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

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.

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].
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.

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.

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).

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].

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.
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.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.
The multigene phylogenetic analyses showed that the 13 fresh collections clustered within Lentitheciaceae.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.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.
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.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.
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 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.
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.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 Lentitheciumlike 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.
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.
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 •    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.
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.

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

Figure 1 .
Figure 1.(a) Sample with a label (arrow indicates sample number); (b) Samples in the plastic box; (c) Plastic box with labels (arrows indicate labels documenting detailed sampling sites and sample order); (d) The samples were incubated on the culture rack.

Figure 2 .
Figure 2. Maximum likelihood (ML) tree is based on combined LSU, SSU, ITS, and tef 1-α sequ data.Bootstrap support values with an ML greater than 70% and Bayesian posterior probabi (PP) greater than 0.97 are given above the nodes, shown as "ML/PP".The tree is rooted to Ple nodictys capensis (CBS 968.97) and P. descalsii (CBS 142298).New species are indicated in red b new strains are indicated in blue, and type strains are in black bold.

Figure 2 .
Figure 2. Maximum likelihood (ML) tree is based on combined LSU, SSU, ITS, and tef 1-α sequence data.Bootstrap support values with an ML greater than 70% and Bayesian posterior probabilities (PP) greater than 0.97 are given above the nodes, shown as "ML/PP".The tree is rooted to Pleomonodictys capensis (CBS 968.97) and P. descalsii (CBS 142298).New species are indicated in red bold, new strains are indicated in blue, and type strains are in black bold.

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