Phragmites (Poaceae) culm in Europe. Phylogenetic analysis combined with ITS, LSU, SSU, and tef 1-α sequence data showed that our new collection (KUN-HKAS 127181) clustered with two strains of H. phragmitis (MFLUCC 20–0223 and MFLUCC 20–0225). The comparison of ITS, LSU, SSU, and tef 1-α sequences between our new collection (KUN-HKAS 127181) and the ex-type of H. phragmitis (MFLUCC 20–0226) showed 8 bp, 1 bp, 3 bp, and 3 bp differences, respectively. Morphologically, our new collection is similar to other sexual members of Halobyssothecium in having immersed or semi-immersed, subglobose or ellipsoidal, dark brown, subcarbonaceous or coriaceous ascomata, clavate to subcylindrical, bitunicate asci and 3-septate, fusiform ascospores [14,15,45,71]. Based on phylogenetic analysis and morphological evidence, we identified our new collection as H. phragmitis, and described its asexual morph. This is the first report of this species in China [14].

Halobyssothecium unicellulare (Abdel-Aziz) M.S. Calabon, K.D. Hyde & E.B.G. Jones, Mycological Progress 20: 715 (2021). Figure 5.

Index Fungorum number: IF558094; Facesoffungi number: FoF 09437

Saprobic on submerged decaying wood in a freshwater lake. Sexual morph: Undetermined. Asexual morph: Coelomycetes. Conidiomata 135–178 µm high, 205–242 µm wide, immersed to semi-immersed, most immersed, clustered, sometimes solitary, scattered, subglobose or ellipsoidal, uniloculate, dark brown to black, carbonaceous, short ostiolate, papillate, rounded. Conidiomatal walls 14–31 µm thick, composed of several layers of hyaline to black–brown cells of textura angularis. Conidiophores are reduced to conidiogenous cells. Conidiogenous cells 5–12 × 3–5 µm ($\overline{x}$ = 8 × 4 µm, n = 30), hyaline, thin-walled, holoblastic, smooth, subglobose to pear-shaped, swollen at the base, sometimes one conidiogenous cell producing two conidia. Conidia 9–11 × 4–5 µm ($\overline{x}$ = 10 × 5 µm, n = 60), subglobose, ovate, clavate, ellipsoid, allantoid or irregular, hyaline, aseptate, several small to one big guttulate, smooth-walled.

Culture characteristics: Conidia germinating on PDA within 12 h and germ tubes produced from one end of the conidia. Colonies on PDA, circular, reaching 5 cm in one month at room temperature, flat surface, pale brown to brown in PDA medium. Mycelium superficial, white to brown, hairy, effuse with wavy edge, dense, circular, raised, undulate to filiform with age; reverse light brown in the middle, with a dark brown deposit on the outside.

Material examined: China, Yunnan Province, Dali City, Eryuan County, Xihu Lake, 26°00′33″ N, 100°03′35″ E (1970 m), on unknown submerged decaying wood, 8 May 2021, S.P. Huang and L.L Li, L412 (KUN-HKAS 124589), living cultures, KUNCC 22-12413.

Known host and distribution: EGYPT, Sohag City, on decayed wood submerged in the River Nile, CBS H-22674 (holotype) [72].

Notes: The multigene phylogenetic analysis showed that our new collection (KUNCC 22-12413) clustered with the ex-type strain of Halobyssothecium unicellulare (MD 6004) with 91% ML/1.00 PP support (Figure 2). Morphologically, our new collection fits well with the original description of H. unicellulare [72]. The nucleotide comparison of LSU and SSU sequence data between our new collection (KUNCC 22-12413) and H. unicellulare (MD 6004) revealed 2 bp (including one gap) and 1 bp (including one gap) differences, respectively. We therefore identified it as H. unicellulare and it was reported from China for the first time.

Lentithecium pseudoclioninum Kaz. Tanaka & K. Hiray, Studies in Mycology 82: 99 (2015). Figure 6.

Index Fungorum number: IF811309; Facesoffungi number: FoF12785.

Saprobic on submerged decaying wood in a freshwater lake. Asexual morph: Undetermined. Sexual morph: Ascomata 201–310 µm high, 227–274 µm wide, black, semi-immersed, gregarious, erumpent, globose or subglobose, uniloculate, ostiolate. Ostiolar neck central, papillate, 92–110 µm long, 100–107 µm wide. Peridium 20–32 µm, thick-walled, brown to dark brown cells, composing several layers of pseudoparenchymatous cells of textura angularis, outer layers heavily pigmented, inner layers hyaline to pale brown, flattened. Pseudoparaphyses 2–3 µm wide, filamentous, branched septate. Asci 98–118 × 14–16 µm ($\overline{x}$ = 108 × 15 µm, n = 30), 8-spored, bitunicate, fissitunicate, cylindric-clavate, slightly curved, pedicellate, apex rounded with a minute ocular chamber. Ascospores 28–32 × 8–10 µm ($\overline{x}$ = 30 × 9 µm, n = 30), overlapping uni- to biseriate, narrowly fusiform, with a nearly median primary septum, constricted at the septum, hyaline, guttulate, usually with 2–4 larger guttules, asymmetrical, broadly fusiform, narrowly rounded at the ends, with a mucilaginous sheath.

Culture characteristics: Ascospore germinating on PDA within 12 h and germ tubes produced from the ends of the spore. Colonies on PDA, circular, reaching 5 cm in one month at room temperature, smooth surface, papillae, brown to dark brown. Mycelium superficial, hairy, smooth, circular, reverse grayish; reverse pale to brown, crack at the middle, flocculent at the edge.

Material examined: China, Yunnan Province, Dali City, Eryuan County, Xihu Lake, 26°00′33″ N, 100°03′35″ E (1970 m), on unknown submerged decaying wood, 8 May 2021, S.P. Huang and L.L Li, L413 (KUN-HKAS 124590), living cultures (KUNCC 22-12414); ibid., Erhai Lake, 26°00′32″ N, 100°03′35″ E (1970 m), on unknown submerged decaying wood, 01 April 2021, Z.Q. Zhang, L445 (KUN-HKAS 124593), living cultures (KUNCC 22-12415)

Known host and distribution: JAPAN, Aomori, Hirosaki, Aoki, Mohei pond, on submerged twigs of woody plant, KT 1113 (holotype) and KT 1111 (paratype); China, Guizhou Province, Weining City, Caohai National Nature Reserve, near 26.817° N, 104.217° E, on submerged decaying aquatic plants in Caohai lake, GZAAS 20-0378 [15].

Notes: Our two new collections are morphologically consistent with the holotype of Lentithecium pseudoclioninum [34]. In addition, phylogenetic analysis revealed that these two collections clustered with L. pseudoclioninum (Figure 2). Based on morphological and phylogenetic evidence, we identified our new collection as L. pseudoclioninum. Lentithecium pseudoclioninum has been collected on submerged twigs of woody plants in China and Japan [15,34]. Our two specimens were collected from a freshwater plateau lake in Yunnan, China.

Lentithecium yunnanensis W.H. Lu, Karun. & Tibpromma, Phytotaxa 554: 108 (2022). Figure 7.

Index Fungorum number: IF559622; Facesoffungi number: FoF 10778.

Saprobic on submerged decaying wood in a freshwater lake. Asexual morph: Undetermined. Sexual morph: Ascomata 246–285 µm high, 179–229 µm wide, immersed to semi-immersed, clustered, sometimes solitary, scattered, subglobose or ellipsoidal, dark brown to black, carbonaceous, uni- to bi-loculate, with indistinct ostiolate. Ostiolar neck central, papillate, 127–156 µm long, 96–110 µm wide. Peridium 11–21 µm thick, composed of several layers of pseudoparenchymatous cells, outer layer composed of back brown to brown cells, arranged in textura angularis, inner layer composed of hyaline cells, arranged in textura angularis. Pseudoparaphyses about 2 µm wide, hyaline, filamentous, branched, septate, globose to subglobose swollen at the apex, sometimes swollen at the septum, anastomosing at the apex, embedded in a hyaline gelatinous matrix. Asci 98–117 × 14–15 µm ($\overline{x}$ = 108 × 15 µm, n = 15), 8-spored, clavate to subcylindrical, bitunicate, apex rounded, short pedicellate with an ocular chamber. Ascospores 27–30 × 5–6 µm ($\overline{x}$ = 28 × 6 µm, n = 30), overlapping, uniseriate to biseriate, hyaline, 1-septate, smooth, constricted at the septa, slightly curved, guttulate, lacking gelatinous sheaths or appendages.

Culture characteristics: Ascospore germinating on PDA within 12 h and germ tubes produced from both ends of the spore. Colonies on PDA, circular, reaching 6 cm in 45 days at room temperature, smooth surface, papillae, brown in PDA medium. Mycelium superficial, brown to dark brown, hairy, smooth, circular; reverse brown to dark brown, crack at the middle, flocculent at the edge.

Material examined: China, Yunnan Province, Dali City, Eryuan County, Xihu Lake, 26°17′37″ N, 99°58′33″ E (2100 m), on unknown submerged decaying wood, 22 July 2021, L.L. Li, L680 (KUN-HKAS 124598), living cultures (KUNCC 22-12420 = KUNCC 22-12422); ibid., 26°17′24″ N, 99°57′56″ E (2100 m), on unknown submerged decaying wood, 22 July 2021, X.J. Yuan, L679 (KUN-HKAS 124597), living culture (KUNCC 22-12421).

Known host and distribution: China, Yunnan, Kunming, Songhua Dam Reservoir, on dead culms of Artemisia sp., HKAS 123192 (holotype) [73].

Notes: Lentithecium yunnanensis is a terrestrial species introduced by Lu et al. [73] that occurs on dead culms of Artemisia sp. near humid places. We collected two Lentithecium-like collections from decaying wood submerged in Xihu Lake, Dali, Yunnan Province. Phylogenetic analysis showed that our two new collections clustered with two strains of L. yunnanensis (KUNCC 22-10776 and KUNCC 22-10776). In addition, the morphology of our two collections is similar to the holotype of L. yunnanensis in having semi-immersed to immersed, subglobose to globose ascomata with short ostioles, and hyaline, clavate to fusiform, septate ascospores. Therefore, the two new collections were identified as L. yunnanensis, which was reported from the freshwater habitat for the first time.

Paralentithecium H.W. Shen, K.D. Hyde & Z.L. Luo gen. nov.

MycoBank number: 849738.

Etymology: referring to the comparable morphological characters to that of Lentithecium.

Saprobic on submerged decaying wood in a freshwater lake. Asexual morph: Undetermined. Sexual morph: Ascomata immersed to semi-immersed, clustered, sometimes solitary, scattered, subglobose or ellipsoidal, dark brown to black, carbonaceous, uni- to bi-loculate, with indistinct ostiolate. Peridium thick, composed of several layers of pseudoparenchymatous cells, an outer layer composed of back brown to brown cells, arranged in textura angularis, and an inner layer composed of hyaline cells, arranged in textura angularis. Pseudoparaphyses thick, hyaline, filamentous, branched, septate, globose to subglobose swollen at the apex, sometimes swollen at the septum, anastomosing at the apex, embedded in a hyaline gelatinous matrix. Asci 8-spored, clavate to subcylindrical, bitunicate, apex rounded, short pedicellate with an ocular chamber. Ascospores overlapping, uniseriate to biseriate, hyaline, 1-septate, smooth, constricted at the septa, slightly curved, with gelatinous sheaths.

Type species: Paralentithecium aquaticum (Yin. Zhang, J. Fourn. & K.D. Hyde) H.W. Shen & Z.L. Luo.

Paralentithecium aquaticum (Yin. Zhang, J. Fourn. & K.D. Hyde) H.W. Shen & Z.L. Luo, comb. nov.

MycoBank number: MB 512791.

Basionym: Lentithecium aquaticum Yin. Zhang, J. Fourn. & K.D. Hyde, Fungal Diversity 38: 234 (2009).

Known host and distribution: FRANCE, Ariège, Rimont, Peyrau, on submerged wood of Fraxinus excelsior; on submerged wood of Alnus glutinosa; Le Baup brook, along D 18, on submerged wood of Platanus sp. [35].

Notes: Lentithecium aquaticum was introduced by Zhang et al. [35] based on phylogenetic analysis and morphological characteristics. The placement of this species was not stable and has been changed by several studies [10,45,71,74]. Previous phylogenetic analyses indicated that Lentithecium aquaticum did not cluster with other Lentithecium species, and it formed an individual lineage basal to Darksidea, Halobyssothecium and Lentithecium [10,14,34,41,48,71]. Furthermore, phylogenetic studies of Dayarathne et al. [45] and Devadatha et al. [71] showed that L. aquaticum clustered within Setoseptoria. Several other studies excluded L. aquaticum from Lentithecium [10,74]. The latest phylogenetic analysis based on combined ITS, LSU, SSU, and tef 1-α genes showed that L. aquaticum formed a separate lineage outside of Lentithecium [48]. Our phylogenetic analysis shows that L. aquaticum clusters with our new collection KUNCC 22-12412 and forms a distinct lineage within Lentitheciaceae with 100 ML/1.00 PP support (Figure 2). Therefore, we propose a new genus, Paralentithecium to accommodate Paralentithecium aquaticum (Lentithecium aquaticum) and a new species P. suae.

Paralentithecium suae H.W. Shen, K.D. Hyde & Z.L. Luo sp. nov. Figure 8.

MycoBank number: 849739; Facesoffungi number: FoF 14876.

Etymology: “suae” (Lat.) in memory of the Chinese mycologist Prof. Hong-Yan Su (4 April 1967–3 May 2022).

Holotype: KUN-HKAS 124587.

Saprobic on submerged decaying wood in a freshwater lake. Asexual morph: Undetermined. Sexual morph: Ascomata 212–253 µm high, 175–204 µm wide, immersed to semi-immersed, clustered, sometimes solitary, scattered, subglobose or ellipsoidal, dark brown to black, carbonaceous, uni- to bi-loculate, with indistinct ostiolate. Peridium 17–32 µm thick, composed of several layers of pseudoparenchymatous cells, outer layer composed of bark brown to brown cells, arranged in textura angularis, inner layer composed of hyaline cells, arranged in textura angularis. Pseudoparaphyses 2–3 µm wide, hyaline, filamentous, branched, septate, globose to subglobose swollen at the apex, sometimes swollen at the septum (6–10 µm wide), anastomosing at the apex, embedded in a hyaline gelatinous matrix. Asci 104–134 × 24–28 µm ($\overline{x}$ = 119 × 26 µm, n = 25), 8-spored, clavate to subcylindrical, bitunicate, apex rounded, short pedicellate with an ocular chamber. Ascospores 28–34 × 11–14 µm ($\overline{x}$ = 31 × 13 µm, n = 40), overlapping, uniseriate to biseriate, hyaline, 1-septate, broadly fusiform, smooth, constricted at the septa, slightly curved, guttulate, with gelatinous sheaths.

Culture characteristics: Ascospore germinating on PDA within 12 h and germ tubes produced from both ends of the spore. Colonies on PDA, circular, reaching 4–5 cm in one month at room temperature, smooth surface, papillae, brown to dark brown, olive green in PDA medium. Mycelium superficial, brown to dark brown, hairy, smooth, circular; reverse dark brown, crack at the middle, flocculent at the edge, dark brown with greenish.

Material examined: China, Yunnan Province, Lijiang City, Ninglang County, Luguhu Lake, 27°44′15″ N, 100°45′16″ E (2700 m), on unknown submerged decaying wood, 5 March 2021, Z.Q. Zhang and L. Sha, L184 (KUN-HKAS 124587, holotype), ex-type living cultures (CGMCC 3.24265 = KUNCC 22–12412).

Notes: In our phylogenetic analysis, Paralentithecium suae clustered with P. aquaticum with 100% ML/1.00 PP support (Figure 2). Comparison of ITS, LSU, SSU, and tef 1-α sequences between Paralentithecium suae and P. aquaticum revealed 11 bp, 4 bp, 4 bp, and 22 bp differences, respectively. Paralentithecium suae resembles P. aquaticum in having hyaline, 1-septate, broadly fusiform ascospores with gelatinous sheaths [35]. However, P. suae is distinct from P. aquaticum in having globose to subglobose pseudoparaphyses that are swollen at the apex and sometimes swollen at the septum. In contrast, the pseudoparaphyses of P. aquaticum are not swollen. In addition, ascospores of P. aquaticum contain four refractive globules, while P. suae has ascospores with many small guttules [35]. Therefore, we introduce P. suae as a new species.

Setoseptoria bambusae J. Yang, Jian K. Liu & K.D. Hyde, Fungal Diversity 119: 44 (2022). Figure 9.

Index Fungorum number: IF559452; Facesoffungi number: FoF12786.

Saprobic on submerged decaying wood in a freshwater lake. Asexual morph: Undetermined. Sexual morph: Ascomata 245–375 µm high, 194–296 µm wide, black, superficial to semi-immersed, gregarious, fully or partly erumpent, globose, uniloculate, ostiolate. Peridium 26–39 µm wide, thick, multi-layered, outer layer most heavily pigmented, comprising blackish to dark brown amorphous layer, middle layer heavily pigmented, inner layer, pale brown to hyaline, cells towards the inside lighter, flattened, thick-walled. Pseudoparaphyses 2–3 µm wide, filamentous, branched septate. Asci 113–128 × 15–19 µm ($\overline{x}$ = 120 × 17 µm, n = 30), 8-spored, bitunicate, fissitunicate, clavate to cylindric-clavate, pedicellate, apex rounded with a minute ocular chamber. Ascospores 32–40 × 6–8 µm ($\overline{x}$ = 36 × 7 µm, n = 20), overlapping uni- to biseriate, narrowly fusiform, with a nearly median primary septum, deeply constricted at the septum, hyaline, guttulate, asymmetrical, conical, and narrowly rounded at the ends.

Culture characteristics: Ascospore germinating on PDA within 12 h and germ tubes produced from one end of the spore. Colonies on PDA, circular, reaching 6 cm in 45 days at room temperature, smooth surface, papillae, pale brown in PDA medium. Mycelium superficial, grayish-brown to brown, hairy, smooth, circular; reverse pale brown at the edges, dark brown in the middle, flocculent at the edge.

Material examined: China, Yunnan Province, Yuxi City, Jiangchuan District, Xingyunhu Lake, 24°23′05″ N, 102°48′22″ E (1720 m), on unknown submerged decaying wood, 10 July 2021, H.W. Shen, L511 (KUN-HKAS 124592), living culture (KUNCC 22–12417); ibid., 24°23′05″ N, 102°48′22″ E (1720 m), on the submerged stem of Phragmites sp. (Poaceae), 10 July 2021, S. Luan, L579 (KUN-HKAS 124596), living culture (KUNCC 22–12418); ibid., on submerged stem of Phragmites sp. (Poaceae), 10 July 2021, Y.K. Jiang, L474 (KUN-HKAS 124591), living culture (KUNCC 22–12416).

Known host and distribution: China, Guizhou Province, Anshun City, Gaodang Village, 26.071° N, 105.698° E, Suoluo River, on decaying bamboo culms submerged in a freshwater stream, HKAS 112629 (holotype) [15].

Notes: Setoseptoria bambusae was introduced by Yang et al. [15] to accommodate two collections, GZCC 17–0044 (ex-type strain) and IFRD500-013 (previously identified as S. arundinaceae, without description). In this study, our four new collections clustered with the ex-type strain of S. bambusae with 100% ML/1.00 PP statistical support (Figure 2). Furthermore, our collections fit the morphological characteristics of S. bambusae except for the size of asci and ascospores, our isolate has shorter asci (113–128 vs. 130–180 µm) and longer ascospores (32–40 vs. 28–37 µm). Therefore, we identified them as S. bambusae. Our four new collections were collected from lentic freshwater habitats. The holotype was collected from lotic habitats.

Setoseptoria suae H.W. Shen, K.D. Hyde & Z.L. Luo sp. nov. Figure 10.

MycoBank number: 849740; Facesoffungi number: FoF 14877.

Etymology: “suae” (Lat.) in memory of the Chinese mycologist Prof. Hong-Yan Su (4 April 1967–3 May 2022).

Holotype: KUN-HKAS 124595.

Saprobic on submerged decaying wood in a freshwater lake. Sexual morph: Undetermined. Asexual morph: Conidiomata 383–512 µm high, 173–196 µm wide, solitary, scattered, semi-immersed to immersed in the host, pycnidial, subglobose to ellipsoidal, unilocular, black, ostiolate, apapillate. Ostiole short, centrally located. Conidiomatal wall 33–55 µm wide, thickening at the upper zone, thick-walled, composed of several layers of textura angularis, an outer layer comprising brown to dark brown cells, pigmented; inner layer comprising hyaline cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells (4–)7–15(–26) × 4–6 µm ($\overline{x}$ = 11 × 5 µm, n = 25), arising from the inner layers of conidiomata, hyaline, enteroblastic, phialidic, determinate, ampuliform, subcylindrical to lageniform. Conidia 33–43 × 4–6 µm ($\overline{x}$ = 38 × 5 µm, n = 50), subcylindrical, with obtuse to subobtuse ends, straight or slightly curved, hyaline, (1–)3-septate, euseptate, mostly with one large central guttule per cell when young, with many small guttules in each cell at maturity, slightly constricted at the septum, smooth-walled.

Culture characteristics: Conidia germinated on PDA within 12 h and germ tubes produced from the ends of the spore. Colonies on PDA, circular, reaching 6 cm in one month at room temperature, brown to dark brown. Mycelium superficial, brown to dark brown, hairy, smooth, circular; dark brown from below.

Material examined: China, Yunnan Province, Yuxi City, Tonghai County, Qiluhuhu Lake, 24°08′37″ N, 102°46′24″ E (1800 m), on submerged stem of Phragmites sp. (Poaceae), 11 July 2021, H.W. Shen, L570 (KUN-HKAS 124595, holotype), ex-type living cultures (CGMCC 3.24266 = KUNCC 22–12419).

Notes: Phylogenetic analysis showed that Setoseptoria suae clustered with S. phragmitis with 100% ML/0.99 PP statistical support (Figure 2). The comparison of ITS and LSU sequences between S. suae and S. phragmitis shows that the similarities are 96.9% (538/555 bp) and 99.9% (826/827 bp), respectively. Setoseptoria suae resembles S. phragmitis in having immersed, globose conidiomata, hyaline, subcylindrical, smooth, guttulate, (1–)3-septate conidia [37]. However, Setoseptoria suae can be distinguished from S. phragmitis by its larger conidia (33–43 × 4–6 µm vs. (19–)25–35(–38) × (3.5–)4 µm). In addition, the conidia of S. phragmitis mostly have one large central guttule per cell, while Setoseptoria suae has conidia with many small guttules in each cell. We, therefore, introduce S. suae as a new species.

4. Discussion

Yunnan, located on the Yunnan–Guizhou Plateau, is one of the global biodiversity hotspots with rich biological resources [18,19,75]. In recent years, research on lignicolous freshwater fungi in Yunnan has developed rapidly, and a large number of new species have been reported from lotic freshwater habitats such as streams and rivers [10,13,76,77,78,79,80,81]. A few studies have reported lignicolous freshwater fungi from lentic habitats in Yunnan Province. For example, Cai et al. [17] and Luo et al. [2] investigated lignicolous freshwater fungi in Fuxianhu and Dianchi Lakes, respectively. However, freshwater fungi in lentic habitats have not been updated recently. In this study, we investigate the freshwater fungi in Cibihu, Luguhu, Qiluhu, Xihu, and Xingyunhu lakes in Yunnan Province, one new genus, two new species, and three new records are reported, the results indicate that high undiscovered diversity of lignicolous freshwater fungi in lentic habitats.

Zhang et al. [36] provided the first multigene phylogenetic analysis of Pleosporales and introduced the family Lentitheciaceae which accepted the genera Lentithecium, Katumotoa, and Keissleriella. Dong et al. [10] treated the family with ten genera and this was followed by Wijayawardene et al. [41]. Previous studies based on morphology and phylogenetic analyses showed that the classification of Lentithecium, Keissleriella, and Setoseptoria is confusing as the placement of several taxa was problematic and has been transferred to different genera. For example, Suetrong et al. [82] transferred Keissleriella rara to Lentithecium as L. rarum; however, later studies showed that L. rarum clustered with K. trichophoricola in Keissleriella [14]. Similarly, Zhang et al. [35] transferred Keissleriella linearis to Lentithecium as L. lineare, Singtripop et al. [83] re-examined the type specimen of K. linearis (L. lineare) and transferred it to Keissleriella based on LSU phylogenetic analysis, and this was confirmed by subsequent phylogenetic studies [14,34,72]. The placements of Lentithecium species have been revised in recent years based on multigene phylogenetic studies [14,34,84]. Calabon et al. [14] transferred several Lentithecium species with brown and versicolored ascospores without sheaths and hyaline conidia to Halobyssothecium, including L. cangshanense, L. carbonneanum, L. kunmingense, L. unicellulare, and L. voraginesporum. Currently, 13 species are accepted in Halobyssothecium. In the present study, we report the sexual morph of H. phragmitis and provide detailed morphological descriptions for its sexual morph.