Morpho-Molecular Characterization of Five Novel Taxa in Parabambusicolaceae (Massarineae, Pleosporales) from Yunnan, China

Parabambusicolaceae is a well-studied family in Massarineae, Pleosporales, comprising nine genera and approximately 16 species. The family was introduced to accommodate saprobic bambusicola-like species in both freshwater and terrestrial environments that mostly occur on bamboos and grasses but are also found on different host substrates. In the present study, we surveyed and collected ascomycetes from bamboo and submerged grass across Yunnan Province, China. A biphasic approach based on morphological characteristics and multigene phylogeny demonstrated five new taxa in Parabambusicolaceae. A novel genus Scolecohyalosporium is introduced as a monotypic genus to accommodate S. submersum sp. nov., collected from dead culms of grass submerged in a freshwater stream. The genus is unique in forming filiform ascospores, which differ from other known genera in Parabambusicolaceae. Multigene phylogeny showed that the genus has a close relationship with Multiseptospora. Moreover, the novel monotypic genus Neomultiseptospora, isolated from bamboo, was introduced to accommodate N. yunnanensis sp. nov. Neomultiseptospora yunnanensis formed a separated branch basal to Scolecohyalosporium submersum and Multiseptospora thailandica with high support (100% ML, 1.00 PP). Furthermore, the newly introduced species, Parabambusicola hongheensis sp. nov. was also isolated from bamboo in terrestrial habitats. Parabambusicola hongheensis clustered with the other three described Parabambusicola species and has a close relationship with P. bambusina with significant support (88% ML, 1.00 PP). Parabambusicola hongheensis was reported as the fourth species in this genus. Detailed description, illustration, and updated phylogeny of Parabambusicolaceae were provided.


Collection, Examination, Isolation, and Culture Preservation
Dead branches and culms of bamboo and grass were collected from Yunnan Province, China in 2021. The samples were stored in plastic Ziploc bags and returned to the laboratory for observation and examination. Fungal fruiting bodies on the host substrates were observed under an Olympus SZ61 series stereo microscope. Squash-mount slides were prepared to examine micro-morphologies and captured under a Nikon ECLIPSE Ni compound microscope connected with a Nikon DS-Ri2 camera. Indian ink was stained to check the mucilaginous sheath covered the ascospores, while congo red was stained to observe the centrum. The permanent slides contained important morphological features, were prepared by adding Lacto glycerol, and sealed using nail polish. Measurements of morphological features such as ascomata, peridium, pseudoparaphyses, asci, and ascospores were done via NIS-Elements BR 3.2 software v. 5.11.01. Photographic plates were edited and provided by Adobe Photoshop CS6 software (Adobe Systems Inc., San Jose, CA, USA).
Pure cultures were obtained from single spore isolation based on the guidelines of Senanayake et al. [14] and cultivated on potato dextrose agar (PDA) under normal light at 20-25 °C. Culture characteristics were recorded after one week and four weeks. The type specimens were deposited with permanent slides in the Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (KUN-HKAS), China, and the ex-type living

Collection, Examination, Isolation, and Culture Preservation
Dead branches and culms of bamboo and grass were collected from Yunnan Province, China in 2021. The samples were stored in plastic Ziploc bags and returned to the laboratory for observation and examination. Fungal fruiting bodies on the host substrates were observed under an Olympus SZ61 series stereo microscope. Squash-mount slides were prepared to examine micro-morphologies and captured under a Nikon ECLIPSE Ni compound microscope connected with a Nikon DS-Ri2 camera. Indian ink was stained to check the mucilaginous sheath covered the ascospores, while congo red was stained to observe the centrum. The permanent slides contained important morphological features, were prepared by adding Lacto glycerol, and sealed using nail polish. Measurements of morphological features such as ascomata, peridium, pseudoparaphyses, asci, and ascospores were done via NIS-Elements BR 3.2 software v. 5.11.01. Photographic plates were edited and provided by Adobe Photoshop CS6 software (Adobe Systems Inc., San Jose, CA, USA).
Pure cultures were obtained from single spore isolation based on the guidelines of Senanayake et al. [14] and cultivated on potato dextrose agar (PDA) under normal light at 20-25 • C. Culture characteristics were recorded after one week and four weeks. The type specimens were deposited with permanent slides in the Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (KUN-HKAS), China, and the ex-type living culture was deposited in the Culture Collection of Kunming Institute of Botany (KUMCC). Index Fungorum numbers are provided for the newly described taxa [15].

DNA Extraction, Amplification, and Sequencing
The generated fungal genomic DNA was extracted from fresh fungal mycelia using Biospin Fungus Genomic DNA Extraction Kit (BioFlux ® , Hangzhou, China) following manufacturer protocols (Hangzhou, China). The duplicated strain was also extracted from different fruiting bodies to verify the correctness of DNA sequence data using Forensic DNA Kit (Omega ® , Norcross, GA, USA). The generated fungal genomic DNA was stored at 4 • C for PCR amplification and duplicated at −20 • C for long-term storage.
DNA amplification was conducted by polymerase chain reaction (PCR) using four phylogenetic markers viz. the internal transcribed spacers (ITS: ITS1-5.8S-ITS2), 28S large subunit rDNA (LSU), 18S small subunit rDNA (SSU), and the translation elongation factor 1-alpha (TEF1-α). Primer pairs: ITS5 and ITS4 [16], LR0R and LR5 [17], NS1 and NS4 [16], and EF1-983F and EF1-2218R [18] were used to amplify the PCR fragments of these gene regions. The final volume of the PCR reaction was 25 µL containing 2 µL of DNA template, 1 µL of each forward and reverse primers, 12.5 µL of 2× Power Taq PCR Master Mix (mixture of EasyTaqTM DNA Polymerase, dNTPs, and optimized buffer, Beijing Bio Teke Corporation (Bio Teke), Wuxi, China), and 8.5 µL of double-distilled water (ddH 2 O). The PCR thermal cycle program for ITS, LSU, SSU, and TEF1-α gene regions was set up in the following condition described by Jiang et al. [19]. PCR fragments were purified and sequenced by TsingKe Biological Technology Co., Ltd., Beijing, China. The consensus sequences of the newly generated strains are deposited in GenBank (Table 1). Table 1. Taxa used in this study and their GenBank accession numbers. The ex-type and ex-epitype strains are indicated by superscript "T". The newly generated sequences are indicated in bold.

Species Name
Strain No.

Sequence Alignment and Phylogenetic Analyses
The consensus ITS, LSU, SSU, and TEF1-α sequences of the newly generated strains were blasted in the nucleotide BLAST search tool via the NCBI website (https://blast. ncbi.nlm.nih.gov/Blast.cgi, accessed on 25 November 2021) for searching the similar taxa. The nucleotide BLAST searches showed that the newly generated sequences had the closest similarity with species in Parabambusicolaceae. Hence, the newly generated strains were aligned with representative taxa in Parabambusicolaceae, which were retrieved from GenBank (Table 1). Individual gene alignments were aligned with MAFFT v. 7.475 [20], and the ambiguous sites were trimmed by TrimAl v. 1.3 via the web server phylemon 2 (http://phylemon.bioinfo.cipf.es/utilities.html, accessed on 25 November 2021) and improved manually where necessary using BioEdit v. 6.0.7 [21]. Improved individual gene alignments were prior analyzed by randomized axelerated maximum likelihood (RAxML) analysis via RAxML-HPC v.8 on the XSEDE (8.2.12) tool in the online web portal CIPRES Science Gateway v. 3.3 [22] using default settings but following adjustments described by Jiang et al. [19]. After checking the tree topologies of every individual gene alignment for incongruities, the combined ITS, LSU, SSU, and TEF1-α sequence dataset were then analyzed based on maximum likelihood (RAxML) and Bayesian inference (BI) analyses following commands described by Jiang et al. [19]. Phylogenetic trees were visualized on

Taxonomy
Neomultiseptospora N. Xie, Phookamsak & Hongsanan, gen. nov. Index Fungorum number: IF 559389 Etymology: Referring to the taxon has a close phylogenetic relationship with the genus Multiseptospora.
Type species: Neomultiseptospora yunnanensis Phookamsak, Hongsanan and N. Xie Notes: The novel genus Neomultiseptospora is introduced herein to accommodate the single species N. yunnanensis collected from dead branches of bamboo in Yunnan Province, China. Multigene phylogeny demonstrated that Neomultiseptospora clustered with the genera Multiseptospora and Scolecohyalosporium ( Figure 2). Morphologically, Neomultiseptospora resembles Multiseptospora in having phragmosporous, hyaline, multi-septate ascospores but differs in forming raised, hemispherical to subconical ascomata, while Multiseptospora has globose to subglobose ascomata, covered by dark, hair-like hyphae and embedded in host tissue [10]. Additionally, Neomultiseptospora has a close phylogenetic relationship with Scolecohyalosporium but the genus is distinct from Scolecohyalosporium in having phragmosporous ascospores while Scolecohyalosporium has scolecosporous ascospores. Unfortunately, Neomultiseptospora is morphologically similar to the generic type of Parabambusicola in having hemispherical to subconical ascomata and hyaline, fusiform, 5-septate ascospores and occurring on bamboo (Sasa spp.) [1]. However, multigene phylogenetic analyses based on a combined ITS, LSU, SSU, and TEF1-α sequence dataset indicated the distinctiveness between these two genera. = 25 × 7.1 μm, n = 30), overlapping 1-2-seriate, hyaline, fusiform to ellipsoidal, or oblong, with rounded ends, slightly curved, (4-)5-septate, constricted at the central septum, less constricted at the other septa, smooth-walled, with small guttules, surrounded by a distinct, thick, mucilaginous sheath. Asexual morph: Undetermined. Culture characteristics: Ascospores germinated on PDA within 24 h. Colonies on PDA reaching 27-30 mm diam after 2 weeks at room temperature (15-20 °C). Colonies dense, irregular in shape, convex to umbonate, surface smooth with an undulate edge, floccose to cottony; from above pale grey to white-grey at the margin, grey at the middle towards the center; from below white-brown at the margin, dark brown to black at the middle towards the center; not producing pigmentation on PDA.  Etymology: Referring to the locality, Yunnan Province, China, where the species was collected.
Culture characteristics: Ascospores germinated on PDA within 24 h. Colonies on PDA reaching 27-30 mm diam after 2 weeks at room temperature (15-20 • C). Colonies dense, irregular in shape, convex to umbonate, surface smooth with an undulate edge, floccose to cottony; from above pale grey to white-grey at the margin, grey at the middle towards the center; from below white-brown at the margin, dark brown to black at the middle towards the center; not producing pigmentation on PDA.
Type species: Neomultiseptospora yunnanensis Phookamsak, Hongsanan and N. Xie Notes: The novel genus Neomultiseptospora is introduced herein to accommodate the single species N. yunnanensis collected from dead branches of bamboo in Yunnan Province, China. Multigene phylogeny demonstrated that Neomultiseptospora clustered with the genera Multiseptospora and Scolecohyalosporium ( Figure 2). Morphologically, Neomultiseptospora resembles Multiseptospora in having phragmosporous, hyaline, multi-septate ascospores but differs in forming raised, hemispherical to subconical ascomata, while Multiseptospora has globose to subglobose ascomata, covered by dark, hair-like hyphae and embedded in host tissue [10]. Additionally, Neomultiseptospora has a close phylogenetic relationship with Scolecohyalosporium but the genus is distinct from Scolecohyalosporium in having phragmosporous ascospores while Scolecohyalosporium has scolecosporous ascospores. Unfortunately, Neomultiseptospora is morphologically similar to the generic type of Parabambusicola in having hemispherical to subconical ascomata and hyaline, fusiform, 5septate ascospores and occurring on bamboo (Sasa spp.) [1]. However, multigene phylogenetic analyses based on a combined ITS, LSU, SSU, and TEF1-α sequence dataset indicated the distinctiveness between these two genera.
Saprobic on stems of unidentified grass submerged in a small stream. Sexual morph: Ascomata solitary, scattered. sometimes clustered, erumpent through host cortex, becoming superficial, uni-loculate, conical to ovoid, glabrous, rough-walled, ostiole central, with truncate apex. Peridium thick-walled, of unequal thickness, thick at sides towards the apex, thin at the base, composed of several layers of small, dark brown to black, pseudoparenchymatous cells, arranged in a textura angularis, with carbonaceous cells at the ostiole, outer layers intermixed with the host tissues. Hamathecium composed of dense, filamentous, branched, septate, cellular pseudoparaphyses, constricted at the septa, anastomosed among the asci, embedded in a gelatinous matrix. Asci 8-spored, bitunicate, fissitunicate, long cylindrical, shortly pedicellate, apically rounded with the indistinct ocular chamber. Ascospores spirally arranged within the ascus, hyaline, filiform, narrower towards the end cells, multi-septate, not constricted at the septa, smooth-walled. Asexual morph: Undetermined.
Type species: Scolecohyalosporium submersum Phookamsak, Hongsanan and N. Xie Notes: Scolecohyalosporium is introduced herein as a monotypic genus to accommodate S. submersum sp. nov. The species was isolated from dead stems of grass submerged in a freshwater stream in Yunnan, China. Scolecohyalosporium has a unique character that can be distinguished from other sexual genera in Parabambusicolaceae in forming scolecosporous ascospores. Scolecohyalosporium morphological resembles genera Neoophiosphaerella and Poaceascoma in Lentitheciaceae. However, Neoophiosphaerella can be distinguished from Scolecohyalosporium in having subglobose to hemispherical ascomata, with thin-walled, brown cells of peridium, and broader cylindrical asci [1], while the type of Poaceascoma formed setose ascomata [23].
Scolecohyalosporium submersum Phookamsak, Hongsanan and N. Xie, sp. nov. Index Fungorum number: IF 559393, Figure 5. Etymology: Referring to the habitat of the taxon, submerged in a small freshwater stream.

Discussion
Parabambusicolaceae is a well-studied family based upon the morphological characterization and molecular phylogeny. Even though the family was introduced in 2015 by K. Tanaka and his colleagues to initially accommodate two novel monotypic genera in Massarineae, the number of genera and species in Parabambusicolaceae has continually increased in recent years [4,[6][7][8][9][11][12][13]. In this study, we establish the other two novel genera, Neomultiseptospora and Scolecohyalosporium as well as the novel species Parabambusicola hongheensis in Parabambusicolaceae based on multigene phylogeny coupled with morphological differences. Hence, there are 11 genera and 19 phylogenetic species in this family.
Most genera in Parabambusicolaceae were introduced as monotypic genera, and many genera are currently accommodated as putative species, including Aquastroma, Multilocularia, Neomultiseptospora, Paramonodictys, Paratrimmatostroma, Pseudomonodictys, and Scolecohyalosporium. Aquastroma, Multilocularia, Neomultiseptospora, and Scolecohyalosporium are only represented by the sexual morph, while Paramonodictys, Paratrimmatostroma, and Pseudomonodictys are represented by the hyphomycetous asexual morph. Due to these genera being represented by a single species, the phylogenetic relationships with the related genera are sometimes not well-resolved. This may be because of a few genetic information and taxon sampling.
Multigene phylogenetic analyses demonstrated that Lonicericola, Multilocularia, Neoaquastroma, Neomultiseptospora, Parabambusicola, Paramonodictys, Paratrimmatostroma, and Scolecohyalosporium form well-resolved subclade (up to 70% ML and 0.95 PP support; Figure 2) in this study. However, the phylogenetic relationships among Lonicericola, Paratrimmatostroma, and Pseudomonodictys are not well-resolved. These three genera clade together with low supported values in this study that concurred with Hyde et al. [7]. Paratrimmatostroma (trimmatostroma-like), and Pseudomonodictys (monodictys-like) are represented by their hyphomycetous asexual morph, whereas Lonicericola is represented by massarina-like sexual morph. Hence, the morphological features of Lonicericola cannot be compared with Paratrimmatostroma, and Pseudomonodictys, while Paratrimmatostroma, and Pseudomonodictys have morphological differences. Moreover, Aquastroma (sexual morph) also clusters with Paramonodictys (asexual morph), and "Monodictys sp. JO 10" with low support. There-fore, more described holomorph species are necessary in these genera to support their generic status.
Multiseptospora was introduced as a monotypic genus by Liu et al. [10] to accommodate M. thailandica, isolated from the dead stem of grass (Thysanolaena maxima) in Thailand. Subsequently, Li et al. [8] introduced the second species, M. thysanolaenae, to the genus. Multiseptospora thysanolaenae morphological resembles the type species M. thailandica but differs in forming brown ascospores [8]. Phylogenetic relationships between M. thysanolaenae and M. thailandica were not well-resolved in Li et al. [8] and the species always formed a separated branch basal to Parabambusicola in subsequent studies [4,6,7,9,11]. Due to most sexual genera in Parabambusicolaceae having similar morphology, M. thysanolaenae may not be congeneric with M. thailandica based on phylogenetic evidence. However, the genetic information of M. thysanolaenae is not well-verified (R. Phookamsak, pers. comm.). The ex-type strain of this species needs to be re-sequenced for further evaluating its generic status.
Most sexual genera in Parabambusicolaceae have similar morphology of the ascospores. However, these genera can be distinguished from each other based on the other morphological features such as the shape of ascomata, peridial structure, host occurrences, and habitats, and the affinities of these genera were also supported by phylogenetic evidence. We thus provide the keys to genera in Parabambusicolaceae for better clarifying the generic resolution.
Key to genera of Parabambusicolaceae