﻿Taxonomic and phylogenetic characterisations of six species of Pleosporales (in Didymosphaeriaceae, Roussoellaceae and Nigrogranaceae) from China

﻿Abstract Pleosporales comprise a diverse group of fungi with a global distribution and significant ecological importance. A survey on Pleosporales (in Didymosphaeriaceae, Roussoellaceae and Nigrogranaceae) in Guizhou Province, China, was conducted. Specimens were identified, based on morphological characteristics and phylogenetic analyses using a dataset composed of ITS, LSU, SSU, tef1 and rpb2 loci. Maximum Likelihood (ML) and Bayesian analyses were performed. As a result, three new species (Neokalmusiakarka, Nigrogranaschinifolium and N.trachycarpus) have been discovered, along with two new records for China (Roussoellaneopustulans and R.doimaesalongensis) and a known species (Roussoellapseudohysterioides). Morphologically similar species and phylogenetically close taxa are compared and discussed. This study provides detailed information and descriptions of all newly-identified taxa.


Introduction
The order Pleosporales was formally established by Luttrell and Barr (1987) and is characterised by perithecioid ascomata with a papillate apex, ostioles with or without periphyses, cellular pseudoparaphyses, bitunicate asci and ascospores of varying shapes, pigmentation and septation (Zhang et al. 2012).
As one of the largest orders in the Dothideomycetes, it comprises a quarter of all dothideomycetous species (Ahmed et al. 2014b).Species in this order are found in various habitats and can be epiphytes, endophytes or parasites of living leaves or stems, hyperparasites on fungi or insects, lichenised or saprobes of dead plant stems, leaves or bark (Ramesh 2003;Kruys et al. 2006).In this study, we identified six species belonging to the order Pleosporales from the families Didymosphaeriaceae Munk, Nigrogranaceae Jaklitsch & Voglmayr and Roussoellaceae Jian et al. in Guizhou, China (Wijayawardene et al. 2022).
The family Didymosphaeriaceae, introduced by Munk (1953) and typified by Didymosphaeria fuckeliana, can be placed in the order Pleosporales.Neokalmusia was introduced to Didymosphaeriaceae by Ariyawansa et al. (2014a).Currently, only eight Neokalmusia species are listed in Index Fungorum (accession date: 25 July 2023).Members of Didymosphaeriaceae are known to form numerous different types of life modes, including saprobes, pathogens or endophytes and can be found both on land and in water (Gonçalves et al. 2019;Hongsanan et al. 2020).In the study of this paper, Neokalmusia karka is taken from the dead culms of the Phragmites karka (Retz.)Trin.ex Steud.Shilihe Beach Park, Huaxi, Guizhou Province, China.
Roussoellaceae was established to accommodate three genera, Neoroussoella Jian K. Liu et al., Roussoella Sacc. and Roussoellopsis I. Hino & Katum., based on molecular phylogenetic studies (Liu et al. 2014).The genus Roussoella has cylindrical asci with Cytoplea asexual morphs, which distinguishes it from other genera (Liu et al. 2014).Another feature reported for the genus Roussoella is the high stability of the ascal exotunica, particularly in 3% potassium hydroxide (KOH).This is quite common for nearly all fungi treated here, while only in Nigrograna can fissitunicate ascus dehiscence be seen rather frequently (Jaklitsch and Voglmayr 2016).Nigrogranaceae was established to accommodate Nigrograna, with N. mackinnonii (Borelli) Gruyter et al. as the type species (Jaklitsch and Voglmayr 2016).As the only genus in the family Nigrogranaceae, Nigrograna was established despite lacking strong bootstrap values support in ITS/tef1-based phylogenetic trees (Kolařík et al. 2017;Mapook et al. 2020;Zhang et al. 2020a;Wijayawardene et al. 2020).Species of Nigrograna may be interpreted as a result of cryptic speciation, as, morphologically, they show only subtle differences (Jaklitsch and Voglmayr 2016).Twenty-three Nigrograna species are listed in Index Fungorum (accession date: 25 July 2023).
In this study, we collected dead branches in Guizhou Province, China.Examination of the wood revealed three novel fungal species, two species that are newly recorded in China and one known species of Pleosporales.To elucidate their taxonomic placement and relationships with related species, we conducted morphological observations and phylogenetic analyses, based on combined ITS, LSU, SSU, tef1, and rpb2 sequences.Detailed descriptions of the morphological features of these species along with their molecular characterisation are provided.

Fungal sampling, isolating and morphology
Fresh fungal specimens were collected in Duyun, Zunyi, Qiannan Prefecture and Guiyang, Guizhou Province and were brought back to the laboratory in self-seal-ing bags.The specimens were then examined for their macroscopic characteristics using a Nikon SMZ 745 series stereomicroscope and photographed, using a Canon 700D digital camera.Micro-morphological structures were photographed using a Nikon digital camera (Canon 700D) that was attached to a light microscope (Nikon Ni).Melzer's iodine reagent was used to test the apical apparatus structures for amyloid reaction.Measurements of the specimens were registered using Tarosoft (R) Image FrameWork 80 software.The photo plates were arranged and improved using Adobe Photoshop CS6 software.Pure cultures were obtained with the single spore isolation method (Long et al. 2019) and the cultures were grown on potato dextrose agar (PDA) for preservation and observation of the anamorph (Rogers and Ju 1996).The specimens were deposited in the Herbaria of Guizhou Medical University (GMB) and Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS).Living cultures were deposited at the Guizhou Medical University Culture Collection (GMBC).

DNA extraction, polymerase chain reaction (PCR) amplification
The pure cultures were cultivated on potato dextrose agar (PDA) medium (Weigh 40.1g of potato dextrose agar (Shanghai Bowei Microbial Technology Co., Ltd.), add 1L of sterile water, and dissolve by heating until boiling.After dissolution, distribute the solution into conical flasks and place them in a high-pressure sterilizer for sterilization.Sterilization conditions are set at 121 degrees Celsius for 30 minutes.After sterilization, add a small amount of injectable potassium penicillin (Huamu) and injectable streptomycin sulfate (Huamu) into the culture medium and mix well.Pour the mixture into disposable culture dishes for later use.This step should be performed in aseptic conditions inside a laminar flow hood.) at 25 °C in the dark for 15-20 days.Fresh mycelium was collected by scraping it with a surgical knife and then transferred to a 1.5 ml centrifuge tube.DNA extraction was performed according to the instructions provided in the Biospin Fungus Genomic DNA Extraction Kit (BIOMIGA®).
Bayesian Inference (BI) analysis was conducted using MrBayes v.3.2.7a (Ronquist et al. 2012) and posterior probabilities (PP) were determined through Markov Chain Monte Carlo sampling (MCMC).Six simultaneous Markov chains for 3,000,000 generations were run and trees were sampled every 1,000 th generation.
The trees were visualised using FigTree v,1.4.4,and formatted using Adobe Illustrator CS v.6.Branches with Maximum-Likelihood bootstrap values (MLBP) equal to or greater than 75% and Bayesian posterior probabilities (BYPP) greater than 0.95 are indicated.The combined loci alignment and resulting phylogenetic trees were submitted to TreeBASE (https://www.treebase.org,submission number: ID 30482; ID 30483; ID 30484).

Neokalmusia karka
Culture characters.After 4 weeks of cultivation at 25 °C, the colonies on PDA measure around 2-2.5 cm in diameter.The surface appears smooth to velvety with an entire or slightly irregular margin, ranging from white to grey olivaceous.The colour is white near the margin with dense circular to filamentous growth.The reverse side of the colonies black to greenish-olivaceous.
Notes.Phylogenetic analyses of the combined ITS, LSU, tef1 and rpb2 gene sequences showed that the sequence from our 2022SLZH6 collection clusters together with Roussoella pseudohysterioides (MFLU 15-1209), with strong support (100% ML, 1 BYPP; Fig. 2).The morphological characteristics of our specimen are also consistent with those of R. pseudohysterioides, which was originally described from decaying bamboo culms in Thailand (Dai et al. 2017).In China, it had previously been reported from Yunnan Province (Jiang et al. 2019).This is the second report of this species in China, representing a new record for Guizhou Province.Descriptions.See Liu et al. (2014).
Notes.The sequence of our Roussoella neopustulans (2022HX25) forms a well-supported clade (85% ML, 0.92 BYPP; Fig. 2) with R. neopustulan .Roussoella neopustulans was originally introduced by Liu et al. (2014), with a description of the sexual morph only.Dai et al. (2017) provided a comprehensive description and illustrations for both the sexual and asexual morphs of this species.Our collection exhibits identical morphological characteristics to those detailed by Dai et al. (2017).This is the first report of this species in China.Descriptions.See Thambugala et al. (2017).

Nigrogranaceae
Culture characters.After 4 weeks at 25 °C, colonies on PDA have a diameter of 2-2.5 cm and are circular, slightly raised to umbonate and dull with an entire edge.They appear floccose and smooth and droplets can be observed due to cellular respiration, water formation or antibiotic production.Colonies from the upper region have brown to cream-coloured margins and blackish-brown centres, while their reverse is white to yellowish-brown at the margin and blackish-brown in the centre.

Nigrograna trachycarpus
Etymology.Named after the host genus Trachycarpus from which the fungus was isolated.
Description.Saprobic or parasitic on dead culms of Trachycarpus sp.
Culture characteristics.After 4 weeks at 25 °C on PDA, colonies typically reach 2-2.5 cm in diameter.They present a circular shape with a dense and elevated centre, while appearing sparse and radiating at the margin.The colonies exhibit colours ranging from dark grey to pale olivaceous when viewed from above and from dark olivaceous to black on reverse.

Discussion
In this study, based on phylogenetic trees of combined ITS, LSU, SSU, tef1 and rpb2 sequences and morphology, we described and illustrated three new species of micro-fungi on dead woody litter, viz., Neokalmusia karka (Didymosphaeriaceae), Nigrograna schinifolium and N. trachycarpus (Nigrogranaceae) and records of three species of Roussoella (Roussoellaceae).Didymosphaeriaceae was introduced by Munk (1953) and is one of the most diverse families within the Pleosporales, with a total of 33 genera (Thambugala et al. 2015;Haridas et al. 2020).We included all of these Didymosphaeriaceae genera in our phylogenetic analysis.We used a dataset that combines ITS, LSU, SSU, tef1 and rpb2 genes for this purpose.Neokalmusia formed a well-supported monophyletic clade within Didymosphaeriaceae, while the newly-discovered species, N. karka, exhibited a distinct separation from other known Neokalmusia species, supported by strong phylogenetic values.
Nigrograna, which is the only genus within Nigrogranaceae, is globally distributed and ecologically diverse.Amongst its species, N. mackinnonii is the most widely distributed species, mainly found in deciduous forests in Canada and northern USA.Nigrograna bergmaniae is mainly distributed in Europe, while N. novae-zelandiae was discovered in New Zealand.Approximately one-quarter of existing species live as saprotrophs on the bark or corticated twigs of various hardwoods (Phukhamsakda et al. 2018;Jayasiri et al. 2019).Nigrograna schinifolium was collected from rotten wood, while N. trachycarpus was obtained from decaying culms.Notably, several Nigrograna species have been established in recent studies without strong bootstrap value support.This finding suggests that these two species, N. schinifolium and N. trachycarpus, belong to the genus Nigrograna with strong evidence supporting this classification.
This study unveils valuable insights about saprophytic fungi, shedding light on their distribution and diversity within the Guizhou Region.It also identified three new species, which are important for the study of fungal taxonomy and further enriches our understanding of these microscopic organisms.Moreover, the study highlights the ongoing instability within the existing taxonomic system, emphasising the necessity for addressing these taxonomic challenges through processes such as re-collection, confirmation and sequencing of samples.

Figure 1 .
Figure 1.RAxML phylogram of Didymosphaeriaceae, based on a combined dataset of partial ITS, LSU, SSU and tef1 DNA sequences.The tree is rooted by Bimuria novae-zelandiae (CBS 107.79) and Bimuria omanensis (SQUCC 15280).Bootstrap supports ML (MLB ≥ 75%) and Bayesian posterior probabilities (BYPP ≥ 0.95) are given as MLB/BYPP above the branches.Sequences from newly-generated isolates are in red, bold letters, while those of ex-type isolates are shown in black, bold letters.

Figure 2 .
Figure 2. RAxML phylogram of Roussoellaceae, based on a combined dataset of partial ITS, LSU, tef1 and rpb2 DNA sequences.The tree is rooted by Torula hollandica (CBS 220.69) and T. herbarum (CBS 111855).Bootstrap supports ML (MLB ≥ 75%) and Bayesian posterior probabilities (BYPP ≥ 0.95) are given as MLB/BYPP above the branches.Sequences from newly-generated isolates are in red, bold letters, while those of ex-type isolates are shown in black, bold letters.

Figure 3 .
Figure 3. RAxML phylogram of Nigrogranaceae, based on a combined dataset of ITS and tef1 DNA sequences.The tree is rooted by Occultibambusa pustula (MFLUCC 11-0502) and O. bambusae (MFLUCC 13-0855).Bootstrap supports ML (MLB ≥ 75%) and Bayesian posterior probabilities (BYPP ≥ 0.95) are given as MLB/BYPP above the branches.Sequences from newly-generated isolates are in red, bold letters, while those of ex-type isolates are shown in black, bold letters.

Figure 8 .
Figure 8. Nigrograna schinifolium (GMB0498) A specimen B appearance of ascomata on substrate C cross-section of ascomata D longitudinal section of an ascoma E peridium F pseudoparaphyses G-I asci J-L ascospores M, N culture on PDA.Scale bars: 0.5 mm (B-C); 10 μm (D-L).

Table 2 .
Composition of PCR reaction system.

Table 3 .
Taxa and corresponding GenBank accession numbers of sequences used in the phylogenetic analysis of Didymosphaeriaceae, Roussoellaceae and Nigrogranaceae.Hongmin Hu et al.: Taxonomic and phylogenetic characterizations of six species of Pleosporales (Dissanayake et al. 2021)MycoKeys 100: 123-151 (2023), DOI: 10.3897/mycokeys.100.109423Notes: Type specimens or Ex-type specimens are marked with T; "-": indicates no sequence available in GenBank; newly-generated sequences are indicated in bold.Abbreviations: CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CPC: Culture collection of Pedro Crous, housed at the Westerdijk Fungal Biodiversity Institute; GMB: Culture collection of Guizhou Medical University; HKAS: Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica, Chinese Academy of Sciences, Kunming, China; HKUCC: Hong Kong University Culture Collection; KT: K. Tanaka; KUMCC: Kunming Institute of Botany Culture Collection, Chinese Science Academy, Kunming, China; MAFF: Ministry of Agriculture, Forestry and Fisheries, Japan; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; NFCCI: National Fungal Culture Collection of India; Others: information not available.