﻿Two novel freshwater hyphomycetes, in Acrogenospora (Minutisphaerales, Dothideomycetes) and Conioscypha (Conioscyphales, Sordariomycetes) from Southwestern China

﻿Abstract Freshwater fungi are highly diverse in China and frequently reported from submerged wood, freshwater insects, herbaceous substrates, sediments, leaves, foams, and living plants. In this study, we investigated two freshwater species that were collected from Yunnan and Guizhou provinces in China. Detailed morphological analysis complemented by multi-gene phylogenetic analyses based on LSU, SSU, ITS, RPB2 and TEF1-α sequences data revealed them to be two new saprobic species, namely Acrogenosporaalangiisp. nov. and Conioscyphayunnanensissp. nov. in their asexual morphs. Additionally, Acrogenosporaalangiisp. nov. is reported for the first time as a freshwater ascomycete associated with the medicinal plant Alangiumchinense (Alangiaceae). Detailed morphological descriptions, illustrations and updated phylogenetic relationships of the new taxa are provided herein.

Species of Conioscypha are mostly reported from freshwater and terrestrial habitats and primarily recorded in their asexual morph.Only few species are reported in sexual morph (Shearer 1973;Shearer and Motta 1973;Kirk 1984;Zelski et al. 2015).The asexual morph is characterized by the enteroblastic percurrent conidiogenesis in distinct conidiogenous cells that retain successive wall layers at the same level as multi-collaretted as each conidium ruptures through the apex with dematiaceous aseptate conidia of various shapes (Shearer 1973;Shearer and Motta 1973;Kirk 1984;Zelski et al. 2015).The sexual morph is characterized by perithecial ascomata that are immersed to superficial, globose to subglobose, cylindrical-clavate asci with a pronounced non-amyloid apical annulus, transversely multi-septate and hyaline ascospores (Luo et al. 2019).
Guizhou and Yunnan provinces are mostly referred as part of the Southwestern China (Feng and Yang 2018;Jiang et al. 2022).This region is a center of biodiversity for freshwater fungi (Shen et al. 2022).Many new freshwater fungi have been reported in Yunnan and Guizhou provinces in recent years (Su et al. 2016;Wang et al. 2016;Li et al. 2017Li et al. , 2020;;Luo et al. 2018aLuo et al. , b, 2019;;Zhao et al. 2018;Dong et al. 2020;Wan et al. 2021;Shen et al. 2022).In particular, Yunnan province stands out as a hotspot for freshwater fungal research (Luo et al. 2019;Dong et al. 2020;Shen et al. 2022).The diversity of freshwater fungi in streams and rivers in northwestern Yunnan has been intensely studied, resulting in the discovery of a large number of new species and new records in some highly diverse genera e.g.Acrogenospora, Dictyosporium, Distoseptispora, Pleurotheciella, Sporidesmium and Sporoschisma (Su et al. 2016;Wang et al. 2016;Li et al. 2017Li et al. , 2020;;Luo et al. 2018aLuo et al. , b, 2019;;Zhao et al. 2018;Bao et al. 2020;Wan et al. 2021;Shen et al. 2022).
In this study, two collections were obtained from decaying submerged wood and dead branches of Alangium chinense in freshwater habitat in Southwestern China.Multi-gene phylogenetic analyses based on Maximum likelihood (ML) and Bayesian analyses along with morphological characters support the establishment of the new species.We also provided a comparative synoptic table for Conioscypha.This study adds new data to our knowledge on fungal diversity of freshwater streams in Southwestern China.

Sample collection, isolation and morphological studies
Submerged decaying wood and branches were collected from Guizhou and Yunnan provinces, China.Fresh specimens were studied following the methods described by Luo et al. (2018b).The samples were incubated in plastic boxes at room temperature for one week.Micromorphological characters were observed using a stereomicroscope (SteREO Discovery.V12, Carl Zeiss Microscopy GmBH, Germany) and photographed using a Nikon ECLIPSE 80i compound microscope fitted with a NikonDS-Ri2 digital camera.Microscopic structures were measured using Tarosoft (R) Image Frame Work program and the photo-micrographs were processed using Adobe Photoshop CS6 version 10.0 software (Adobe Systems, USA).
Single spore isolation was performed following the method described by Luo et al. (2018b).The germinated conidia were transferred to fresh PDA plates and incubated at room temperature.The specimens were dried under natural light, wrapped in absorbent paper, and placed in a Ziplock bag with mothballs.Herbarium specimens were deposited in the Herbarium of Cryptogams, Kunming Institute of Botany Academia Sinica (KUN-HKAS), Kunming, China, and Herbarium, University of Electronic Science and Technology (HUEST), Chengdu, China.The cultures were deposited in Kunming Institute of Botany, Chinese Academy of Sciences (KUNCC), Kunming, Yunnan, China and the University of Electronic Science and Technology Culture Collection (UESTCC), Chengdu, China.The novel species were registered in Faceoffungi (Jayasiri et al. 2015) and MycoBank databases (https://www.mycobank.org/mycobank-deposit;accessed on 22 September 2023).

DNA extraction, PCR amplification and sequencing
Fresh mycelia were scraped from colonies grown on potato dextrose agar (PDA) medium.DNA extraction was carried out using DNA extraction kit following the manufacturer's instructions (TOLOBIO Plant Genomic DNA Extraction Kit, Tsingke Company, Beijing, P.R. China).PCR amplification was performed using primers pairs LR0R/LR5 (Vilgalys and Hester 1990) for the nuclear ribosomal large subunit 28S rDNA gene (LSU); NS1/NS4 (White et al. 1990) for the nuclear ribosomal small subunit 18S rDNA gene (SSU); ITS5/ITS4 (White et al. 1990) for the internal transcribed spacer rDNA region (ITS); fRPB2-5F/fRPB2-7cR (Liu et al. 1999) for the RNA polymerase second largest subunit (RPB2); and EF1-983F/EF1-2218R (Rehner and Buckley 2005) for the translation elongation factor 1-alpha (TEF1-α).The PCR amplification was carried out in a 25 μL reaction volume containing 12.5 μL of 2× Power Taq PCR Master Mix, 1 μL of each forward and reward primer (10 μM), 1 μL of genomic DNA template (30-50 ng/μL) and 9.5 μL of sterilized double-distilled water.Amplifications were carried out using the BioTeke GT9612 thermocycler (Tsingke Company, Beijing, P.R. China).The PCR amplification conditions for ITS, LSU, and SSU consisted of initial denaturation at 98 °C for 3 minutes, followed by 35 cycles of denaturation at 98 °C for 20 seconds, annealing at 53 °C for 10 seconds, an extension at 72 °C for 20 seconds, and a final extension at 72 °C for 5 minutes.The PCR amplification condition for RPB2 consisted of initial denaturation at 95 °C for 5 minutes, followed by 40 cycles of denaturation at 95 °C for 1 minute, annealing at 52 °C for 2 minutes, an extension at 72 °C for 90 seconds, and a final extension at 72 °C for 10 minutes.The amplification condition for TEF1-α consisted of initial denaturation at 94 °C for 3 minutes, followed by 35 cycles of 45 seconds at 94 °C, 50 seconds at 55 °C and 1 minute at 72 °C, and a final extension period of 10 minutes at 72 °C.Quality of PCR products were checked using 1% agarose gel electrophoresis and distinct bands were visualized in gel documentation system (Compact Desktop UV Transilluminator analyzer GL-3120).The PCR products were purified and obtained Sanger sequences by Tsingke Company, Beijing, P.R. China.
The evolution model for the Bayesian inference (BI) analyses was performed using MrModeltest v2.3 (Ronquist et al. 2012).GTR+I+G was selected as the best-fit model for LSU, SSU, ITS, RPB2 and TEF1-α dataset.Markov Chain Monte Carlo sampling (MCMC) was computed to estimate Bayesian posterior probabilities (BPP) using MrBayes v.3.2.7 (Ronquist et al. 2012).Six simultaneous Markov chains were run for random trees for 1,000,000 generations and trees were sampled every 200 th generation.The first 10% of the total trees were set as burn-in and were discarded.The remaining trees were used to calculate Bayesian posterior probabilities (BPP) in the majority rule consensus tree (when the final average standard deviation of split frequencies reached below 0.01).Phylograms were visualized using FigTree v1.4.0 (Rambaut 2006) and rearranged in Adobe Photoshop CS6 software (Adobe Systems, USA).

Phylogenetic analyses
Two phylogenetic analyses were conducted to resolve the phylogenetic affinities of the two new freshwater species, one each, within the genera Acrogenospora (Acrogenosporaceae/ Minutisphaerales/ Dothideomycetes; Analysis 1), and the other within Conioscypha (Conioscyphaceae/ Conioscyphales/ Sordariomycetes; Analysis 2), as follows: Analysis 1: The phylogram generated from ML analysis based on combined LSU, SSU, ITS, RPB2 and TEF1-α sequences data was selected to represent the relationship between the new species and other known species in Acrogenospora.Twenty-six strains were included in the combined dataset which comprised 4,527 characters (LSU: 987 bp, SSU: 1007 bp, ITS: 535 bp, RRB2: 1044 bp, TEF1-α: 954 bp) after alignment (including gaps).Minutisphaera aspera (DSM29478) and M. japonica (HHUF30098) were selected as the outgroup taxa.The best RAxML tree with a final likelihood value of -15211.062629 is presented in Fig. 1.RAxML analysis yielded 1,028 distinct alignment patterns and 43.09% of undetermined characters or gaps.Estimated base frequencies were as follows: A = 0.260065, C = 0.232516, G = 0.268900, T = 0.238519, with substitution rates AC = 1.050467,AG = 3.191516, AT = 1.485302,CG = 1.086194,CT = 7.658416, GT = 1.000000; gamma distribution shape parameter alpha = 0.180026.The final average standard deviation of split frequencies at the end of total MCMC generations for BI analysis was 0.009674 (the critical value for the topological convergence diagnostic is below 0.01).
Phylogenetic analyses retrieved from ML and BI analyses were not significantly different and showed similar topologies.Phylogenetic analyses showed that our new collection (KUNCC23-14553 and UESTCC 23.0140) formed an independent subclade with strong statistical support (100% MLBS/ 1.00 BPP) and shared the same clade with Acrogenospora.terricola and A. thailandica with moderate statistical support (71% MLBS/ 0.95 BPP; Fig. 1).
Analysis 2: The phylogram generated from ML analysis based on combined LSU, ITS, SSU and RPB2 sequences data was selected to represent the relationship between the new species and other known species in Conioscypha.Twenty-three strains were included in the combined dataset which comprised 3,679 characters (LSU: 904 bp, ITS: 696 bp, SSU: 1026 bp, RPB2: 1053 bp) after alignment (including gaps).Parafuscosporella garethii (BCC79986) and P. moniliformis (MFLUCC 15-0626) were selected as the outgroup taxa.The best RAxML tree with a final likelihood value of -14285.072957 is presented in Fig. 2. RAxML analysis yielded 1,112 distinct alignment patterns and 40.07% of undetermined characters or gaps.Estimated base frequencies were as follows: A = 0.236438, C = 0.267389, G = 0.295788, T = 0.200385, with substitution rates AC = 1.738303,AG = 2.933990, AT = 1.389088,CG = 1.593182,CT = 7.181256, GT = 1.000000; gamma distribution shape parameter alpha = 0.453781.The final average standard deviation of split frequencies at the end of total MCMC generations for BI analysis was 0.003901 (the critical value for the topological convergence diagnostic is below 0.01).
Phylogenetic analyses retrieved from ML and BI analyses were not significantly different and showed similar topologies.Phylogenetic analyses showed that our new collections (KUNCC 23-13319 and KUNCC 23-13172) formed an Etymology.The epithet 'alangii' refers to the host genus Alangium on which the holotype was collected.
Culture characteristics.Conidia germinating on PDA within 24 h and germ tubes produced from the conidial base.Colonies reaching 16 mm diam at the room temperature in natural light for one month.Colonies on PDA medium dense, irregular in shape, slightly raised to umbonate or convex, surface rough, radially striated with lobate edge, fairy fluffy to floccose, white at the center, white-gray to gray sparse towards the margin; in reverse, white to white-gray at the center, with dark gray to brown-gray in the middle, white to pale yellowish at the edge, radiating outwards with irregular ring; no pigmentation on PDA.
Notes.In the combined multi-locus phylogenetic analyses, Acrogenospora alangii formed a distinct clade with A. terricola and A. thailandica with significant support (71% MLBS/ 0.95 BPP; Fig. 1).The nucleotide base pair comparison between A. alangii (KUNCC 23-14553) and A. terricola (PS 3610) revealed the differences as 25/829 bp (3.0%) of LSU and 5/1006 bp (0.50%) of SSU.While the differences between A. alangii (KUNCC 23-14553) and A. thailandica (MFLUCC 17-2396) showed 30/834 bp (3.6%) of LSU and 2/1029 bp (0.2%) of SSU and 131/1045 bp (12.5%) of RPB2.Acrogenospora alangii can be distinguished from A. terricola in having conidia that are hyaline to pale gray when young, becoming pale brown to dark brown when mature, while A. terricola has olive green to dark brown conidia.Additionally, A. thailandica differs from A. alangii in having deep brown to black conidia (Hyde et al. 2019;Harrington et al. 2022).Furthermore, A. alangii also differs from the type species A. sphaerocephala in conidial color which is dark reddish brown, or pale to mid brown in A. sphaerocephala (Hughes 1978).Both A. alangii and A. guizhouensis were collected from Guizhou Province.However, morphological comparison of A. alangii with A. guizhouensis shows their differences in conidial color (hyaline, to pale gray, becoming pale brown to dark brown vs. brown) and position of conidial development (acropleurogenous vs. acrogenous) (Hyde et al. 2023).Etymology.The specific epithet "yunnanensis" refers to the name of the region, Yunnan Province (China), from where the holotype was collected.
Culture characteristics.Conidia germinating on PDA within 48 h and germ tubes produced from the conidial base.Colonies reaching 4.3 mm diam at room temperature in natural light for three months.Colonies on PDA medium dense to dense, circular, white and gray in the center, with packed mycelium, becoming black mycelial patch in the middle, white to cream at the margin, slightly radiating with irregular edge, radially furrowed aspect; in reverse, dark brown to black at the center, radiated with pale yellowish and dark greenish furrowed ring, white to cream at the margin with furrows aspects; no pigmentation on PDA. ) of SSU differences.The new taxon shares similar morphology with C. peruviana in having cup-like phialidic conidiogenous cells, and brown conidia but differing by varied shapes (globose to subglobose vs. ellipsoidal to allantoid or fabiform), the size (18-26 × 17-22 µm vs. 13.5-18 × 5-8.5 µm) and absence of lipid droplets (Zelski et al. 2015).Conioscypha yunnanensis also resembles C. minutispora in having subglobose conidia but differs in the size measurement (18-26 × 17-22 µm vs. 6-9 × 5-6 µm) (Crous et al. 2014).Furthermore, C. yunnanensis shares similar morphology to the type species C. lignicola in having micronematous conidiophores and globose to subglobose conidia that are brown.However, C. yunnanensis differs by the absence of dark brown ring surrounded in the conidia and presence of guttules periphery of conidia (Shearer 1973).The morphological comparison with other Conioscypha species is also provided in Table 3.

Discussion
Dothideomycetes and Sordariomycetes are the two largest classes of lignicolous freshwater fungi (Luo et al. 2019;Calabon et al. 2022;Shen et al. 2022).In this study, two new freshwater species belonging to Dothideomycetes and Sordariomycetes were introduced which add to the fundamental knowledge on the diversity of freshwater fungi in Southwestern China.Furthermore, an updated phylogenetic information was provided and thus we attempted to resolve the taxonomic ambiguities of the genus Acrogenospora (Acrogenosporaceae, Minutisphaerales, Dothideomycetes) and Conioscypha (Conioscyphaceae, Conioscyphales, Sordariomycetes).The study will provide a better understanding of the taxonomic boundaries of these two genera with the illustration of two new species.Acrogenospora species are mostly reported from freshwater habitats (Bao et al. 2020;Hyde et al. 2023).Recent studies have revealed that more than half of the new and interesting Acrogenospora species were observed from freshwater habitats in China, including A. alangii (in this study), A. aquatica, A. basalicellularispora, A. ellipsoidea, A. guizhouensis, A. guttulatispora, A. hainanensis, A. obovoidspora, A. olivaceospora, A. ovalia, A. sphaerocephala, A. submersa, A. subprolata, A. verrucispora and A. yunnanensis (Goh et al. 1998;Ho et al. 2001;Zhu et al. 2005;Hu et al. 2010;Bao et al. 2020;Hyde et al. 2023).Previous studies revealed that the highest number of Acrogenospora species were reported from Yunnan Province whereas a few species have been reported from Guizhou, Hainan, Hongkong, and Xizang.This suggests a high diversity of freshwater fungi in Yunnan Province, especially of the genus Acrogenospora.Simultaneously, Guizhou Province is located in southwestern China that shares similar biogeographical environments with Yunnan Province and therefore, the province may also offer a potential diversity of Acrogenospora.
Morphologically, species of Acrogenospora are distinguished from each other with difficulty and previous studies made efforts to segregate them based on shape, size, and color of the conidia and the degree of pigmentation of the conidiophores (Hughes 1978;Bao et al. 2020).A comprehensive study of Acrogenospora was carried out by Bao et al. (2020) who provided an updated taxonomic treatment of Acrogenospora and introduced seven new Acrogenospora species from Yunnan, China.Bao et al. (2020) and Hughes (1978) also provided a synoptic table of morphological comparison for all known Acrogenospora species.No significant morphological differences have been observed among known Acrogenospora species according to the species delineation provided by Bao et al. (2020).However, phylogenetic evidence and nucleotide pairwise comparison provide adequate justification for our species novelty following the recommendation of Jeewon and Hyde (2016).
The host association of freshwater fungi is difficult to identify.Besides, Acrogenospora species were mostly reported on submerged wood.Interestingly, the host associations of some Acrogenospora species (e.g., A. altissima, A. gigantospora, A. sphaerocephala, and A. verrucispora) have been identified.In this study, we reported A. alangii from freshwater habitat and associated with the medicinal plant Alangium chinense for the first time.
Present phylogenetic analyses also showed that Acrogenospora carmichaeliana (CBS 206.36) formed a separated clade with other strains of A. carmichaeliana (CBS 164.76, CBS 179.73, FMR 11021).Acrogenospora carmichaeliana (CBS 206.36) was identified as Farlowiella carmichaeliana (sexual morph) by E.W. Mason (https://wi.knaw.nl/fungal_table;accessed on 17 October 2023).While strain CBS 164.76 was priorly identified as Acrogenospora sphaerocephala (on decaying wood in Belgium), strain CBS 179.73 was identified as Farlowiella carmichaeliana (on decaying wood in Germany) and FMR 11021 was identified as Farlowiella carmichaeliana (unknown source).Hyde et al. (2019) introduced a new species A. thailandica and designated the reference specimen for the type species of Acrogenospora, A. sphaerocephala.Based on their phylogenetic analyses, these four unpublished strains were identified as A. carmichaeliana.However, the molecular data from the ex-type strain of A. carmi-quate to justify the species' novelty.Similarly, there are only ITS and LSU sequence data of C. pleiomorpha currently available.These two genes may be inadequate to resolve the phylogenetic relationship of C. pleiomorpha and C. verrucosa.
The phylogenetic relationship of Conioscypha boutwelliae and C. japonica is not well-resolved in the present study.This also may be affected by the available genes used in the analyses.There are only ITS and LSU sequences available for C. boutwelliae whereas LSU, SSU, and RPB2 are available for C. japonica.Unfortunately, the nucleotide pairwise comparison between C. boutwelliae and C. japonica could not be done due to the LSU sequence of C. japonica is too short (531 bp) and lacking needful genetic information compared with C. boutwelliae (1,053 bp).Notably, C. boutwelliae was introduced by Crous et al. (2018).The species was isolated from soil in the Netherlands (holotype CBS H-23743, cultures ex-type CBS 144928 = JW203008, GenBank no.LR025182 (ITS) and LR025183 (LSU), MycoBank: MB828023).The search results of LR025182 (ITS) and LR025183 (LSU) via NCBI nucleotide search brought us to the species C. pleiomorpha.We have rechecked the detailed source information of LR025182 (ITS) and LR025183 (LSU) and resolved that the source information belongs to C. boutwelliae, resulting that the sequence name of C. boutwelliae is incorrect in NCBI database and the name "C. boutwelliae" should instead be referred to as "C.pleiomorpha" for the GenBank no.LR025182 (ITS) and LR025183 (LSU).

Figure 1 .
Figure1.Phylogenetic tree constructed from RAxML analysis of LSU, SSU, ITS, RPB2 and TEF1-α sequences data.Bootstrap support values for ML equal or greater than 50% and Bayesian posterior probabilities greater than 0.95 BPP are indicated at the nodes.The tree is rooted to Minutisphaera aspera (DSM29478) and Minutisphaera japonica (HHUF30098).The new isolates are in red bold.

Figure 2 .
Figure 2. Phylogenetic tree constructed from RAxML analysis of LSU, ITS, SSU and RPB2 sequences data.Bootstrap support values for ML equal or greater than 50% and Bayesian posterior probabilities greater than 0.95 BPP are indicated at the nodes.The tree is rooted to Parafuscosporella moniliformis (MFLUCC 15-0626) and P. garethii (BCC79986).The new isolates are in red bold.

Table 1 .
Taxon names, strain numbers and GenBank accession numbers of the ITS, LSU, SSU, RPB2 and TEF1-α sequences used in the phylogenetic analyses.Newly generated sequences are highlighted in black bold font.The ex-type strains are indicated by superscript T. "-" stands for no sequence data in GenBank.

Table 2 .
Taxon names, strain numbers and GenBank accession numbers of the LSU, ITS, SSU and RPB2 sequences used in the phylogenetic analyses.The newly generated sequences are highlighted in black bold font.The ex-type strains are indicated by superscript T. "-" stands for no sequence data in GenBank.
Abbreviation: AMI-SPL: Collection of A. Mateos & S. De la Peña, Azores, Terceira, Portugal; BCC: BIOTEC Culture Collection, Thailand; CBS: CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; DSM: Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Science Campus Braunschweig-Süd, Germany; FMR: Facultat de Medicina i Ciencies de la Salut, Reus, Spain; JCM: Japan Collection of Microorganism, RIKEN BioResource Center, Japan; HHUF: Herbarium of Hirosaki University, Japan; KUNCC: Kunming Institute of Botany, Chinese Academy of Sciences Culture Collection, Kunming, Yunnan, China; MFLU: the herbarium of Mae Fah Luang University, Chiang Rai, Thailand; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; PS: the R. L. Gilbertson Mycological Herbarium at the University of Arizona (MYCO-ARIZ); UESTCC: University of Electronic Science and Technology Culture Collection, Chengdu, China.et al. 2011; Katoh and Standley 2013).Phylogenetic relationships of the new species were performed based on Maximum likelihood (ML) and Bayesian inference (BI) analyses.

Table 3 .
Synopsis and distribution of Conioscypha species.The new species is indicated by black bold.