Exploring ascomycete diversity in Yunnan, China I: resolving ambiguous taxa in Phaeothecoidiellaceae and investigating conservation implications of fungi

Yunnan, located in southwestern China, is known for its high fungal diversity, and many of which are endemic to the region. As part of our ongoing studies on fungi in Yunnan, we introduce two new genera in Phaeothecoidiellaceae (Mycosphaerellales), to accommodate one Repetophragma-like and another Stomiopeltis-like taxa. Pseudorepetophragma gen. nov. is introduced herein as a monotypic genus to accommodate P. zygopetali comb. nov.(≡ Repetophragma zygopetali), whereas Pseudostomiopeltis gen. nov. is introduced to accommodate Ps. xishuangbannaensis gen. et sp. nov. and Ps. phyllanthi comb. nov.(≡ Stomiopeltis phyllanthi), based on a new collection from Yunnan. In addition, Stomiopeltis sinensis is transferred to Exopassalora as E. sinensis comb. nov. due to its phylogenetic affinity and grouped with E. zambiae, the generic type of Exopassalora. This study provides new insights into the biodiversity of fungal species in this region and adds to our understanding of their ecological roles, as well as the resolution to ambiguous taxa in Phaeothecoidiellaceae.


Introduction
China is home to diverse climates and environments containing four of the world's 36 biodiversity hotspots; of which, three hotspots, the mountain ranges of Southwest China, Eastern Himalaya, and Indo-Burma, intersect with the Yunnan Province (Feng and Yang, 2018;Cai et al., 2019).Yunnan has diverse climate types and environments and is significantly affected by the monsoon rains (abundant rainfall and resultant humid tropical evergreen rainforests).Diverse environments, complex topography and geography, and highly variable plant species allow fungi to specialize and flourish, and these also affect the fungal growth and distribution (Yang et al., 2004;Zhu et al., 2006;Feng and Yang, 2018;Wanasinghe et al., 2020).Furthermore, Yunnan is an agricultural province that cultivates a wide variety of agricultural and horticultural crops such as coffee (Arabica), commercial flowers, fruits (e.g., grapes, passion fruits, bananas, and mangoes), grains (e.g., rice), rubber, sugarcane, tea (Pu'er), tobacco, plantation trees (e.g., Yunnan pine, bamboo, and teak), vegetables as well as wild edible mushrooms (Li et al., 2011;Frayer et al., 2014;Zhang et al., 2014;Shen et al., 2017;ORO Yunnan Commerce, 2023).This also led Yunnan to host a high diversity of fungi that has not been studied so far.
According to Feng and Yang (2018), there may be approximately 104,000 fungal species existing in Yunnan, but only 6,000 are described, including roughly 3,000 species of higher fungi (Ascomycota and Basidiomycota), indicating that fewer than 5% of known species have been described in this province.Two prominent regions of Yunnan have been well documented on fungal diversity, viz., the Eastern Himalayas and Hengduan Mountains in northwestern Yunnan and the tropical region in southern and southwestern Yunnan, while the other parts remain a huge challenge (Feng and Yang, 2018).
Stomiopeltis was introduced by Theissen (1914) to initially accommodate a single species S. aspersa which was collected on the leaves of Laurus sp. in India.Subsequently, many speciesmostly in the 19th century-that lacked molecular data to clarify their phylogenetic placement were included in the genus (Luttrell, 1946;Müller and von Arx, 1962;Index Fungorum, 2023).Stomiopeltis has been reported as a pathogen causing sooty blotch/flyspeck disease but has also been found as a saprobe on fruits (Mayfield et al., 2013;Ajitomi et al., 2017;Jayasiri et al., 2019;Batzer et al., 2022).The genus is scarcely known, and only a few species have molecular data available in GenBank (Crous et al., 2019;Jayasiri et al., 2019;Renard et al., 2020).Most Stomiopeltislike isolates were treated as Stomiopeltis sp.(Ajitomi et al., 2017;Batzer et al., 2022).An updated taxonomic treatment of Stomiopeltis was carried out by Zeng et al. (2019) who treated the genus in Capnodiales incertae sedis, and this was followed by Hongsanan et al. (2020b) and Wijayawardene et al. (2022).Whereas Renard et al. (2020) demonstrated that Stomiopeltis is polyphyletic, forming clades within the orders Microthyriales and Venturiales.Since the type species of Stomiopeltis, S. aspersa, has not yet been sequenced, the phylogenetic status of Stomiopeltis remains doubtful and also pending further study.
Several taxonomic studies of Dothideomycetes in Yunnan have been published in the past few years (Tibpromma et al., 2018;Phookamsak et al., 2019;Dong et al., 2020;Hyde et al., 2020;Li et al., 2020;Mortimer et al., 2021;Wanasinghe et al., 2021;Yang et al., 2022).Even though these studies resulted in a substantial increase in the number of described microfungi in Yunnan, there is still a glaring knowledge gap in our understanding of the fungi in this region.The present study aims to introduce two novel genera, one novel species, and three new combinations of Phaeothecoidiellaceae in Yunnan, based on molecular phylogeny coupled with morphological characteristics.

Sample collection, morphological examination, isolation, and preservation
The sample was collected from Xishuangbanna, Yunnan Province, China in 2021.The sample was stored in a plastic Ziploc bag and returned to the laboratory for observation and examination.Fungal fruiting bodies on host substrates were observed using an Olympus SZ61 series stereomicroscope.Micromorphologies on squash-mount slides were observed and photographed using a Nikon ECLIPSE Ni-U compound microscope equipped with a Nikon DS-Ri2 camera.Congo red was used to stain the conidiomatal centrum for clarity of conidiophores and conidiogenous cells.Lacto-glycerol was added to preserve important morphological features on permanent slides and edges of the coverslip were sealed with nail polish.All morphological features were measured using Tarosoft (R) Image FrameWork version 0.9.7., and the photographic plate was processed using Adobe Photoshop CS6 software (Adobe Systems Inc., San Jose, CA, USA).
Fungal pure culture was obtained by single spore isolation, according to the methods described in Senanayake et al. (2020).The germinated spores were transferred to freshly sterilized potato dextrose agar (PDA) and incubated under normal light at 20°C-25°C.Culture characteristics were observed and recorded after one-and four-week intervals.The specimen was deposited in the Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (KUN-HKAS), China.Axenic living cultures were preserved at the collection of Rungtiwa Phookamsak housed at Honghe Center for Mountain Futures (RPC) and duplicated in the Culture Collection of the Herbarium of Cryptogams Kunming Institute of Botany, Academia Sinica (KUNCC) Kunming, China.Index Fungorum numbers are provided for the newly described taxa.

DNA extraction, PCR amplification, and sequencing
Fungal genomic DNA was extracted from mycelia that grow on PDA for four weeks by using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux ® , Hangzhou, China) according to the manufacturer's protocol.The conditions for the polymerase chain reaction (PCR) were determined using the primer pairs LR0R/LR5 (Vilgalys and Hester, 1990) to amplify the 28S large subunit region (LSU), and ITS4/ITS5 (White et al., 1990) to amplify the internal transcribed spacer region (ITS: ITS1-5.8S-ITS2).The amplification of ITS and LSU was carried out with setting times and temperatures for the initialization, denaturation, annealing, and final extension periods following Phookamsak et al. (2017).
The final PCR reaction component was 25 µl, containing 12.5 ml Master Mix (mixture of EasyTaqTM DNA Polymerase, dNTPs, and optimized buffer; Beijing TransGen Biotech Co., Ltd., Chaoyang District, Beijing, China), 8.5 µl of double-distilled water (ddH 2 O), 1 ml of each forward and reverse primer (10 mm), and 2 ml DNA template.The PCR products were sent to TsingKe Biological Technology, Kunming City, Yunnan Province, China, for purification and Sanger sequencing.The consensus sequences of the newly generated strains are available to the scientific community via submission to GenBank.

Sequence alignment and phylogenetic analyses
The chromatograms of sequence results were checked, manually edited, trimmed, and assembled into consensus sequences using SeqMan Pro version 11.1.0(DNASTAR, Inc.Madison, WI, USA).The consensus sequences of the newly generated strains were blasted using the nucleotide BLAST search tool on the NCBI website to search for closely related species in the GenBank database.Sequence data obtained from this study, the closely related species from nucleotide BLAST search, and previous studies were downloaded from GenBank to supplement the datasets (Table 1).The dataset was prepared to generate the phylogenetic trees for taxa in Mycosphaerellales.Each sequence dataset was aligned using MAFFT (Katoh et al., 2019) and checked manually in Bioedit (Hall, 2004).Maximum likelihood (ML) and Bayesian analysis (BI) were conducted based on the individual datasets.
Maximum likelihood analyses were performed in RAxML-HPC v.8 on the XSEDE (8.2.12) tool in the online web portal CIPRES Science Gateway v. 3.3 using default settings but following adjustments with 1,000 bootstrap replications.The BI analyses were conducted via the same web portal as in ML, with two different runs, and six chains were executed.The initial 25% of sample trees were treated as burn-in and discarded.The trees were visualized using Figtree v. 1.4.0 (Rambaut, 2014) and edited in Adobe Illustrator version 20.0.0.

Molecular phylogeny
The phylogenetic tree which represented novel taxa in Phaeothecoidiellaceae was constructed using sequence data from ITS and LSU genes.A total of 66 strains of taxa in the family Phaeothecoidiellaceae, representative of other related families in Capnodiales, Cladosporiales, Microthyriales, and Mycosphaerellales, were included, with two strains of Botryosphaeria fusispora (MFLUCC 10-0098) and Lasiodiplodia gonubiensis (CBS 115812) as the outgroup.The aligned dataset contained 1,808 characters, including gaps.The best-scoring RAxML tree was selected to represent the relationships among taxa, with a final likelihood value of -17,660.527024.The matrix contained 1,121 distinct alignment patterns, with estimated base frequencies of A = 0.240165, C = 0.248028, G = 0.296115, T = 0.215692; substitution rates AC = 1.209829,AG = 1.862907,AT = 1.368661,CG = 1.131373,CT = 4.503418, GT = 1.000000; and gamma distribution shape parameter a = 0.495210 (Figure 1).For BI analysis, GTR + I + G was selected as the best-fit model by AIC in MrModeltest for each gene (ITS and LSU).Six simultaneous Markov chains were run for 3,000,000 generations, and trees were sampled every 100 generations.The first 25% of trees were discarded as the burn-in phase of the analyses, and the remaining trees were used for calculating posterior probabilities in the majority rule consensus tree (the critical value for the topological convergence diagnostic is 0.01), of which the final average standard deviation of split frequencies at the end of total MCMC generations was 0.009273.
Index Fungorum number: IF 900624 Etymology: The generic epithet "Pseudorepetophragma" refers to the genus that is morphologically resembling Repetophragma.
(2011) re-illustrated the genus by providing the synopsis table of morphological features and key to the species of Repetophragma.Based on this comprehensive study, Castañeda-Ruiz et al. ( 2011) introduced a novel species, R. paracambrense, and 12 new combinations in the genus.Of these, most species were previously known in Endophragmiella and Sporidesmium.Considering the species of Repetophragma, most of the accepted species have annellidic, percurrent proliferations of the conidiogenous cells bearing euseptate conidia with apically rounded, well-defined, and without rostrate or appendiculate apical cell (Iturriaga et al., 2008;Silvera-Simoń et al., 2009).This led to the inclusion of morphologically diverse species in Repetophragma.The phylogenetic analyses inferred by Shenoy et al. (2006) and Hernańdez-Restrepo et al. ( 2017) also revealed the status of some Repetophragma to be polyphyletic.Unfortunately, the phylogenetic affinity of Repetophragma is uncertain due to the lack of molecular data for the type species of Repetophragma.Furthermore, R. zygopetali formed an independent lineage within the Phaeothecoidiellaceae.Morphologically, R. zygopetali is similar to R. biseptatum in having monoblastic, enteroblastic, conspicuously percurrent conidiogenous cells.However, R. zygopetali can be distinguished from the rest of Repetophragma in having integrated, monoblastic, conspicuously percurrent but irregularly distanced conidiogenous cells with wavy or uneven apices after each conidial secession [Figure 29a in Buyck et al. (2017)] rather than equidistantly laid annellidic conidiogenous cells with even apices after each conidial secession (Subramanian, 1992;Seifert et al., 2011).Based on the morphological differences in the conidiogenous cells and phylogenetic analyses, we introduce the new genus Pseudorepetophragma to accommodate R. zygopetali as P. zygopetali comb.nov.2017) due to its morphological resemblance with R. dennisii, but differing in the size of conidiophores and conidia (Castañeda-Ruiz et al., 2011;Buyck et al., 2017).The species was reported as a sooty blotch fungus that occurred on Zygopetalum mackayi in Brazil, while other species of Repetophragma have been mostly reported as hyperparasites or saprobes (Ellis, 1963;Castañeda-Ruiz et al., 2011;Index Fungorun, 2023).Its life mode is fitted well with the genera in Phaeothecoidiellaceae, and this was confirmed by phylogenetic evidence.
Type species: Pseudostomiopeltis xishuangbannaensis Phookamsak, Hongsanan, Wanas. and Bhat Notes: Luttrell (1946) re-circumscribed the genus Stomiopeltis based on morphological studies and divided the species of Stomiopeltis into two groups based on the difference in the types of upper wall cell arrangements.The first group included the type species (S. aspersa) that has non-radiating upper wall cells, composed of disorderly arranged, irregularly lobed pseudoparenchymatous cells, while the second group has radiating upper wall cells, somewhat obscured by the curving and twisting of the radiating hyphae and by the irregularly lobed cells, which may be termed as "meandering plectenchyma".Luttrell (1946) mentioned that the second group should be placed in Microthyriaceae.The phylogenetic analyses conducted by Renard et al. (2020) also showed that Stomiopeltis is polyphyletic due to S. betulae forming a clade within Microthyriales, while two Stomiopeltis-like species formed a clade with Tothia fuscella in Venturiales.The present phylogenetic analyses of a concatenated ITS and LSU sequence dataset demonstrated that our new isolate formed a well-resolved subclade with other Stomiopeltis sensu lato in Phaeothecoidiellaceae, Mycosphaerellales.Besides, the type species of Stomiopeltis, S. aspersa, has not yet been sequenced, and hence the phylogenetic affinity of Stomiopeltis sensu stricto is still uncertain.Zeng et al. (2018) re-examined the holotype of Stomiopeltis aspersa and provided an updated morphological description that is characterized by superficial, brown, reticulate hyphae, flattened, circular, brown, thyriothecia with an irregular central ostiole.The upper wall comprises brown, meandrous, compact hyphae, lacking a basal plate.Asci are 8-spored, ellipsoidal, short-pedicellate, with an ocular chamber and ascospores are overlapping 2-3-seriate, hyaline, cylindrical, 1-septate, not constricted at the septum, with the upper cell shorter and broader than the lower cell.Morphologically, our new isolate could not be compared with the type of S. aspersa because they form different morphs.However, the new isolate is clearly distinguished from S. aspersa by the absence of superficial, brown, reticulate hyphae that penetrate the host and form hemispherical, dimidiate-scutate thyriothecia.Additionally, the upper wall of the thyriothecia radiates, and the cells at the margin are loosely and irregularly lobed, while S. aspersa has a nonradiating upper wall composed of sinuous, irregularly lobed cells [Figures 20c, d in Zeng et al. (2018)].Furthermore, S. phyllanthi which formed a clade with our new isolate, is also different from S. aspersa in lacking superficial, reticulate hyphae on the host and pseudoparaphyses (Jayasiri et al., 2019).Based on phylogenetic evidence and morphological distinctiveness with the type of Stomiopeltis, we introduced the new genus Pseudostomiopeltis to accommodate the new species, Ps. xishuangbannaensis, while Stomiopeltis phyllanthi is also transferred to the new genus as Pseudostomiopeltis phyllanthi comb.nov.2019) which was found as a saprobe on the fruits of Phyllanthus emblica.The species formed a sexual morph and is characterized by black, superficial, rounded thyriothecia, with the upper wall neatly lined by dark cells of textura angularis, lacking pseudoparaphyses, with 4-spored, fissitunicate, oblong to subglobose asci, and hyaline, obovoid to ellipsoid, 1-septate ascospores (Jayasiri et al., 2019).The present phylogenetic analyses of a concatenated ITS and LSU sequence dataset demonstrated that the species formed a separate branch and is basal to the clade Pseudostomiopeltis with significant support (90% ML, 0.98 PP; Figure 1).Therefore, we transferred S. phyllanthi to Pseudostomiopeltis as Ps.phyllanthi comb.nov.

Discussion
In the present study, two new genera, one new species, and three new combinations are described and illustrated based on morphology and phylogeny.The new genus, Pseudostomiopeltis, is introduced to accommodate the type species, Ps. xishuangbannaensis sp.nov.and Ps.phyllanthi com.nov.This genus belongs to Phaeothecoidiellaceae (Mycosphaerellales).The members of Pseudostomiopeltis share certain similar characteristics with Stomiopeltis which has been classified as a genus incertae sedis in Capnodiales by Wijayawardene et al. (2022).Stomiopeltis is a polyphyletic genus, and the sequence data of the type species are not available (Renard et al., 2020).It is interesting to note that the morphology of Stomiopeltis shows remarkable similarities to the species in Micropeltidaceae (Micropeltidales, Lecanoromycetes).However, molecular analysis has revealed that the majority of Stomiopeltis strains are classified in Phaeothecoidiellaceae (Mycosphaerellales), and most of these strains have not yet been identified at the species level.In the phylogenetic tree constructed using ITS and LSU sequence data (Figure 1), our new isolate clustered together with Cf.Stomiopeltis sp.RS7.2.Since the strain Cf.Stomiopeltis sp.RS7.2 has not been identified as its morphology is not available, we thus establish our strain as a new species.Whereas, S. phyllanthi is grouped with the clade of Pseudostomiopeltis with significant support (90% ML, 0.98 BYPP; Figure 1).Therefore, we transfer S. phyllanthi to Pseudostomiopeltis based on phylogenetic evidence, although the upper wall structure of S. phyllanthi could not be determined in this study.
Exopassalora was established to accommodate Passalora zambiae (Crous et al., 2004).The species was isolated from leaf spots of Eucalyptus globulus in Zambia.Crous et al. (2004) classified the species into Passalora due to its being phylogenetically distinct from other Mycosphaerella spp.known from Eucalyptus.Later, Videira et al. (2017) introduced many novel genera to accommodate Passalora sensu lato, including Exopassalora, based on multigene phylogenetic evidence.The sexual morph of E. zambiae is known only for its asci and ascospores, prepared onto the slide (Crous et al., 2004).In the present phylogenetic analyses, Stomiopeltis sinensis formed a separate branch basal to Exopassalora.Hence, the species is transferred to Exopassalora, as E. sinensis comb.nov., based on phylogenetic evidence.Morphologically, E. sinensis could be only compared with E. zambiae in ascospore characters that are similar in having hyaline, 1-septate ascospores (Crous et al., 2004;Jayasiri et al., 2019).Moreover, E. zambiae was found on leaf spots of Eucalyptus globulus, while Stomiopeltis spp.clustered in the subclade of Exopassalora were found as sooty blotch and flyspeck fungi on apples and pears (Batzer, 2005;Ismail et al., 2016).In contrast, E. sinensis was isolated from decaying fruit pericarp of Harpephyllum sp.(wild plum).It is presumable that the species may occur on fresh fruits of Harpephyllum as parasites and continue living on dead fruits as a saprobe, similar to Pseudostomiopeltis xishuangbannaensis.However, the change in life mode may need further study for a better understanding of the pathogenic capabilities of these species.
Yunnan is a region known for its high biodiversity, serving as a critical habitat for numerous species.However, this region is under threat from habitat loss, climate change, and various human activities.Among these factors, climate change is a major threat to the biodiversity in Yunnan.As global temperatures continue to rise, the region may experience changes in precipitation patterns and temperature regimes that could potentially impact the distribution and survival of numerous species, including fungi.Despite these challenges, there are also opportunities for biodiversity conservation in Yunnan.Scientific research and monitoring play crucial roles in providing valuable information for the biodiversity conservation efforts in the region.The discovery of novel taxa highlights the rich fungal diversity in Yunnan and contributes to our understanding of the ecological roles of fungi in forest ecosystems.The description and documentation of these new taxa not only provide important information on fungi useful in future research but also enhance our knowledge of conservation efforts in the region.By expanding our understanding of the biodiversity of Yunnan, we can better protect and manage its unique ecosystems and ensure the long-term survival of its species.

FIGURE 1
FIGURE 1Phylogram of the best-scoring maximum likelihood (ML) consensus tree based on a combined dataset (ITS and LSU) of Pseudostomiopeltis.The new species is indicated in blue, and the new combination species are indicated in green.Isolates from type materials are in bold.The ML ultrafast bootstrap and Bayesian PP values greater than 70% and 0.95 are shown at the nodes.The tree is rooted with Botryosphaeria fusispora (MFLUCC 10-0098) and Lasiodiplodia gonubiensis (CBS 115812).

TABLE 1 Continued
The newly generated sequences are indicated in red, and the ex-type strains are in bold.The missing sequences are indicated by "-".
1-2 µm], subcylindrical conidia, whereas Ps. xishuangbannaensis sporulated in vitro has subglobose to ellipsoidal or oblong conidia.The phylogenetic analyses demonstrated that S. syzygii (CPC 36323, ex-type strain) formed a subclade wi th Stomiop el tis sp.RS1 PEC6a basal to Pseudostomiopeltis with low support.Hence, the species is tentatively excluded from Pseudostomiopeltis until taxon samplings are increased, providing a better phylogenetic resolution of Pseudostomiopeltis with uncertain Stomiopeltis spp.within Phaeothecoidiellaceae.It is notable that S. betulae clustered within Microthyriales.However, the phylogenetic placements of Stomiopeltis and its relationships with other genera remain unclear.Further sequence data and morphological studies are needed to confirm the placement of this genus.Besides the phylogenetic investigation of Stomiopeltis sensu lato in Phaeothecoidiellaceae, Repetophragma zygopetali formed an independent lineage within Phaeothecoidiellaceae in the present study.Buyck et al. (2017) treated R. zygopetali in Micropeltidaceae (Micropeltidales, Lecanoromycetes) based on phylogenetic evidence that R. zygopetali formed a basal clade with Sporidesmajora pennsylvaniensis (CPC 16112), Houjia pomigena (CPC 16109), and H. yanglingensis