Pseudobactrodesmium (Dactylosporaceae, Eurotiomycetes, Fungi) a Novel Lignicolous Genus

During our ongoing surveys of fungi on submerged wood in the Greater Mekong Subregion, we collected two new species similar to Bactrodesmium longisporum. Pseudobactrodesmium gen. nov. is introduced to accommodate the new species, P. aquaticum, P. chiangmaiensis and B. longisporum is transferred to this genus. Fasciculate conidiophores, enteroblastic conidiogenous cells and subulate to fusiform, phragmoseptate conidia with a tapering apical cell and sheath characterize the genus. Pseudobactrodesmium aquaticum has longer conidia than P. chiangmaiensis. The placement of Pseudobactrodesmium in Dactylosporaceae (Eurotiomycetes) is a novel finding based on analyses of combined LSU, SSU, ITS and RPB2 sequence data. Our study reveals that Pseudobactrodesmium is likely to be a speciose genus with different species in streams around the world.

Aquatic hyphomycetes are a morphologically diverse and polyphyletic group (Shenoy et al., 2006;Baschien et al., 2013;Su et al., 2016). Species with similar morphological characters are difficult to identify without molecular data. Previously, identification was mostly carried out based on morphology and only a few asexual taxa have been subjected to phylogenetic studies (Goh and Hyde, 1996;Cai et al., 2002;Cai and Hyde, 2007). With more molecular data becoming available for phylogenetic analyses, numerous new combinations have been proposed to accommodate poorly documented hyphomycetous species (Lu et al., 2018;Yang et al., 2018a,b). Molecular data also demonstrated that some previously known congeneric species are now distributed in different families, e.g., Monodictys arctica in Leptosphaeriaceae (Day et al., 2006), M. capensis in Pleomonodictydaceae (Hernández-Restrepo et al., 2017), and some other Monodictys species in Parabambusicolaceae (Tanaka et al., 2015). Although the polyphyletic nature of some hyphomycetous genera were partially resolved, e.g., Dendryphion, Sporidesmium and torula-like species , fresh collections with molecular data are still needed to obtain a natural classification of hyphomycetes.
We are studying the freshwater fungi on submerged wood along a north-south latitudinal gradient in the Asian/Australian region  and have published several papers on the Greater Mekong Subregion (Zhang et al., 2011(Zhang et al., , 2012(Zhang et al., , 2013(Zhang et al., , 2014(Zhang et al., , 2016(Zhang et al., , 2017Dong et al., 2018;Wei et al., 2018;Yu et al., 2018;Wang et al., 2019). In this study, two taxa morphologically similar to Bactrodesmium longisporum were collected from submerged wood. To clarify the classification of the two new collections, we analyzed a combined LSU, SSU, ITS and RPB2 sequence dataset and compared their morphological characters. Pseudobactrodesmium, a new genus with two new species, 1 http://www.indexfungorum.org/names/Names.asp and one new combination are introduced. Morphologically similar genera are compared with Pseudobactrodesmium and the taxonomic placements of Bactrodesmium species are discussed.

Isolation and Morphology
The decaying wood samples were collected from freshwater streams in Chiang Mai Province, Thailand and Yunnan Province, China. Specimens were placed in zip-lock plastic bags with moist cotton or tissue paper and taken to the laboratory.
Morphological observations were carried out after incubation at room temperature for 1-2 weeks. Colonies were examined using a Nikon SMZ-171 dissecting microscope. Photomicrographs were made with a Nikon ECLIPSE Ni compound microscope fitted with a Canon EOS 600D digital camera. Measurements were made with the Tarosoft (R) Image Frame Work program. Images used for figures were processed with Adobe Photoshop CS5 software (Adobe Systems, United States). Single spore isolations were made from conidia onto potato dextrose agar (PDA) at room temperature, as detailed in Chomnunti et al. (2014) and cultured as outlined by Vijaykrishna et al. (2004) and Liu et al. (2010). Herbarium specimens (dry wood with fungal material) were deposited in the herbarium of Mae Fah Luang University (MFLU), Chiang Rai, Thailand and herbarium of Cryptogams, Kunming Institute of Botany Academia Sinica (HKAS), Kunming, China. Living cultures were deposited in Mae Fah Luang University Culture Collection (MFLUCC) and Kunming Institute of Botany Culture Collection (KUMCC). Facesoffungi and Index Fungorum numbers were registered as in Jayasiri et al. (2015) and Index Fungorum (2020), respectively.
A ML analysis was performed with RAxML-HPC v.8 on XSEDE in CIPRES Science Gateway (Miller et al., 2010 with 1000 rapid bootstrap replicates. The model selected for ML was GTRGAMMA. Maximum likelihood bootstrap values equal to or greater than 60% are given above or below the nodes (first value, Figure 1). Bayesian inference was conducted with MrBayes v. 3.1.2 (Huelsenbeck and Ronquist, 2001) to evaluate posterior probabilities (BPP) (Rannala and Yang, 1996) by Markov chain Monte Carlo (MCMC) sampling. The bestfit model was GTR + I + G for LSU, SSU and RPB2, and SYM + I + G for ITS. Six simultaneous Markov chains were run for one million generations and trees were sampled every 100 generation (resulting in 10000 trees). The first 2500 trees, representing the burn-in phase of the analyses, were discarded and the remaining 7500 trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree (Larget and Simon, 1999). Bayesian posterior probabilities (BPP) equal to or greater than 0.95 are given above or below the nodes (second value, Figure 1).
Phylogenetic trees were viewed with FigTree v1.4.0 3,4 and edited using Microsoft Office PowerPoint 2007 (Microsoft Corporation, WA, United States). The new sequences were deposited in GenBank (Table 1).
Culture characteristics: On PDA, colony circular, slow growing, reaching 10 mm in 50 days at 25 • C, gray to brown from above, dark gray from below, surface rough, dry, raised, entire at edge.
Notes: Bactrodesmium longisporum was described by Ellis (1976) with a line-drawing. It was subsequently synonymized with Stigmina longispora by Hughes (1978) who observed percurrently proliferating conidiophores in old specimens from New Zealand. Bactrodesmium stilboideum is another synonym listed in Index Fungorum database. However, they can be distinguished by the aggregation of conidiophores (synnematous in B. stilboideum vs. mononematous, fasciculate conidiophores in B. longisporum) (Ellis, 1976;Castañeda Ruiz and Arnold, 1985).
A Thai strain of B. longisporum (NBRC 104945) clustered with Pseudobactrodesmium chiangmaiensis (MFLUCC 18-0982) in our phylogenetic tree (Figure 1). A comparison of sequence data between NBRC 104945 and MFLUCC 18-0982 shows a difference of 2, 281, 5 nucleotides in LSU, SSU and ITS gene regions, respectively (NBRC 104945 has 3 major insertions spanning over 281 nucleotides in SSU gene). In this study, we name NBRC 104945 as Pseudobactrodesmium sp. until its morphological characters are established to formally name this isolate. Five additional strains with only ITS2 sequence data are named as Bactrodesmium longisporum in GenBank. However, their status should be treated with caution as they represent OTUS from a metagenomic study of a heap leaching system (Hu et al., 2015) and further evidence of conspecificity is needed.
Unfortunately, the holotype specimen of B. longisporum (IMI 63746 B), does not exist in herbarium IMI. 5 According to protologue description of the holotype (Ellis, 1976), B. longisporum (IMI 63746 B) has similar conidial size to P. chiangmaiensis (MFLU 18-0994) (50-80 × 7-8 µm in former vs. 40-90 × 5.5-8.5 µm in latter). However, P. chiangmaiensis has elongated apical cells (up to 16 µm long) with subglobose tuberculate ends, which were not described and drawn in protologue of B. longisporum (Ellis, 1976). The conidiophores of B. longisporum are up to 50 µm long, but only 15-23 µm long in P. chiangmaiensis. The size of apical sheath of B. longisporum is also unclear. Thus, we treat them as different species and synonymize B. longisporum under Pseudobactrodesmium as the third species in the genus. Epitypification of Pseudobactrodesmium longisporum is needed using a collection from its type locality.

DISCUSSION
Bactrodesmium longisporum has been recorded as having a worldwide distribution, however these records have not been verified with molecular data. The type of B. longisporum also appears to be lost and therefore its identity cannot be verified. We therefore designate our new species of Pseudobactrodesmium from China as the generic type, describe a second species from Thailand and transfer Bactrodesmium longisporum to the new genus. However, it is likely that many collections of this species have been misidentified and as more collections are made from different countries, we would expect Pseudobactrodesmium to become speciose.
Bactrodesmium is a complex genus in need of extensive taxonomic reassessment. Pem et al. (2019) reviewed the holotype material of Bactrodesmium abruptum (≡ Sporidesmium abruptum) and tentatively placed the generic type in Dothideomycetes incertae sedis based on morphology. Both B. cubense and B. obovatum produce clavate or obovate conidia with darker septa and unequal cells, similar to the type species B. abruptum (Ellis, 1971;Zucconi and Lunghini, 1997). However, our phylogenetic study shows that they belong to different classes, Dothideomycetes and Sordariomycetes, respectively (Figure 1). Bactrodesmium gabretae differs from B. abruptum by its transversely or occasionally oblique, distoseptate conidia, and phylogenetically clustered in Leotiomycetes (Figure 1). Bactrodesmium pallidum is different from B. abruptum but similar to our new genus Pseudobactrodesmium in conidial shape (Ellis, 1959), and phylogeny places this species in Sordariomycetes (Figure 1). Our phylogenetic study is in agreement with the studies of Koukol and Kolárová (2010) and Hernández-Restrepo et al. (2017).
Although molecular data of B. abruptum is still missing, the working hypothesis of Bactrodesmium sensu stricto in Dothideomycetes provides further evidence for the introduction of Pseudobactrodesmium. Pseudobactrodesmium shares some morphological characters with Digitodesmium in having acrogenous, long, transversely septate conidia with a hyaline sheath at the apex (Kirk, 1981;Boonmee et al., 2016). However, the semi-macronematous, moniliform conidiophores and digitate conidia of Digitodesmium are clearly distinguishable from the macronematous, subcylindrical conidiophores and subcylindrical to narrowly fusiform, or subulate conidia of Pseudobactrodesmium. Phylogeny also segregates them into different classes, viz. Pseudobactrodesmium in Eurotiomycetes, and Digitodesmium in Dothideomycetes (Tsui et al., 2006;Boonmee et al., 2016;this study). The conidia of Scolecostigmina are superficially similar to those of Pseudobactrodesmium, but the former is characterized by conspicuously annellate conidiogenous cells, thick-walled, smooth to verrucose conidia occasionally with a few longitudinal or oblique septa or a few intermixed distosepta, contrasting with inconspicuously proliferating conidiogenous cells and thin-walled, smooth, transversely phragmoseptate conidia with a hyaline, spherical sheath at the apex in Pseudobactrodesmium (Braun et al., 1999;Crous et al., 2013). Scolecostigmina, typified by S. mangiferae, clustered in Capnodiales (Dothideomycetes) (Crous et al., 2013), while Pseudobactrodesmium clustered in Dactylosporaceae (Eurotiomycetes). Pseudobactrodesmium longisporum is superficially similar to Gangliostilbe malabarica in the conidial shape and apical sheath, but the synnemata and apically rounded conidia of the latter can easily be separated from the former (Xia et al., 2015). These characters of G. malabarica are also distinguished from those in the collection of Castañeda Ruiz and Arnold (1985) bearing the name Bactrodesmium stilboideum.
It is challenging to reconstruct the phylogeny of Bactrodesmium considering lack of living cultures of B. abruptum. The species having clavate or obovate, long or short, transversely septate conidia with or without apical sheath are common and scattered in different groups (Ellis, 1976;Hughes, 1978;Holubová-Jechová, 1984;Braun et al., 1999;Koukol and Kolárová, 2010;Crous et al., 2013;Xia et al., 2015;Boonmee et al., 2016;Su et al., 2016;Hernández-Restrepo et al., 2017;Videira et al., 2017). These groups of fungi are morphologically similar and therefore molecular characters are of crucial importance to clarify their taxonomy. The sequence data of B. abruptum is needed in the future to clarify the natural classification of Bactrodesmium.

DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in the NCBI GenBank: MN335230, MN335226, MN335228, MN335229, MN335225, and MN335227.

AUTHOR CONTRIBUTIONS
WD conducted the experiments, analyzed the data, and wrote the manuscript. KH planned the experiments. MD analyzed the data. X-DY conducted the experiments. DB and RJ revised the manuscript. SB funded the experiments. G-NW conducted the experiments. SN planned the experiments. HZ planned the experiments, analyzed the data, and wrote the manuscript. All authors revised the manuscript.

FUNDING
This work was mainly supported by National Natural Science Foundation of China (Project ID: NSF 31500017 to HZ), Yunnan young and middle aged academic and technical leaders reserve talents (Project ID: 2018HB008). KH thanks the Foreign Experts Bureau of Yunnan Province, Foreign Talents Program (2018; grant no. YNZ2018002), Thailand Research grants entitled Biodiversity, phylogeny and role of fungal endophytes on above parts of Rhizophora apiculata and Nypa fruticans (grant no: RSA5980068), the future of specialist fungi in a changing climate: baseline data for generalist and specialist fungi associated with ants, Rhododendron species and Dracaena species (grant no: DBG6080013), Impact of climate change on fungal diversity and biogeography in the Greater Mekong Subregion (grant no: RDG6130001). KH also thanks Chiang Mai University for the award of visiting Professor. MD would like to thank the 5th batch of Postdoctoral Orientation Training Personnel in Yunnan Province (grant no: Y934283261) and the 64th batch of China Postdoctoral Science Foundation (grant no: Y913082271). SB would like to thank the National Research Council of Thailand (No. 61215320023).