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Research Article
Taxonomic and phylogenetic characterisations of six species of Pleosporales (in Didymosphaeriaceae, Roussoellaceae and Nigrogranaceae) from China
expand article infoHongmin Hu‡, Minghui He‡, Youpeng Wu‡, Sihan Long‡, Xu Zhang‡, Lili Liu‡, Xiangchun Shen‡, Nalin N. Wijayawardene§|, Zebin Meng, Qingde Long‡, Jichuan Kang#, Qirui Li‡
‡ Guizhou Medical University, Guiyang, China
§ Qujing Normal University, Qujing, China
| Tropical Microbiology Research Foundation, Pannipitiya, Sri Lanka
¶ Guizhou Education University, Guiyang, China
# Guizhou University, Guiyang, China

Corresponding author: Qingde Long ( longqingde@gmc.edu.cn )

Corresponding author: Qirui Li ( lqrnd2008@163.com )

Academic editor: Jennifer Luangsa-ard
© 2023 Hongmin Hu, Minghui He, Youpeng Wu, Sihan Long, Xu Zhang, Lili Liu, Xiangchun Shen, Nalin N. Wijayawardene, Zebin Meng, Qingde Long, Jichuan Kang, Qirui Li.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Hu H, He M, Wu Y, Long S, Zhang X, Liu L, Shen X, Wijayawardene NN, Meng Z, Long Q, Kang J, Li Q (2023) Taxonomic and phylogenetic characterisations of six species of Pleosporales (in Didymosphaeriaceae, Roussoellaceae and Nigrogranaceae) from China. MycoKeys 100: 123-151. https://doi.org/10.3897/mycokeys.100.109423
Open Access

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 (Neokalmusia karka, Nigrograna schinifolium and N. trachycarpus) have been discovered, along with two new records for China (Roussoella neopustulans and R. doimaesalongensis) and a known species (Roussoella pseudohysterioides). Morphologically similar species and phylogenetically close taxa are compared and discussed. This study provides detailed information and descriptions of all newly-identified taxa.

Key words

phylogeny, saprophytic fungi, taxonomy, three new 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.

Materials and methods

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-sealing 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®).

The amplification of internal transcribed spacers (ITS), small subunit rDNA (SSU), large subunit rDNA (LSU), translation elongation factor 1-gene region (tef1) and RNA polymerase II second largest subunit (rpb2) was achieved using ITS5/ITS4, NS1/NS4, LR0R/LR5, EF1-938F/EF1-2218R and fRPB2-5f/fRPB2-7cr primers (Tibpromma et al. 2018; Vu et al. 2019; Wijesinghe et al. 2020; Dissanayake et al. 2021). The polymerase chain reaction (PCR) for the amplification of ITS, SSU, LSU, tef1 and rpb2 loci were performed using the Eppendorf Mastercycler nexus (SimpliAmp Thermal Cycler, A24811, SimpliAmp, China) gradient under the conditions specified in Table 1. Subsequently, the PCR fragments were sent to Sangon Biotech (Shanghai) Co., China, for sequencing. Amplification conditions using the Polymerase Chain Reaction is shown in Table 2. The obtained sequences were deposited in GenBank and are listed in Table 3.

Table 1.
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PCR conditions used for ITS, SSU, LSU, tef1 and rpb2 loci.

Genes Initial period Cycles, denaturation, annealing and elongation Final extension
ITS, LSU, SSU, tef1 95°C for 5 min 35 cycles of denaturation at 94 °C for 1 min, annealing at 52°C for 1 min, elongation at 72°C for 1.5 min 72°C for 10 minutes
rpb2 95°C for 5 min 35 cycles of denaturation at 95°C for 1 minute, annealing at 54°C for 2 minutes, elongation at 72°C for 1.5 minutes 72°C for 10 minutes
Table 2.
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Composition of PCR reaction system.

Components Volumetry Concentration
2× Tap PCR Mix 12.5 μl 1×
Primer 1 1 μl 10μM μl-1
Primer 1 μl 10μM μl-1
DNA template 1 μl 0.1-0.2 μg μl-1
ddH2O Up to 25 μl
Table 3.
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Taxa and corresponding GenBank accession numbers of sequences used in the phylogenetic analysis of Didymosphaeriaceae, Roussoellaceae and Nigrogranaceae.

Species Strain GenBank Accession Numbers References
ITS SSU LSU tef1 rpb2
Alloconiothyrium camelliae NTUCC 17-032-1T MT112294 MT071221 MT071270 MT232967 — (Kolařík et al. 2017)
Arthopyrenia sp. UTHSC DI16–362 LT796905 LN907505 — LT797145 LT797065 (Crous et al. 2015)
Austropleospora ochracea KUMCC 20-0020T MT799859 MT808321 MT799860 MT872714 — (Dissanayake et al. 2021)
A. keteleeriae MFLUCC 18-1551T NR_163349 MK347910 NG_070075 MK360045 — (Mapook et al. 2020)
Biatriospora antibiotica CCF 1998 LT221894 — — — — (Kolařík et al. 2017)
B. carollii CCF 4484T LN626657 — — LN626668 — (Kolařík et al. 2017)
B. mackinnonii E9303e — — — LN626673 — (Kolařík et al. 2017)
B. peruviensis CCF 4485T LN626658 — — LN626671 — (Kolařík et al. 2017)
Bimuria omanensis SQUCC 15280T NR_173301 — NG_071257 MT279046 — (Wijesinghe et al. 2020)
B. novae-zelandiae CBS 107.79T MH861181 AY016338 AY016356 DQ471087 — (Vu et al. 2019)
Chromolaenicola nanensis MFLUCC 17-1477 MN325014 MN325008 MN325002 MN335647 — (Liu et al. 2014)
C. siamensis MFLUCC 17-2527T NR_163337 MK347866 NG_066311 MK360048 — (Mapook et al. 2020)
C. thailandensis MFLUCC 17-1475 MN325019 MN325013 MN325007 MN335652 — (Liu et al. 2014)
C. lampangensis MFLUCC 17-1462T MN325016 MN325010 MN325004 MN335649 — (Liu et al. 2014)
Cylindroaseptospora leucaenae MFLUCC 17-2424 NR_163333 MK347856 NG_066310 MK360047 — (Mapook et al. 2020)
Deniquelata hypolithi CBS 146988T MZ064429 — NG_076735 MZ078250 — (Ariyawansa et al. 2020b)
D. barringtoniae MFLUCC 16-0271 MH275059 — MH260291 MH412766 — (Tibpromma et al. 2018)
Didymocrea sadasivanii CBS 438.65 MH858658 DQ384066 DQ384103 — — (Vu et al. 2019)
Didymosphaeria rubi-ulmifolii MFLUCC 14-0023T — NG_063557 KJ436586 — — (Jayasiri et al. 2019)
Kalmusia erioi MFLU 18-0832T MN473058 MN473046 MN473052 MN481599 — (Vu et al. 2019)
K. italica MFLUCC 13-0066T KP325440 KP325442 KP325441 — — (Vu et al. 2019)
K. variisporum CBS 121517T NR_145165 — JX496143 — — (Wijesinghe et al. 2020)
K. ebuli CBS 123120T KF796674 JN851818 JN644073 — — (Dissanayake et al. 2021)
Kalmusibambusa triseptata MFLUCC 13-0232 KY682697 KY682696 KY682695 — — (Tibpromma et al. 2018)
Karstenula rhodostoma CBS 690.94 — GU296154 GU301821 GU349067 — (Crous et al. 2021)
Laburnicola hawksworthii MFLUCC 13-0602T KU743194 KU743196 KU743195 — — (Ariyawansa et al. 2014)
Letendraea helminthicola CBS 884.85 MK404145 AY016345 AY016362 MK404174 — (Tibpromma et al. 2018)
L. muriformis MFLUCC 16-0290T KU743197 KU743199 KU743198 KU743213 — (Ariyawansa et al. 2014)
L. padouk CBS 485.70 — GU296162 AY849951 — — (Zhang et al. 2013)
L. cordylinicola MFLUCC 11 0148T NR_154118 KM214001 NG_059530 — — (Wijayawardene et al. 2020)
Montagnula chromolaenicola MFLUCC 17-1469T NR_168866 NG_070157 NG_070948 MT235773 — (Liu et al. 2014)
M. cirsii MFLUCC 13 0680 KX274242 KX274255 KX274249 KX284707 — (Hyde et al. 2020)
M. krabiensis MFLUCC 16-0250T MH275070 MH260343 MH260303 MH412776 — (Tibpromma et al. 2018)
M. thailandica MFLUCC 17-1508T MT214352 NG_070158 NG_070949 MT235774 — (Liu et al. 2014)
M. bellevaliae MFLUCC 14-0924T NR_155377 KT443904 KT443902 KX949743 — (Ariyawansa et al. 2014)
Neoroussoella alishanense FU31016 MK503816 MK503822 — MK336181 MN037756 (Verkley et al. 2014)
N. bambusae MFLUCC 11–0124 KJ474827 KJ474839 — KJ474848 KJ474856 (Dissanayake et al. 2021)
N. brevispora KT2313T LC014574 AB524460 AB524601 AB539113 — (Tanaka et al. 2015)
N. brevispora KT1466 LC014573 AB524459 AB524600 AB539112 — (Tanaka et al. 2015)
N. heveae MFLUCC 17–1983 MH590693 MH590689 — — — (Wanasinghe et al. 2018)
N. jonahhulmei KUMCC 21-0819 ON007044 ON007040 ON007049 ON009134 — (Wanasinghe et al. 2016)
N. karka GMB0494T OR120445 OR120442 OR120432 OR150020 — This study
N. karka GMB0500 OR120438 OR120433 OR120443 OR150021 — This study
N. kunmingensis KUMCC 18-0120T MK079886 MK079887 MK079889 MK070172 — (Vu et al. 2019)
N. lenispora GZCC 16-0020T — KX791431 — — — (Hyde et al. 2020)
N. scabrispora KT1023 LC014575 AB524452 AB524593 AB539106 — (Tanaka et al. 2015)
N. solani CPC 26331T KX228261 KX228312 — — — (Wijayawardene et al. 2014)
N. thailandica MFLUCC 16-0405T NR_154255 KY706137 NG_059792 KY706145 — (Thambugala et al. 2015)
Neokalmusia arundinis MFLUCC 15-0463T NR_165852 NG_068372 NG_068237 KY244024 — (Thambugala et al. 2015)
Nigrograna antibiotica CCF 4378T JX570932 — — JX570934 — (Kolařík et al. 2018)
Nigrograna cangshanensis MFLUCC15-0253T KY511063 — — KY511066 — (Crous et al. 2015)
N. chromolaenae MFLUCC 17-1437T MT214379 — — MT235801 — (Liu et al. 2014)
N. didymospora MFLUCC 11-0613 — KP091435 KP091434 — — (Haridas et al. 2020)
N. fuscidula CBS 141556T KX650550 — — KX650525 — (Feng et al. 2019)
N. fuscidula CBS 141476 KX650547 — — KX650522 — (Feng et al. 2019)
N. hydei GZCC 19-0050T NR_172415 — — MN389249 — (Zhang et al. 2020)
N. impatientis GZCC 19-0042T NR_172416 — — MN389250 — (Zhang et al. 2020)
N. leucaenae MFLUCC 18–1544 MK347767 MK347984 — MK360067 MK434876 (Mapook et al. 2020)
N. locuta-pollinis CGMCC 3.18784 MF939601 — — MF939613 — (Ahmed et al. 2014)
N. locuta-pollinis LC11690 MF939603 — — MF939614 — (Ahmed et al. 2014)
N. mackinnonii CBS 674.75T NR_132037 — — KF407986 — (Ariyawansa et al. 2015)
N. mackinnonii E5202H JX264157 — — JX264154 — (Phukhamsakda et al. 2018)
N. magnoliae GZCC 17-0057 MF399066 — — MF498583 — (Zhang et al. 2020)
N. magnoliae MFLUCC 20-0020T MT159628 — — MT159605 — (Liu et al. 2014)
N. mycophila CBS 141478T KX650553 — — KX650526 — (Feng et al. 2019)
N. mycophila CBS 141483 KX650555 — — KX650528 — (Feng et al. 2019)
N. norvegica CBS 141485T KX650556 — — — — (Feng et al. 2019)
N. obliqua CBS 141477T KX650560 — — KX650531 — (Feng et al. 2019)
N. obliqua CBS 141475 KX650558 — — KX650530 — (Feng et al. 2019)
N. rhizophorae MFLUCC 18-0397T MN047085 — — MN077064 — (Poli et al. 2020)
N. samueliana NFCCI-4383T MK358817 — — MK330937 — (Poli et al. 2020)
N. schinifolium GMB0498T OR120434 — — OR150022 — This study
N. schinifolium GMB0504 OR120441 — — OR150023 — This study
N. thymi MFLUCC 14-1096T KY775576 — — KY775578 — (Crous et al. 2015)
N. trachycarpus GMB0499T OR120437 — — OR150024 — This study
N. trachycarpus GMB0505 OR120440 — — OR150025 — This study
N. yasuniana YU.101026T HQ108005 — — LN626670 — (Kolařík et al. 2018)
Occultibambusa pustula MFLUCC 11-0502T KU940126 — — — — (Crous et al. 2014)
O. bambusae MFLUCC 13-0855T KU940123 — — KU940193 — (Crous et al. 2014)
Paracamarosporium fagi CPC 24890T NR_154318 — NG_070630 — — (Ariyawansa et al. 2014)
P. cyclothyrioides CBS 972.95 JX496119 AY642524 JX496232 — — (Schoch et al. 2009)
P. estuarinum CBS 109850T JX496016 AY642522 JX496129 — — (Verkley et al. 2014)
P. hawaiiense CBS 120025T JX496027 EU295655 JX496140 — — (Verkley et al. 2014)
P. robiniae MFLUCC 14–1119T KY511142 KY511141 — KY549682 — (Crous et al. 2015)
P. rosarum MFLUCC 17–6054T NR_157529 NG_059872 — MG829224 — (Hyde et al. 2016)
P. rosicola MFLUCC 15-0042 NR_157528 MG829153 MG829047 — — (Hyde et al. 2016)
Paramassariosphaeria anthostomoides CBS 615.86 MH862005 GU205246 GU205223 — — (Vu et al. 2019)
Paraphaeosphaeria rosae MFLUCC 17-2547T MG828935 MG829150 MG829044 MG829222 — (Hyde et al. 2016)
Pararoussoella mukdahanensis KUMCC 18-0121 MH453489 MH453485 — MH453478 MH453482 (Flakus et al. 2019)
Parathyridaria ramulicola CBS 141479T KX650565 KX650565 — KX650536 KX650584 (Feng et al. 2019)
Phaeodothis winteri CBS 182.58 — GU296183 GU301857 — — (Zhang et al. 2013)
Pseudocamarosporium propinquum MFLUCC 13-0544T KJ747049 KJ819949 KJ813280 — — (Thambugala et al. 2017)
Pseudodidymocyrtis lobariellae KRAM Flakus 25130T NR_169714 NG_070349 NG_068933 — — (Tanaka et al. 2015)
Pseudoneoconiothyrium euonymi CBS 143426T MH107915 MH107961 — — MH108007 (Valenzuela-Lopez et al. 2017)
Pseudopithomyces entadae MFLUCC 17-0917T — MK347835 NG_066305 MK360083 — (Mapook et al. 2020)
Pseudoroussoella chromolaenae MFLUCC 17–1492T MT214345 MT214439 — MT235769 — (Liu et al. 2014)
P. elaeicola MFLUCC 15–0276a MH742329 MH742326 — — — (Liu et al. 2014)
P. kunmingnensis MFLUCC 17-0314 MF173607 MF173606 MF173605 — — (Mapook et al. 2020)
P. pteleae MFLUCC 17-0724T NR_157536 MG829166 MG829061 MG829233 — (Hyde et al. 2016)
P. rosae MFLUCC 15-0035T MG828953 MG829168 MG829064 — — (Hyde et al. 2016)
P. ulmi-minoris MFLUCC 17-0671T NR_157537 MG829167 MG829062 — — (Hyde et al. 2016)
Roussoella acaciae CBS:138873T KP004469 KP004497 — — — (Karunarathna et al. 2019)
R. aquatic MFLUCC 18-1040T NR171975 NG073797 — — — (Liu et al. 2014)
R. chiangraina MFLUCC 10-0556T NR155712 NG059510 — — — (Dissanayake et al. 2021)
R. doimaesalongensis MFLUCC 14-0584T NR165856 NG068241 — KY651249 KY678394 (Thambugala et al. 2015)
R. doimaesalongensis GMB0497 OR116188 OR117732 — OR150026 — This study
R. doimaesalongensis GMB0503 OR120435 OR120444 — OR150027 — This study
R. elaeicola MFLUCC 15-15-0276a MH742329 MH742326 — — — (Crous et al. 2015)
R. euonymi CBS:143426T MH107915 MH107961 — — MH108007 (Valenzuela-Lopez et al. 2017)
R. guttulata MFLUCC 20-0102T NR172428 NG075383 — — (Senwanna et al. 2018)
R. hysterioides CBS 546.94 MH862484 MH874129 — KF443399 KF443392 (Vilgalys et al. 1990)
R. intermedia CBS 170.96 KF443407 KF443382 — KF443398 KF443394 (Crous et al. 2013)
R. japanensis MAFF 239636T NR155713 — — — — (Dissanayake et al. 2021)
R. kunmingensis HKAS 101773T MH453491 MH453487 — MH453480 MH453484 (Flakus et al. 2019)
R. magnatum MFLUCC 15-0185T — KT281980 — — — (Jiang et al. 2019)
R. mangrovei MFLU 17-1542T MH025951 MH023318 — MH028246 MH028250 (Jaklitsch and Voglmayr 2016)
R. margidorensis MUT 5329T NR169906 MN556322 — MN605897 MN605917 (Tibpromma et al. 2017)
R. mediterranea MUT5369T KU314947 MN556324 — MN605899 MN605919 (Tibpromma et al. 2017)
R. mexicana CPC 25355T KT950848 KT950862 — — — (Crous et al. 2015a)
R. mukdahanensis MFLU 11-0237T NR155722 — — — — (Crous et al. 2014)
R. multiplex GMB0316T ON479891 — ON479892 — — (Dong et al. 2020)
R. neopustulans MFLUCC 11-0609T KJ474833 KJ474841 — KJ474850 — (Dissanayake et al. 2021)
R. neopustulans GMB0496 OR120436 OR120446 This study
R. neopustulans GMB0502 OR116176 OR117714 This study
R. nitidula MFLUCC 11-0634 KJ474834 KJ474842 — KJ474851 KJ474858 (Dissanayake et al. 2021)
R. padinae MUT 5503T — MN556327 — MN605902 MN605922 (Tibpromma et al. 2017)
R. percutanea CBS 868.95 KF322118 KF366449 — KF407987 KF366452 (Ahmed et al. 2014a)
R. pseudohysterioides GMBC0009T MW881445 MW881451 — — MW883345 (Zhang et al. 2020)
R. pseudohysterioides GMB0495 OR116175 OR117737 — OR150028 — This study
R. pseudohysterioides GMB0501 OR120447 OR120439 — OR150029 — This study
R. pustulans KT 1709 — AB524623 — AB539116 AB539103 (Zhang et al. 2020)
R. scabrispora MFLUCC 14-0582 KY026583 KY000660 — — — (Zhang et al. 2020)
R. siamensis MFLUCC 11-0149T KJ474837 KJ474845 — KJ474854 KJ474861 (Dissanayake et al. 2021)
R. thailandica MFLUCC 11-0621T KJ474838 KJ474846 — — — (Dissanayake et al. 2021)
R. tuberculata MFLUCC 13-0854T KU940132 KU863121 — KU940199 (Crous et al. 2014)
R. verrucispora CBS 125434T KJ474832 — — — — (Dissanayake et al. 2021)
R. yunnanensis HKAS 101762 MH453492 MH453488 — MH453481 — (Flakus et al. 2019)
Roussoellopsis macrospora MFLUCC 12-0005 — KJ474847 — KJ474855 KJ474862 (Dissanayake et al. 2021)
R. tosaensis KT 1659 — AB524625 — AB539117 AB539104 (Zhang et al. 2020)
Setoarthopyrenia chromolaenae MFLUCC 17–1444 MT214344 MT214438 — MT235768 MT235805 (Liu et al. 2014)
Spegazzinia deightonii yone 212 — AB797292 AB807582 AB808558 — (Tanaka et al. 2015)
S. radermacherae MFLUCC 17-2285T MK347740 MK347848 MK347957 MK360088 — (Mapook et al. 2020)
S. tessarthra NRRL 54913 JQ673429 AB797294 AB807584 AB808560 — (Tanaka et al. 2015)
Thyridaria acaciae CBS 138873 KP004469 KP004497 — — — (Liu et al. 2014)
T. broussonetiae CBS 141481 NR_147658 KX650568 — KX650539 KX650586 (Karunarathna et al. 2019)
Torula herbarum CBS 111855 KF443409 KF443386 — KF443403 KF443396 (Crous et al. 2013)
T. hollandica CBS 220.69 KF443406 KF443384 — — KF443393 (Crous et al. 2013)
Tremateia arundicola MFLU 16-1275 KX274241 KX274254 KX274248 KX284706 — (Hyde et al. 2020)
T. chromolaenae MFLUCC 17-1425T NR_168868 NG_070160 NG_068710 MT235778 — (Tanaka et al. 2015)
T. guiyangensis GZAAS01 KX274240 KX274253 KX274247 KX284705 — (Hyde et al. 2020)
T. murispora GZCC 18-2787 NR_165916 MK972750 MK972751 MK986482 — (Feng et al. 2019)
T. thailandensis MFLUCC 17-1430T NR_168869 NG_070161 NG_068711 MT235781 — (Liu et al. 2014)
Verrucoconiothyrium nitidae CBS:119209 EU552112 — EU552112 — — (Wanasinghe et al. 2018)
Xenocamarosporium acaciae CPC 24755T NR_137982 — NG_058163 — — (Crous et al. 2015b)
Xenoroussoella triseptata MFLUCC 17–1438 MT214343 MT214437 — MT235767 MT235804 (Liu et al. 2014)

Notes: 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.

Phylogenetic analysis

BioEdit v.7.0 was used to verify the quality of sequences (Hall TA 1999) and MAFFT v.7.215 (http://mafft.cbrc.jp/alignment/server/index.html) was employed to generate single gene alignments (Katoh and Standley 2013). The file format was converted using ALTER (Alignment Transformation Environment) (http://www.sing-group.org/ALTER/). Maximum Likelihood (ML) analyses and Bayesian posterior probabilities (BYPP), based on a combination of ITS, LSU, tef1 and rpb2 sequence data, were performed using RAxML-HPC BlackBox and MrBayes v. 3.2.7a tools in the CIPRES Science Gateway platform (Liang et al. 2020). GTR+I+G was estimated as the best-fit substitution model by ModelTest2 on XSEDE v.2.1.6. (Posada and Crandall 1998).

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 1,000th 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).

Results

Phylogenetic analyses

Phylogenetic analyses of Didymosphaeriaceae (Fig. 1), Roussoellaceae (Fig. 2), and Nigrogranaceae (Fig. 3) were performed separately, with corresponding parameters presented in Table 4.

Table 4.
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Results of Maximum-Likelihood (ML) and Bayesian (BI) analyses for each sequenced dataset.

Analyses Didymosphaeriaceae Roussoellaceae Nigrogranaceae
Number of taxa 64 59 32
Gene regions ITS, LSU, SSU and tef1 ITS, LSU, tef1 and rpb2 ITS and tef1
Number of character positions (including gaps) 2423 2267 868
ML optimisation likelihood value -13324.603084 -16237.062124 -3695.409391
Distinct alignment patterns in the matrix 584 773 240
Number of undetermined characters or gaps (%) 14.26% 27.45% 7.87%
Estimated base frequencies A 0.237970 0.240773 0.229686
C 0.246811 0.255815 0.293625
G 0.277468 0.276383 0.242370
T 0.237752 0.227030 0.234319
Substitution rates AC 1.764988 2.186105 1.598706
AG 2.187844 5.410475 2.533043
AT 1.416956 2.441301 1.640025
CG 1.132266 1.384067 0.752494
CT 7.848138 11.885781 8.062830
GT 1.000000 1.000000 1.000000
Proportion of invariable sites (I) 0.595845 0.544120 0.487317
Gamma distribution shape parameter (a) 0.516792 0.502253 0.634309
Number of generated trees in BI 14806 10678 9932
Average standard deviation of split frequencies 0.006852 0.004431 0.004939

Taxonomy

Didymosphaeriaceae Munk, 1953

Neokalmusia Ariyawansa & K.D. Hyde, Fungal Diversity 68: 92 (2014b)

MycoBank No: 550700

Notes

Neokalmusia was established by Ariyawansa et al. (2014b) to accommodate two bambusicolous taxa, N. brevispora and N. scabrispora, previously referred to Kalmusia. Members of Neokalmusia are characterised by solitary sphaeroid ascomata, a peridium of small pseudoparenchymatous cells, clavate basal asci with very long pedicels, very thin pseudoparaphyses and distoseptate, smooth-walled ascospores (Ariyawansa et al. 2014b; Zhang et al. 2020a). In this study, we introduce a new species of Neokalmusia, based on a combination of morphological and molecular analyses (Fig. 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.

Neokalmusia karka H. M. Hu & Q. R. Li, sp. nov.

MycoBank No: 851046
Fig. 4

Type material

Holotype : GMB0494.

Etymology

In reference to the host, Phragmites karka (Retz.) Trin. ex Steud.

Description

Saprobic on dead culms of P. karka.

Sexual morph: Clypeus visible as black dots on the host surface, breaking through slightly raised cracks at the centre. Ascomata 241–386 × 161–231 μm (average = 375 × 197 μm, n = 5), smooth, semi-immersed, scattered, solitary or in small groups, black, oval, with ostiole. Peridium 12–20 μm wide, composed of a few layers of thin-walled, brown to dark brown, cells of textura angularis. Hamathecium comprising 1.5–2.8 μm wide, numerous, cellular, pseudoparaphyses, embedded in a mucilaginous matrix. Asci 80–109 × 10–14 μm (average = 95 × 11.4 μm, n = 15), 8-spored, bitunicate, fissitunicate, cylindrical-clavate, with bulbous pedicel, apically rounded with an indistinct ocular chamber, with a J-subapical ring. Ascospores 14–17 × 4–6 μm (average = 15.8 × 5.3 μm, n = 30), overlapping 1–2-seriate, fusiform, pale brown to brown, 1-septate, constricted at the septum, often enlarged near septum in the upper cell, distinctly verrucose on the surface, without a mucilaginous sheath. Asexual morph: undetermined.

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.

Specimens examined

China, Guizhou Province, Zunyi City, Suiyang County, Kuanqwashui Nature Reserve (28°31'51.04"N, 107°9'33.65"E), 1544 m elev., on decaying culms, 12 October 2022, Y.P Wu and H.M Hu, 2022KKS49 (GMB0494, holotype; GMBC0494, ex-type; KUN-HKAS 129179, isotype).

Other examined material

China, Guizhou Province, Huaxi District, Shilihetan Wetland Park (26°41'34.3"N, 106°67'68.8"E), 1500 m elev., on decaying culms, 8 October 2022, Y.P Wu and H.M Hu, 2022SLZH11 (GMB0500; GMBC0500, living culture).

Notes

This fungus shares morphological characters similar to Neokalmusia in having immersed ascomata, a clypeus-like structure composed of thin-walled cells and verrucose ascospores (Tanaka et al. 2009; Ariyawansa et al. 2014b). Other than Neokalmusia karka, only two species, N. arundinis Thambug. & K.D. Hyde and N. didymospora D.Q. Dai & K.D. Hyde have been reported with 1-septate ascospores. However, N. karka can be distinguished, based on differences in asci size (N. karka, 80–109 × 10–14 μm; N. arundinis 60–85 × (7.5–) 8.5–10.5 μm; N. didymospora 125–160 × 9.5–14 μm) and the obvious oval shape of its ascomata (Wanasinghe et al. 2018; Flakus et al. 2019). In our phylogram, Neokalmusia karka formed a well-supported separate clade (100% ML, 1 BYPP; Fig. 1) in a sister relationship with N. arundinis and N. didymospora. The macro and micro-morphological differences and phylogenetic analyses support the recognition of N. karka as a new species (Fig. 1).

Roussoellaceae Jian K. Liu, Phook., D.Q. Dai & K.D. Hyde 2014

Roussoella Sacc., Atti Inst. Veneto Sci. lett., ed Arti, Sér. 6 6: 410 (1888)

MycoBank No: 541317

Notes

the genus Roussoella was introduced by Saccardo et al. (1888), with R. nitidula Sacc. & Paol. as the type species, which was collected from bamboo in Malaysia. This family is characterised as having semi-immersed to immersed, solitary or gregarious, clypeate ascostromata containing trabeculate pseudoparaphyses embedded in a gel matrix, long cylindrical to clavate bitunicate asci with or without obvious fissitunicate dehiscence and brown, 2-celled ornamented ascospores (Liu et al. 2014). In this study, we introduce three new records of Roussoella species, based on morpho-anatomical and molecular analyses (Fig. 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. 

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 4. 

Neokalmusia karka (GMB0494, holotype) A type specimen B, C appearance of ascomata on substrate D, E longitudinal section of an ascoma F peridium G pseudoparaphyses H–K asci L–N ascospores O, J ascus subapical ring in Melzer’s Reagent. Scale bars: 0.5 mm (B–D); 10 μm (E–O).

Roussoella pseudohysterioides D.Q. Dai & K.D. Hyde, in Dai et al., Fungal Diversity 82(1): 37 (2017)

MycoBank No: 552026
Fig. 5

Descriptions

See Dai et al. (2017).

Specimen examined

China, Guizhou Province, Huaxi District, Shilihetan Wetland Park (26°43'34.3"N, 106°67'68.8"E), 1542 m elev., on decaying bamboo, 8 October 2022, Y.P Wu and H.M Hu, 2022SLZH6 (GMB0495; GMBC0495, living culture).

Figure 5. 

Roussoella pseudohysterioides (GMB0495) A specimen B, C appearance of ascomata on substrate D cross-section of ascostromata E longitudinal section of an ascoma F peridium G pseudoparaphyses H–I asci J–M ascospores. Scale bars: 0.5 mm (B–D); 10 μm (E–M).

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.

Roussoella neopustulans D.Q. Dai, J.K. Liu & K.D. Hyde, in Liu et al. Phytotaxa 181(1): 15 (2014)

MycoBank No: 550664
Fig. 6

Descriptions

See Liu et al. (2014).

Specimens examined

China, Guizhou Province, Huaxi District, Guiyang Huaxi National Urban Wetland Park (26°2'2.34"N, 106°34'16.22"E), on dead branch of bamboo, 12 October 2022, 1130 m elev., Y.P Wu and H.M Hu, 2022HX25 (GMB0496; GMBC0496, living culture).

Figure 6. 

Roussoella neopustulans (GMB0496) A specimen B, C appearance of ascomata on substrate D cross-section of ascostromata E longitudinal section of an ascoma F peridium G pseudoparaphyses H–K asci L–O ascospores. Scale bars: 0.5 mm (B–D); 10 μm (E–O).

Notes

The sequence of our Roussoella neopustulans (2022HX25) forms a well-supported clade (85% ML, 0.92 BYPP; Fig. 2) with R. neopustulan (MFLUCC 11-0609). 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.

Roussoella doimaesalongensis Thambug. & K.D. Hyde, Mycosphere 8 (4): 782 (2017)

MycoBank No: 553169
Fig. 7

Descriptions

See Thambugala et al. (2017).

Specimen examined

China, Guizhou Province, Huaxi District, Shilihetan Wetland Park (26°23'23.4"N, 106°67'56.4"E), 1511 m elev., on dead bamboo branches, 8 October 2022, Y.P Wu and H.M Hu, 2022SLHT14 (GMB0497; GMBC0497, living culture).

Figure 7. 

Roussoella doimaesalongensis (GMB0497) A specimen B appearance of ascomata on substrate C cross-section of ascostromata D longitudinal section of an ascoma E peridium F pseudoparaphyses G–I asci J–M ascospores. Scale bars: 0.5 mm (B–C); 10 μm (D–M).

Notes

In our phylogram (Fig. 2), the sequence of our collection clustered with Roussoella doimaesalongensis with robust support (100% ML, 1 BYPP). Roussoella doimaesalongensis was originally found on decaying bamboo culms in Thailand (Thambugala et al. 2017). Morphologically, our specimens match the description provided by Thambugala et al. (2017) and this species was first reported in China by Seong et al. (2022).

Nigrogranaceae Jaklitsch & Voglmayr, 2016

Nigrograna Gruyter, Verkley & Crous, Stud. Mycol. 75: 31 (2012) [2013]

MycoBank No: 564794

Notes

Nigrograna was described by De Gruyter et al. (2012) as a monotypic genus. Nigrograna is characterised by black ascomata, clavate, short pedicellate asci and pale to chocolate brown, asymmetric, fusoid to narrowly ellipsoid, septate ascospores (Zhang et al. 2020a).

Nigrograna schinifolium H. M. Hu & Q. R. Li, sp. nov.

MycoBank No: 849204
Fig. 8

Type material

Holotype. GMB0498.

Etymology

With reference to the host, Zanthoxylum schinifolium Sieb. & Zucc.

Description

Saprobic on dead stem of Z. Schinifolium.

Sexual morph

Ascomata 198–320 μm wide, 105–160 μm high, solitary or aggregated in small groups, black, semi-immersed, appearing as slightly raised regions. Ostioles are black, lined with paraphyses. Peridium 26–39 μm wide, comprising several fused layers of "textura angularis", thin-walled and pale brown at the interior, becoming darker and thicker-walled to the outside. Hamathecium comprising 1–2 μm wide, cylindrical to filiform, septate, branched, pseudoparaphyses, embedded in a gelatinous matrix. Asci 44–59 × 8–10 μm (average = 51.5 × 9.3 μm, n = 25), 8-spored, bitunicate, fissitunicate, cylindrical to broadly filiform, with a short stipe and knob-like base, apically rounded with a minute ocular chamber. Ascospores 10–14 × 2.8–4 μm (average = 11.6 × 3.3 μm, n = 40), broadly fusiform to inequilaterally ellipsoid, with the second cell slightly enlarged, straight or slightly curved, with obtuse to rounded ends, hyaline when immature, becoming brown to dark brown at maturity, 3-euseptate, slightly constricted at the median septum. Asexual morph: undetermined.

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).

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.

Specimen examined

China, Guizhou Province, Qiannan Prefecture, Sandu Shui Autonomous County, Yao Man Mountain National Forest Park (25°94′18.76"N, 107°95′70.09"E), 563 m elev., on branches of Zanthoxylum schinifolium, 28 September 2022, Y.P. Wu, 2022YRS36 (GMB0498, holotype, GMBC0498, ex-type; KUN-HKAS 12983, isotype).

Other examined material

China, Guizhou Province, Huaxi District, Shilihetan Wetland Park (26°23'13.4"N, 106°66'56.4"E), 1501 m elev., on branches of Zanthoxylum schinifolium, 8 October 2022, Y.P Wu and H.M Hu, 2022SLHT44 (GMB0504; GMBC0504, living culture).

Notes

Nigrograna schinifolium and N. thymi Mapook et al. form a monophyletic clade with moderate support (MPBP 48%, BYPP 0.83, Fig. 3). However, N. schinifolium is distinguished by having 3-septate ascospores (Hyde et al. 2017). Morphologically, N. schinifolium can be distinguished from other species of Nigrograna by its shorter asci and ascospores (Hyde et al. 2017; Zhao et al. 2018; Zhang et al. 2020a). Our research confirms N. schinifolium is a new species.

Nigrograna trachycarpus H. M. Hu & Q. R. Li, sp. nov.

MycoBank No: 849205
Fig. 9

Type material

Holotype : GMB0499.

Etymology

Named after the host genus Trachycarpus from which the fungus was isolated.

Description

Saprobic or parasitic on dead culms of Trachycarpus sp.

Sexual morph: Ascomata 160–380 μm wide, 100–210 μm high, pyriform to globose, scattered or clustered in small groups, black, immersed, the base remaining immersed in the substrate, smooth, with ostiole. Ostiole single, central, flattened, with a short neck, without paraphyses. Peridium 22–34 μm wide, multi-layered, composed of 4–6 rows of heavily pigmented, light brown to dark brown cells of textura angularis. Hamathecium comprising numerous 1.4–2.2 μm diameter, filamentous, unbranched, anastomosing, septate pseudoparaphyses. Asci 86–126 × 11–13 μm (average = 99 × 12 μm, n = 25), 8-spored, bitunicate, with fissitunicate dehiscence occurring rarely, elliptical, shortly pedicellate, apically rounded, with an ocular chamber, with a J-subapical ring. Ascospores 15–17 × 5–7 μm (average = 16.3 × 6.1 μm, n = 40), hyaline to yellow brown, 2–3-septate, deeply constricted at second septum, tapering to each end, the widest point at second cell from apex, smooth-walled, distinctly guttulate, without a sheath or appendages. Asexual morph: undetermined.

Figure 9. 

Nigrograna trachycarpus (GMB0499) A specimen B, C appearance of ascomata on substrate D, E longitudinal section of an ascoma F peridium G–I asci J pseudoparaphyses K J-ascus subapical ring in Melzer’s L–O ascospores. Scale bars: 0.5 mm (B–D); 10 μm (E–O).

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.

Specimen examined

China, Guizhou Province, Guiyang Huaxi National Urban Wetland Park (26°2'2.34"N, 106°34'16.22"E), 1130 m elev., on decaying culms of Trachycarpus sp., 12 October 2022, Y.P Wu and H.M Hu, 2022 HXGY11 (GMB0499, holotype, GMBC0499, ex-type; KUN-HKAS 12984, isotype).

Other examined material

China, Guizhou Province, Qiannan Prefecture, Sandu Shui Autonomous County, Yao Man Mountain National Forest Park (25°93′18.76"N, 107°95′15.66"E), 540 m elev., on decaying bamboo culms of Trachycarpus sp.; 28 September 2022; Y.P. Wu, 2022YRS50 (GMB050; GMBC0505, living culture).

Notes

In the phylogenetic analysis, Nigrograna trachycarpus and N. locuta-pollinis F. Liu & L. Cai formed a monophyletic branch within the Nigrograna genus, with a bootstrap support value of 31% (Fig. 3). However, this relationship remained consistent in repeated phylogenetic analyses. Sequences generated from the cultures of N. trachycarpus are similar to sharing an ITS similarity of 70.7% (with 57/488 gaps) and a tef1 similarity of 89.8% (with 0/481 gaps). Morphologically, N. trachycarpus can be distinguished by its larger ascospores, measuring 16.3 × 6.1 μm, in contrast to N. schinifolium’s ascospores, 11.6 × 3.3 μm. Morphologically, it is close to N. impatientis J.F. Zhang, J.K. Liu & Z.Y. Liu, but the latter typically has ascocarps in groups of 2–6 with ostiole necks penetrating the host surface together. Moreover, the N. trachycarpus a possesses longer asci (measuring 99 × 12 μm) and larger ascospores (measuring 16.3 × 6.1 μm) compared to N. impatientis (asci measuring 48 × 8, ascospores measuring 12 × 4.3 μm) (Zhang et al. 2020a).

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.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This research was supported by National Natural Science Foundation of China (31960005, 32000009 and 32170019); Science and Technology Department Foundation of Guizhou Province ([2018]2322); Qianhe Talents, Science and Technology Department of Guizhou Province ([2015]4029); Guizhou Provincial Education Department Scientific Research Project for Higher Education Institutions ([2022]064); National Natural Science Foundation of China Karst Centre Project U1812403-4-4.

Author contributions

Conceptualization, Jichuan Kang, Qirui Li, Xiangchun Shen; investigation, Hongmin Hu, Youpeng Wu, Qingde Long; morpho-logical examinations, molecular sequencing, and phylogenetic analyses, Xu Zhang, Sihan Long and Youpeng Wu; specimen identification, Hongmin Hu and Qirui Li; writing—original draft preparation, Hongmin Hu, Minghui He; writing—review and editing, Nalin N. Wijayawardene, Zebin Meng; supervision, Qirui Li. All authors have read and agreed to the published version of the manuscript.

Author ORCIDs

Hongmin Hu https://orcid.org/0000-0003-3894-3269

Sihan Long https://orcid.org/0000-0002-8346-3646

Nalin N. Wijayawardene https://orcid.org/0000-0003-0522-5498

Data availability

All of the data that support the findings of this study are available in the main text.

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