Skip to main content
NCBI home page
Biology logoLink to Biology
. 2022 Jun 9;11(6):889. doi: 10.3390/biology11060889

Taxonomic and Phylogenetic Insights into Novel Ascomycota from Forest Woody Litter

Dhanushka N Wanasinghe 1,2,*, Peter E Mortimer 1,2,*
Editor: Adrian Leuchtmann
PMCID: PMC9220210  PMID: 35741409

Abstract

Simple Summary

Studies suggest that fungi belonging to Ascomycota are sensitive to environmental changes which are disrupting ecosystems globally, with numerous extinction events, imbuing fungal diversity research with a sense of urgency. Thus, it is crucial we assess the diversity of Ascomycota across different habitats and substrates, and include programs to seek out novel taxa within Ascomycota. Our study provides some insights into the woody-based saprotrophic microfungi in Yunnan, China by introducing two novel Ascomycota species. Furthermore, these species were discovered by a 5-year-old boy, thus highlighting how young enthusiasts can be involved in field studies and make a significant impact.

Abstract

While surveying the mycobiomes of dead woody litter in Yunnan Province, China, numerous isolates with affinity to Pleosporales (Dothideomycetes, Ascomycota) were recovered. The present work characterizes two species associated with dead woody twigs found in terrestrial habitats in the Kunming area of Yunnan. The novel taxa were recognized based on a polyphasic approach, including morphological examination and multiple gene phylogenetic analyses (non-translated loci and protein-coding regions). Neokalmusia jonahhulmei sp. nov. is introduced in Didymosphaeriaceae (Pleosporales) as a woody-based saprobic ascomycete that possesses multiloculate ascostromata immersed under a black clypeus-like structure, and three-septate, brown, fusiform, guttulate ascospores. Thyridaria jonahhulmei (Thyridariaceae) is introduced with teleomorphic and anamorphic (coelomycetous) characteristics. The teleomorph has the following characteristics: globose to subglobose ascomata with an ostiolum, a pruinose layer of yellow to reddish- or orange-brown material appearing around the top of the ostiolar necks, and brown, ellipsoid to fusoid, two-to-three-septate, euseptate, rough-walled ascospores; the anamorph features pycnidial conidiomata, phialidic, ampulliform to doliiform, conidiogenous cells, and brown, guttulate, ellipsoidal, aseptate conidia.

Keywords: anamorph, Greater Mekong Subregion, Pleosporales, teleomorph, Yunnan

1. Introduction

Forest ecosystems produce a large quantity of litter in various forms, such as leaves, branches, twigs, inflorescence, and other debris. Plant litter maintains pathways involved in nutrient cycling that are crucial to forest productivity [1,2,3,4,5,6]. A great proportion of total forest carbon (C) is contained in these woody debris, providing habitats for thousands of organisms, including fungi. Fungi play a critical role in the decomposition of woody litter as they are capable of actively decomposing lignin and other recalcitrant components found in these materials. However, comprehensive studies of the fungal taxonomic systematics of woody litter are scarce.

Most of the described woody-based fungal species lack biological and ecological information and reliable taxonomic status. Researchers have paid more attention to economic species, such as those producing edible mushrooms, and less attention to the narrowly distributed microfungal species responsible for the decomposition and recycling of woody material. However, in recent years, there have been numerous studies into woody-based microfungal occurrences in Yunnan Province, leading to reports of higher microfungal diversity, especially in Dothideomycetes [7,8,9,10,11,12,13,14,15,16]. At the Centre for Mountain Futures (Kunming Institute of Botany), researchers are investigating microfungal diversity across several substrates in southwest China, including leaf and woody litter, in order to clarify their taxonomy using morphology in conjunction with multigene phylogeny. Accordingly, we have isolated ascomycetes (Pleosporales) from woody litter, collected in a Pinus yunnanensis forest in Yunnan Province (Kunming), China (Figure 1). This study assesses the systematic categorization of two taxonomic novelties in Neokalmusia and Thyridaria, assesses morphological characteristics on natural substrates and in cultures, and conducts phylogenetic analyses.

Figure 1.

Figure 1

Open in a new tab

Habitat. (a–d) Mixed forest (predominantly Fagaceae and Pinus spp., with Pinus yunnanensis being the most abundant tree species); (e) collector.

2. Materials and Methods

2.1. Isolates and Specimens

During fieldwork in the Kunming region, Yunnan, China, characteristic black ascomata/conidiomata on dead woody twigs were collected during the dry season. The local environment features mixed forests (predominantly Fagaceae and Pinus spp., with Pinus yunnanensis being the most abundant tree species) (Figure 1) and a humid temperate climate at elevation 2080 masl. Specimens were stored in Ziploc plastic bags and taken to the laboratory. Isolations were made from single ascospores, according to the methods of Wanasinghe et al. [11]. Voucher specimens were preserved in the herbarium of the Kunming Institute of Botany (KUN-HKAS), and the living cultures were placed in the Culture Collection of Kunming Institute of Botany (KUMCC), Kunming, China. Nomenclatural novelties were deposited in MycoBank as outlined in http://www.MycoBank.org (accessed on 17 March 2022).

2.2. Morphological Observations

Examination of external structures of the fungal specimens were made using a Motic SMZ 168 Series stereo-microscope (Motic Asia, Kowloon, Hong Kong). Micro-morphological characters were examined and evaluated following the protocols provided by Wanasinghe et al. [10]. Macroscopic images of colonies were documented using an iPhone XS Max (Apple Inc., Cupertino, CA, USA) with daylight. Photoplates and images were processed using Adobe Photoshop CS6 (Adobe Systems, San Jose, CA, USA).

2.3. DNA Extraction, PCR Amplifications and Sequencing

The genomic DNA of each isolate was extracted from scraped mycelia following Wanasinghe et al. [10] using the Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux, Shanghai, China). Primers for PCR amplification used were ITS (internal transcribed spacers) = ITS5/ITS4 [17], LSU (partial 28S large subunit rDNA) = LR0R/LR5 [18,19], SSU (partial 18S small subunit rDNA) = NS1/NS4 [17], tef1 (translation elongation factor 1-α) = EF1-983F/EF1-2218R [20,21], and rpb2 (RNA polymerase II second largest subunit) = fRPB2-5f/fRPB2-7cR [22]. The PCR protocols were programmed as described in The PCR protocols of SSU, LSU, ITS, and tef1 and were programmed as described in Wanasinghe et al. [11]. The PCR amplification condition of rpb2 was set as denaturation at 98 °C for 2 min, followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 52 °C for 10 s, and extension at 72 °C for 20 s, with a final extension step at 72 °C for 5 min. DNA sequencing were performed at a private company for sequencing (BGI, Ltd., Shenzhen, China).

2.4. Molecular Phylogenetic Analyses

2.4.1. Sequencing and Sequence Alignment

Sequences generated from SSU, LSU, ITS, tef1, and rpb2 were first used for BLASTn analyses. Based on BLAST similarities and relevant publications [23,24,25], closely related sequences were downloaded from GenBank (Table 1 and Table 2). Loci were aligned using MAFFT v. 7 [26,27]) under default conditions. Final improvements were made when necessary using BioEdit v.7.0.5.2 software [28].

Table 1.

Taxa used in the phylogenetic analyses of Didymosphaeriaceae and their corresponding GenBank numbers of partial ITS, LSU, SSU, tef1, and rpb2 sequences. Isolates/sequences in bold were isolated/sequenced in the present study.

Species Strain GenBank Accession Numbers
ITS LSU SSU tef1 rpb2
Alloconiothyrium camelliae NTUCC 17-032-1 MT112294 MT071221 MT071270 MT232967 -
Alloconiothyrium camelliae NTUCC 17-032-2 MT112295 MT071222 MT071271 MT232965 -
Austropleospora keteleeriae MFLUCC 18-1551 NR_163349 MK347910 NG_070075 MK360045 MK434909
Austropleospora ochracea KUMCC 20-0020 MT799859 MT808321 MT799860 MT872714 -
Austropleospora osteospermi MFLUCC 17-2429 MK347757 MK347863 MK347974 MK360044 MK434884
Bambusistroma didymosporum MFLU 15-0057 KP761733 KP761737 KP761730 KP761727 KP761720
Bambusistroma didymosporum MFLU 15-0058 KP761734 KP761738 KP761731 KP761728 KP761721
Bimuria novae-zelandiae CBS 107.79 MH861181 AY016338 AY016356 DQ471087 DQ470917
Bimuria omanensis SQUCC 15280 NR_173301 - NG_071257 MT279046 -
Chromolaenicola lampangensis MFLUCC 17-1462 MN325016 MN325010 MN325004 MN335649 MN335654
Chromolaenicola nanensis MFLUCC 17-1477 MN325014 MN325008 MN325002 MN335647 MN335653
Chromolaenicola thailandensis MFLUCC 17-1475 MN325019 MN325013 MN325007 MN335652 MN335656
Cylindroaseptospora leucaenae MFLUCC 17-2424 NR_163333 MK347856 NG_066310 MK360047 -
Cylindroaseptospora siamensis MFLUCC 17-2527 NR_163337 MK347866 NG_066311 MK360048 -
Deniquelata barringtoniae MFLUCC 16-0271 MH275059 - MH260291 MH412766 MH412753
Deniquelata hypolithi CBS 146988 MZ064429 - NG_076735 MZ078250 MZ078201
Deniquelata vittalii NFCCI4249 MF406218 MF622059 MF182395 MF182398 MF168942
Didymocrea sadasivanii CBS 438.65 MH858658 DQ384066 DQ384103 - -
Didymosphaeria rubi ulmifolii CBS 100299 MH862698 AY642523 JX496124 - -
Didymosphaeria rubi ulmifolii MFLUCC 140023 KJ436586 NG_063557 KJ436586 - -
Kalmusia ebuli CBS 123120 KF796674 JN851818 JN644073 - -
Kalmusia erioi MFLU 18-0832 MN473058 MN473046 MN473052 MN481599 -
Kalmusia italica MFLUCC 14 0560 KP325440 KP325442 KP325441 - -
Kalmusia variisporum CBS 121517 NR_145165 - JX496143 - -
Kalmusibambusa triseptata MFLUCC 13-0232 KY682697 KY682696 KY682695 - -
Karstenula rhodostoma CBS 690.94 - GU296154 GU301821 GU349067 GU371788
Karstenula rhodostoma CBS 691.94 LC014559 AB797241 AB807531 AB808506 -
Laburnicola hawksworthii MFLUCC 13 0602 KU743194 KU743196 KU743195 - -
Laburnicola muriformis MFLUCC 16 0290 KU743197 KU743199 KU743198 KU743213 -
Laburnicola muriformis MFLUCC 14 0921 KU743200 KU743202 KU743201 - -
Laburnicola rhizohalophila CGMCC 8756 KJ125522 - KJ125523 KJ125525 KJ125524
Letendraea cordylinicola MFLUCC 11 0150 KM213996 KM214002 KM213999 - -
Letendraea cordylinicola MFLUCC 11 0148 NR_154118 KM214001 NG_059530 - -
Letendraea helminthicola CBS 884.85 MK404145 AY016345 AY016362 MK404174 MK404164
Letendraea padouk CBS 485.70 - GU296162 AY849951 - -
Montagnula bellevaliae MFLUCC 14 0924 NR_155377 KT443904 KT443902 KX949743 -
Montagnula chromolaenicola MFLUCC 17-1469 NR_168866 NG_070157 NG_070948 MT235773 MT235809
Montagnula cirsii MFLUCC 13 0680 KX274242 KX274255 KX274249 KX284707 -
Montagnula krabiensis MFLUCC 16-0250 MH275070 MH260343 MH260303 MH412776 -
Montagnula thailandica MFLUCC 17-1508 MT214352 NG_070158 NG_070949 MT235774 MT235810
Neokalmusia arundinis MFLU 17-0754 MT649882 MT649880 MT649878 MT663766 -
Neokalmusia arundinis MFLUCC 15-0463 NR_165852 NG_068372 NG_068237 KY244024 -
Neokalmusia arundinis MFLUCC 14-0222 KX965731 KX986344 KX954400 KY271091 -
Neokalmusia brevispora KT 2313 LC014574 AB524460 AB524601 AB539113 -
Neokalmusia brevispora KT 1466 LC014573 AB524459 AB524600 AB539112 -
Neokalmusia didymospora MFLUCC 11-0613 - KP091435 KP091434 - -
Neokalmusia jonahhulmei KUMCC 21-0818 ON007043 ON007039 ON007048 ON009133 ON009137
Neokalmusia jonahhulmei KUMCC 21-0819 ON007044 ON007040 ON007049 ON009134 ON009138
Neokalmusia kunmingensis KUMCC 18-0120 MK079886 MK079887 MK079889 MK070172 -
Neokalmusia scabrispora KT 1023 LC014575 AB524452 AB524593 AB539106 -
Neokalmusia scabrispora KT 2202 LC014576 AB524453 AB524594 AB539107 -
Neokalmusia thailandica MFLUCC 16-0405 NR_154255 KY706137 NG_059792 KY706145 KY706148
Neokalmusia thailandica MFLUCC 16-0399 KY706141 KY706136 KY706131 - -
Neptunomyces aureus CMG12 MK912121 - - MK948000 -
Neptunomyces aureus CMG13 MK912122 - - MK948001 -
Neptunomyces aureus CMG14 MK912123 - - MK948002 -
Paracamarosporium fagi CPC 24890 NR_154318 - NG_070630 - -
Paracamarosporium fagi CPC 24892 KR611887 - KR611905 - -
Paracamarosporium hawaiiense CBS 120025 JX496027 EU295655 JX496140 - -
Paraconiothyrium cyclothyrioides CBS 972.95 JX496119 AY642524 JX496232 - -
Paraconiothyrium estuarinum CBS 109850 JX496016 AY642522 JX496129 - -
Paramassariosphaeria anthostomoides CBS 615.86 MH862005 GU205246 GU205223 - -
Paramassariosphaeria clematidicola MFLU 16-0172 KU743206 KU743208 KU743207 - -
Paraphaeosphaeria rosae MFLUCC 17-2547 MG828935 MG829150 MG829044 MG829222 -
Paraphaeosphaeria rosae MFLUCC 17-2549 MG828937 MG829152 MG829046 MG829223 -
Paraphaeosphaeria rosicola MFLUCC 15-0042 NR_157528 MG829153 MG829047 - -
Phaeodothis winteri CBS 182.58 - GU296183 GU301857 - -
Pseudocamarosporium propinquum MFLUCC 13 0544 KJ747049 KJ819949 KJ813280 - -
Pseudocamarosporium pteleae MFLUCC 17-0724 NR_157536 MG829166 MG829061 MG829233 -
Pseudocamarosporium ulmi-minoris MFLUCC 17-0671 NR_157537 MG829167 MG829062 - -
Pseudodidymocyrtis lobariellae KRAM Flakus 25130 NR_169714 NG_070349 NG_068933 - -
Pseudopithomyces entadae MFLUCC 17-0917 MK347835 NG_066305 MK360083 MK434899
Pseudopithomyces kunmingnensis MFLUCC 17-0314 MF173607 MF173606 MF173605 - -
Pseudopithomyces rosae MFLUCC 15-0035 MG828953 MG829168 MG829064 - -
Spegazzinia deightonii yone 212 AB797292 AB807582 AB808558 -
Spegazzinia radermacherae MFLUCC 17-2285 MK347740 MK347848 MK347957 MK360088 MK434893
Spegazzinia tessarthra SH 287 JQ673429 AB797294 AB807584 AB808560 -
Tremateia arundicola MFLU 16 1275 KX274241 KX274254 KX274248 KX284706 -
Tremateia chromolaenae MFLUCC 17-1425 NR_168868 NG_070160 NG_068710 MT235778 MT235816
Tremateia guiyangensis GZAAS01 KX274240 KX274253 KX274247 KX284705 -
Tremateia murispora GZCC 18-2787 NR_165916 MK972750 MK972751 MK986482 -
Tremateia thailandensis MFLUCC 17-1430 NR_168869 NG_070161 NG_068711 MT235781 MT235819
Verrucoconiothyrium nitidae CBS 119209 EU552112 - EU552112 - -
Vicosamyces venturisporus CDA1494 MF802825 - MF802828 - -
Vicosamyces venturisporus CDA1495 MF802826 - MF802829 - -
Vicosamyces venturisporus CDA495 MF802827 - MF802830 - -
Xenocamarosporium acaciae CPC 24755 NR_137982 - NG_058163 - -
Xenocamarosporium acaciae MFLUCC 17-2432 MK347766 MK347873 MK347983 MK360093 -
Open in a new tab
Table 2.

Taxa used in the phylogenetic analyses of Thyridariaceae and their corresponding GenBank numbers of partial ITS, LSU, SSU, tef1, and rpb2 sequences. Isolates/sequences in bold were isolated/sequenced in the present study.

Species Strain GenBank Accession Numbers
ITS LSU SSU tef1 rpb2
Arthopyrenia salicis CBS 368.94 KF443410 AY538339 AY538333 KF443404 KF443397
Chromolaenomyces appendiculatus MFLUCC 17-1455 NR_168862 NG_068705 MT214394 MT235770 MT235806
Cycasicola goaensis MFLUCC 17-0754 MG828885 MG829001 MG829112 MG829198 -
Cycasicola leucaenae MFLUCC17-0914 NR_163322 MK347942 MK347833 MK360046 MK434900
Liua muriformis KUMCC 18-0177 MK433599 MK433598 MK433595 MK426798 MK426799
Neoroussoella alishanense AKW 11 FU31018 MK503818 MK503824 MK503830 MK336182 MN037757
Neoroussoella alishanense AKW 03 FU31016 MK503816 MK503822 MK503828 MK336181 MN037756
Neoroussoella entadae MFLUCC 18-0243 MK347786 MK348004 MK347893 MK360065 MK434866
Neoroussoella leucaenae MFLUCC 18-1544 MK347767 MK347984 MK347874 MK360067 MK434876
Occultibambusa bambusae MFLUCC 11-0394 KU940124 KU863113 - KU940194 KU940171
Occultibambusa bambusae MFLUCC 13-0855 KU940123 KU863112 KU872116 KU940193 KU940170
Ohleria modesta MGC KX650562 KX650562 - KX650533 KX650582
Ohleria modesta OM KX650563 KX650563 KX650513 KX650534 KX650583
Parathyridaria clematidis MFLUCC 17-2154 MT310645 MT214601 MT226712 MT394657 MT394712
Parathyridaria clematidis MFLUCC 17-2157 MT310644 MT214600 MT226711 MT394656 MT394711
Parathyridaria clematidis MFLUCC 17-2160 MT310643 MT214599 MT226710 MT394655 MT394710
Parathyridaria clematidis MFLUCC 17-2185 MT310642 MT214598 NG_070668 MT394654 MT394709
Parathyridaria ellipsoidea KNU-JJ-1829 LC552950 LC552952 - - -
Parathyridaria flabelliae MUT 4886 KR014358 KP671720 KT587317 MN605910 MN605930
Parathyridaria flabelliae MUT 4859 KR014355 KP671716 KT587315 MN605909 MN605929
Parathyridaria percutanea CBS 128203 KF322117 KF366448 KF366450 KF407988 KF366453
Parathyridaria percutanea CBS 868.95 KF322118 KF366449 KF366451 KF407987 KF366452
Parathyridaria philadelphi CBS 143432 MH107905 NG_063958 - MH108023 -
Parathyridaria ramulicola MUT 4397 KC339235 KF636775 MN556311 MN605913 MN605933
Parathyridaria ramulicola CBS 141479 NR_147657 KX650565 KX650514 KX650536 KX650584
Parathyridaria robiniae MUT 2452 MG813183 MG816491 MN556312 MN605903 MN605923
Parathyridaria robiniae MUT 4893 KM355998 MN556328 KM355993 MN605904 MN605924
Parathyridaria robiniae MFLUCC 14-1119 KY511142 KY511141 - KY549682 -
Parathyridaria rosae MFLU 17-0623 NR_157530 NG_059873 - - -
Parathyridaria serratifoliae MFLUCC 17-2210 MT310646 MT214602 NG_070669 MT394658 MT394713
Parathyridaria tyrrhenica MUT 4966 KR014366 KP671740 KT587309 MN605911 MN605931
Parathyridaria tyrrhenica MUT 5371 KU314951 MN556329 KU314952 MN605912 MN605932
Parathyridaria virginianae MFLUCC 17-2163 MT310647 NG_073853 NG_070670 MT394659 MT394714
Parathyridariella dematiacea MUT 4419 KC339245 KF636786 MN556313 MN605905 MN605925
Parathyridariella dematiacea MUT 5310 KU255057 MN556330 MN556314 MN605907 MN605927
Parathyridariella dematiacea MUT 5381 KU314959 MN556331 KU314960 MN605908 MN605928
Parathyridariella dematiacea MUT 4884 MN556317 KP671726 KT587329 MN605906 MN605926
Pseudothyridariella chromolaenae MFLUCC 17-1472 NR_168863 NG_068706 MT214395 MT235771 MT235807
Pseudothyridariella mahakoshae NFCCI 4215 MG020435 MG020438 MG020441 MG023140 MG020446
Roussoella hysterioides CBS 546.94 KF443405 KF443381 AY642528 KF443399 KF443392
Roussoella intermedia CBS 170.96 KF443407 KF443382 KF443390 KF443398 KF443394
Roussoella japanensis MAFF 239636 KJ474829 AB524621 AB524480 AB539114 AB539101
Roussoella margidorensis MUT 5329 KU314944 MN556322 MN556309 MN605897 MN605917
Roussoella mediterranea MUT 5306 KU255054 MN556323 MN556310 MN605898 MN605918
Roussoella padinae MUT 5341 KU158153 MN556325 KU158176 MN605900 MN605920
Roussoella pustulans KT 1709 KJ474830 AB524623 AB524482 AB539116 AB539103
Roussoella siamensis MFLUCC 11-0149 KJ474837 KJ474845 KU872125 KJ474854 KJ474861
Thyridaria acaciae CBS 138873 KP004469 KP004497 - - -
Thyridaria aureobrunnea MFLUCC 21-0090 MZ538528 MZ538562 - - -
Thyridaria broussonetiae TB KX650567 KX650567 - KX650538 KX650585
Thyridaria broussonetiae TB1a KX650569 KX650569 - - -
Thyridaria broussonetiae TB2 KX650570 KX650570 - KX650540 KX650587
Thyridaria broussonetiae TB1 KX650568 KX650568 KX650515 KX650539 KX650586
Thyridaria jonahhulmei KUMCC 21-0816 ON007041 ON007037 ON007046 ON009131 ON009135
Thyridaria jonahhulmei KUMCC 21-0817 ON007042 ON007038 ON007047 ON009132 ON009136
Thyridariella mangrovei NFCCI 4214 MG020436 MG020439 MG020442 MG020444 MG020447
Thyridariella mangrovei NFCCI 4213 MG020434 MG020437 MG020440 MG020443 MG020445
Torula herbarum CBS 111855 KF443409 KF443386 KF443391 KF443403 KF443396
Torula herbarum CBS 595.96 KF443408 KF443385 KF443387 KF443402 KF443395
Open in a new tab

2.4.2. Phylogenetic Analyses

The single-gene data sets were examined for topological incongruence among loci for members of the analyses. The conflict-free single alignments for each locus were combined into a multi-locus dataset. Concatenated alignment was used to construct maximum-likelihood (ML) and Bayesian (BI) phylogenetic analyses. MrModeltest v. 2.3 [29] was used under the Akaike Information Criterion (AIC) implemented in PAUP v. 4.0b10 to determine the evolutionary models for Bayesian and maximum-likelihood analyses.

The CIPRES webportal [30] was used to execute RAxML [31] and Bayesian analyses [32]. RAxML-HPC2 on XSEDE v. 8.2.10 [30] was used with default parameters and 1000 bootstrap repetitions to construct the ML analysis.

MrBayes analyses were performed setting GTR+I+G as the evolutionary model, with 2 M generations, sampling every 1000 generations, ending the run automatically when standard deviations of split frequencies dropped below 0.01, and with a burnin fraction of 0.25. ML bootstrap values equal or greater than 70% and BYPP greater than 0.95 are given above each node of every tree.

FigTree v1.4.0 program [33] was used to visualize the phylogenetic trees and reorganized in Microsoft Power Point (2007). The finalized datasets and trees were submitted to TreeBASE, submission ID: 29569 (http://purl.org/phylo/treebase/phylows/study/TB2:S29569 (accessed on 17 March 2022)).

3. Results

3.1. Phylogenetic Analyses

Two analyses were performed in this study. The first is a phylogenetic overview of the genera treated in Didymosphaeriaceae (Figure 2), while the remaining alignment represents the genera in Thyridariaceae (Figure 3). Other details related to both ML and BI analyses from Didymosphaeriaceae and Thyridariaceae datasets are presented in Table 3.

Figure 2.

Figure 2

Open in a new tab

RAxML phylogram generated from combined dataset of partial SSU, LSU, ITS, tef1, and rpb2 DNA sequence analyses for Didymosphaeriaceae. The tree is rooted to Bambusistroma didymosporum (MFLU 15-0057, MFLU 15-0058). Bootstrap supports ML (MLB) ≥ 70% and Bayesian posterior probabilities (BYPP) ≥ 0.95 are given as MLB/BYPP above the branches. Branches with an asterisk (*) indicate MLB = 100% and BYPP = 1.00. The newly generated isolates are in blue.

Figure 3.

Figure 3

Open in a new tab

Phylogram generated from RAxML based on a concatenated sequence dataset of partial SSU, LSU, ITS, tef1, and rpb2 DNA for Thyridariaceae. Bootstrap values equal to or greater than 70% for ML (MLB) and Bayesian posterior probabilities (BYPP) ≥ 0.95 are shown at each node (as MLB/BYPP). An asterisk (*) represents branches with MLB = 100% and BYPP = 1.00. The new isolates are show in in blue.

Table 3.

Maximum-likelihood (ML) and Bayesian (BI) analyses results for each sequenced dataset.

Analyses Didymosphaeriaceae Thyridariaceae
Number of taxa 88 59
Gene regions SSU, LSU, ITS, tef1, and rpb2 SSU, LSU, ITS, tef1, and rpb2
Number of character positions (including gaps) 5016 4529
ML optimization likelihood value −35,672.743881 −30,606.10565
Distinct alignment patterns in the matrix 2249 1796
Number of undetermined characters or gaps (%) 41.88% 19.56%
Estimated base frequencies A 0.240418 0.249274
C 0.253351 0.25578
G 0.270784 0.267476
T 0.235446 0.227469
Substitution rates AC 1.561664 1.486771
AG 3.248718 3.744601
AT 1.433496 1.706836
CG 1.323566 1.014483
CT 7.428045 7.933665
GT 1.0 1.0
Proportion of invariable sites (I) 0.396829 0.505108
Gamma distribution shape parameter (α) 0.454368 0.442817
Number of generated trees in BI 11,301 2501
Number of trees sampled in BI after 25% were discarded as burn-in 8476 1876
Final split frequency 0.009959 0.009966
The total of unique site patterns 2252 1798
Open in a new tab

3.2. Taxonomy

Pleosporales Luttr. ex M.E. Barr, Prodromus to class Loculoascomycetes: 67 (1987)

Didymosphaeriaceae Munk, Dansk botanisk Arkiv 15 (2): 128 (1953)

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

Neokalmusia jonahhulmei Wanas. & Mortimer sp. nov. (Figure 4)

Figure 4.

Figure 4

Open in a new tab

Neokalmusia jonahhulmei (HKAS122910, holotype). (a,b) Ascostromata on the bamboo culms; (c) horizontal section of ascostromata; (d,e) vertical sections of ascomata; (f) vertical section through two locules; (g) close-up of ostiole; (h) peridium; (i) pseudoparaphyses; (j–l) asci; (m–r) ascospores (r germinated ascospore); (s,t) colonies on PDA after 21 days. Scale bars, (d–f) 100 µm; (g) 50 µm; (h,m–r) 10 µm; (i) 5 µm; (j–l) 20 µm.

MycoBank: MB843400

Etymology: The epithet is derived from Jonah Hulme Mortimer, who is the collector of this fungus.

Holotype: HKAS122910

Saprobic on dead bamboo culms. Teleomorph: Ascomata 200–300 μm high × 1200–1500 µm diam. (M = 180 × 1400 µm, n = 5), scattered or in groups, immersed under a black clypeus-like structure, composed of host epidermis and fungal mycelium, hemispherical, dark brown to black, multi-loculate. Locules 100–170 μm high, 150–280 μm diam. (M = 137.6 × 217.8 µm, n = 5), immersed within ascostromata, dark brown to black, subglobose to ampulliform, ostiolate. Peridium 10–12 μm wide at the base, 15–30 μm wide at the sides, comprising several layers; outer layers dark brown to brown, with compressed cells of textura angularis; inner layers hyaline, with compressed pseudoparenchymatous cells, arranged in textura angularis. Hamathecium contains 2–3 μm wide, branched, septate, cellular pseudoparaphyses. Asci 65–85 × 10–15 μm (M = 72.8 × 11.8 μm, n = 15), eight-spored, bitunicate, cylindrical to clavate, curved, short pedicel with slightly furcate ends, apically rounded. Ascospores 15–17 × 6–7 μm (M = 15.5 × 6.4 µm, n = 30), bi-seriate, overlapping and are initially hyaline, turning brown at maturity, fusiform, three-transversely septate, slightly curved, constricted at the septa, conically rounded at the ends, and smooth-walled, guttulated, without a distinct mucilaginous sheath. Anamorph: undetermined.

Culture characteristics: colonies reaching 4 cm diameter on PDA after 2 weeks at 20 °C. Mycelium dense, circular, slightly raised, smooth on surface and undulated floccose edge. Colony grey at the centre and coffee brown near margin from the top and dark brown at the bottom. Hyphae septate, branched, hyaline, thin, and smooth-walled.

Known distribution: Yunnan, China, on dead woody litter.

Material examined: China, Yunnan, Kunming, Wuhua, 25.131198 N, 102.590770 E, 2080 m, on dead bamboo culms (Phyllostachys sp.), 20 March 2021, Jonah Hulme Mortimer, PEM03-6-2-1 (HKAS122910, holotype), ex-holotype culture, KUMCC 21-0818. ibid. 25.131178 N, 102.590749 E, PEM03-6-2-4 (HKAS122911), living culture, KUMCC 21-0819.

Notes: The new fungus was collected from dead bamboo culms in Kunming. It is characterized as a typical Neokalmusia taxon based on its immersed, hemispherical multi-loculate ascomata under a black clypeus-like structure, bitunicate, clavate, apically rounded asci and bi-seriate, fusiform, brown ascospores with transverse septa [34]. It has a close phylogenetic affinity to Neokalmusia brevispora (KT 2313, KT 1466) and N. kunmingensis (KUMCC 18-0120), with 100 MLB and 1.00 BYPP support values (Figure 2). Neokalmusia brevispora and N. kunmingensis are reported as saprobes from Japan on the Sasa sp. and on dead bamboo culms in China, respectively [34,35,36]. These three species are morphologically similar with few dimensional differences in asci and ascospores. Neokalmusia brevispora has comparatively larger asci and ascospores (80–118 × 10.5–15 μm; 18–26.5 × 4–7 μm [34]) than N. kunmingensis (63–77 × 9.6–11.4 μm; 13–15 × 4–5 μm [36]) and our new collection (65–85 × 10–15 μm; 15–17 × 6–7 μm, this study). Further comparison of the ITS regions reveals Neokalmusia brevispora and N. kunmingensis feature nucleotide differences from N. jonahhulmei of 66/695 (9.5%) and 55/560 (9.8%), respectively. Comparison of the tef1 nucleotides of Neokalmusia brevispora and N. kunmingensis with our new strains revealed nucleotide differences of 31/917 (3.4%) and 26/904 (2.9%), respectively. The rpb2 region is not available for Neokalmusia brevispora and N. kunmingensis for comparison with our new species. This is the first time rpb2 sequence data for a Neokalmusia species have been provided (Table 1).

Ariyawansa et al. [34] established Neokalmusia to accommodate N. brevispora and N. scabrispora, which are characterized by immersed and subglobose to oblong ascomata with multiple perithecia, thin-walled clypeus-like structure, and verrucose ascospores. These two were found on dead culms of Sasa and Phyllostachys species in Japan, respectively [34,35]. Subsequent studies added four other species viz. N. arundinis (Italy) [37], N. didymospora (Thailand) [38], N kunmingensis (China) [36], and N. thailandica (Thailand) [37]. In this study, we introduce the seventh species in the genus from Phyllostachys species in China. All specimens of these species were obtained from dead culms of Bambusoideae (Poaceae) in China, Italy, Japan, and Thailand; therefore, Neokalmusia appear to be saprobic on bambusicolous hosts in temperate to tropical environments.

Thyridariaceae Q. Tian & K.D. Hyde, Fungal Diversity 63 (1): 254 (2013)

Thyridaria Sacc., Grevillea 4 (29): 21 (1875)

Thyridaria jonahhulmei Wanas. & Mortimer sp. nov. (Figure 5 and Figure 6)

Figure 5.

Figure 5

Open in a new tab

Sexual morph of Thyridaria jonahhulmei (HKAS122912, holotype). (a,b) Ascomata on the dead woody twigs; (c,d) cross section of ascomata; (e) close-up of ostiole; (f) peridium; (g) pseudoparaphyses; (h–j) asci; (k–p) ascospores. Scale bars, (d) 200 µm; (e) 50 µm; (f,h–j) 20 µm; (f,k–p) 10 µm.

Figure 6.

Figure 6

Open in a new tab

Asexual morph of Thyridaria jonahhulmei (KUMCC 21-081, ex-type culture). (a) Germinated ascospore (b–d) colony on PDA (c from the bottom); (e–g) immersed pycnidia in PDA; (h) squashed conidiomata; (i,j) conidiogenous cells; (k) conidia. Scale bars, (h) 100 µm; (i,j) 5 µm; (k) 10 µm.

MycoBank: MB843401

Etymology: The epithet is derived from Jonah Hulme Mortimer, who is the collector of this fungus.

Holotype: HKAS122912

Saprobic on dead twigs of Fagaceae sp. Teleomorph: Ascomata 550–750 μm high, 200–300 μm diam. (M = 679.3 × 256.4 µm, n = 5), solitary or gregarious, immersed, coriaceous, heavily pigmented, globose to subglobose, ostiolate, yellow to reddish- or orange-brown pruinose layer forming around the apices of the ostiolar necks. Ostiole 100–150 μm long, 40–70 μm diam. (M = 121.1 × 57.8 µm, n = 5), central papillate, comprising hyaline periphyses. Peridium 15–25 μm wide, wider at the apex (60–70 μm), composed with two layers, with outer stratum comprising pale brown to brown, compressed, thick-walled cells of textura angularis, fused with the host tissues, and inner stratum multi-layered and composed with lightly pigmented to hyaline cells of textura angularis. Hamathecium comprised of 2–2.5 μm wide, branched, septate, cellular pseudoparaphyses, situated between and above the asci, embedded in a gelatinous matrix. Asci 150–200 × 18–22 μm (M = 170.2 × 20.1 μm, n = 25), eight-spored, bitunicate, fissitunicate, cylindrical to cylindric-clavate, long pedicellate (30–60 μm), and apex rounded with an ocular chamber. Ascospores 25–35 × 8–12.5 μm (M = 29.3 × 9.7 µm, n = 30), one-to-two-seriate, overlapping, and pale or yellowish brown when young, turning yellowish brown to brown at maturity, narrowly ellipsoid to fusoid, ends narrowly rounded (sometimes pointed), straight or curved, two-to-three-transversely septate, with median euseptum, slightly constricted at the septa, containing several guttules, with surface finely punctate to verruculose. Anamorph: Coelomycetous. Conidiomata (1–1.5 mm diam.), pycnidial, scattered or grouped, immersed and heavily pigmented. Pycnidial wall comprised with several strata, with brown-walled pseudoparenchymatous cells at outer margin becoming hyaline and thin-walled towards the inner conidiogenous cell-layer. Conidiogenous cells phoma-like, phialidic, ampulliform to doliiform, hyaline, flexuous, and smooth, with a short collarette. Conidia 5.5–8 × 2.5–3.5 μm (M = 6.9 × 3.1 μm, n = 50), first hyaline, turning pale brown, one-celled, straight or curved, ellipsoidal, rounded at both ends, thin and smooth-walled, comprising numerous guttules.

Culture characteristics: Colonies spreading on PDA up-to 4 cm diameter after 2 weeks at 20 °C, circular, whitish at the beginning, and becoming slightly raised and greenish-grey after 4 weeks, reverse dark brown. Hyphae septate, branched, hyaline, thin, smooth-walled, producing conidia after six weeks.

Known distribution: Yunnan, China, on dead woody litter.

Material examined: Material examined: China, Yunnan, Kunming, Wuhua, 25.131178 N, 102.590726 E, 2080 m, on dead twigs of woody litter of Fagaceae sp., 30 January 2021, Jonah Hulme Mortimer, PM03-2-1 (HKAS122912, holotype), ex-holotype culture, KUMCC 21-0816. ibid. 25.131157 N, 102.590708 E, 20 March 2021, PM03-2-3 (HKAS122913), living culture, KUMCC 21-0817.

Notes: Based on multi-gene sequence analyses (SSU, LSU, ITS, tef1 and rpb2), isolates KUMCC 21-0816 and KUMCC 21-0817 cluster with Thyridaria acaciae (CBS 138873), T. aureobrunnea (MFLUCC 21-0090) and T. broussonetiae (TB, TB1a, TB1, TB2) with 100 MLB and 1.00 BYPP bootstrap support values (Figure 3). Thyridaria acaciae is reported from Tanzania (on Acacia tortilis) [39], T. aureobrunnea from decaying wood in Thailand [40], and T. broussonetiae from Croatia (Hippocrepis emerus), Hungary (Amorpha fruticosa), and Italy (Broussonetia papyrifera) [41], whereas our new isolate is from dead woody twigs in Yunnan, China. Morphologically Thyridaria jonahhulmei has a close affinity to T. aureobrunnea and T. broussonetiae in its ascomata, asci, and ascospore characteristics. The asci dimensions of Thyridaria jonahhulmei (150–200 × 18–22 μm) are comparatively larger than T. aureobrunnea (45–61.5 × 7–8.5 µm) and are not significantly different from T. broussonetiae (109–183 × 12–19 µm). Comparison of the 546 ITS (+5.8S) nucleotides reveals 43 (7.9%) nucleotide differences between T. aureobrunnea and T. jonahhulmei. Thyridaria aureobrunnea lacks tef1 and rpb2 gene regions for comparison. Comparison of the 514 ITS (+5.8S) nucleotides of Thyridaria broussonetiae and T. jonahhulmei reveals 25 (4.86%) nucleotide differences, and in tef1 and rpb2 there are 8/712 (1.12%) and 18/1025 (1.75%) nucleotide differences, respectively. Thyridaria acaciae is known only from its asexual morph, and therefore it is not possible to compare their sexual morphologies. However, the asexual morph of Thyridaria jonahhulmei is similar to Thyridaria acacia. They both have ampulliform to doliiform conidiogenous cells and pale brown, aseptate conidia. Thyridaria acacia has subcylindrical conidia that lack guttules, whereas Thyridaria jonahhulmei has ellipsoidal conidia with large guttules. Comparison of the 469 ITS (+5.8S) nucleotides of these two strains reveals 23 (4.9%) nucleotide differences while tef1 and rpb2 gene regions were unavailable for comparison. Therefore, we recognize these isolates belong to two distinct species [42].

Excluding Thyridaria acaciae, T. aureobrunnea and T. broussonetiae, our new species resembles T. eminens (30 × 12 µm [43]), T. koae (14–21 × 6–9 µm [44]), T. minor (15–18 × 5–6.5 µm [45]), T. sambucina (12–15.5 µm [46]), T. subrufa (12–15 × 4–5 µm [46]), and T. triseptata (15–18 µm [47]) with its three-septate pigmented ascospores. Among them, Thyridaria eminens (collected from dead Streblus asper in Philippines [43]) is morphologically closely affiliated with T. jonahhulmei by ascospore dimensions. However, all of these species lack molecular data for further phylogenetic characterization.

4. Discussion

In this study, we describe and illustrate two new species of microfungi on dead woody litter, Neokalmusia jonahhulmei (Didymosphaeriaceae) and Thyridaria jonahhulmei (Thyridariaceae), from Kunming, Yunnan, based on morphological and molecular analyses (Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5). Neokalmusia jonahhulmei is introduced with only its sexual characteristics, whereas Thyridaria jonahhulmei is accounted for with both asexual and sexual morphological features.

Didymosphaeriaceae was introduced by Munk [48], and, given that the family is composed of 33 genera, it is considered one of the most specious families in the order Pleosporales [23,25]. 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 or in water [23,49]. We have presented representative sequence data of all currently available genera listed in Hongsanan et al. [23] and Samarakoon et al. [25] for the phylogenetic analyses (except Barria, Curreya, Julella, and Lineostroma, for which no DNA-based sequence data were available). Additionally, we included the sequences of Pseudodidymocyrtis lobariellae that were introduced by Flakus et al. [50] as a lichenicolous fungus from Bolivia on Lobariella pallida. Multi-gene phylogenetic analyses (Figure 2) revealed that Pseudodidymocyrtis clusters with Kalmusia species, and from the morphological perspective, these two genera appear to have a close resemblance. Therefore, generic delimitation needs defining among these two genera, possibly by using more fresh collections with additional morpho–phylo data.

Even though Thyridaria is one of the oldest genera (introduced in 1875) in Ascomycota, no exact family was available to accommodate this genus [51]. More than 30 species are included in Thyridaria [52]; however, for many of these species, data are lacking illustrations, descriptions, or DNA-based molecular data, leading to ambiguous taxonomic relationships. Therefore, Thyridaria species have suffered from uncertain family placement and have been assigned to different families in Dothideomycetes at various times [41,53,54,55,56,57,58,59,60,61]. Hyde et al. [51] considered its unique morphology and the phylogenetic placement of Thyridaria rubronotata in the Dothideomycetes backbone tree and introduced Thyridariaceae to accommodate this genus. Recently, Jaklitsch and Voglmayr [41] studied several thyridaria-like genera and constructed a multi-gene analysis to clarify intergeneric taxonomic affinities of Thyridaria in the Pleosporales. They found that thyridaria-like genera are distributed among at least nine clades of the Pleosporales. Barr [61] thoroughly reviewed various descriptions of Thyridaria and suggested that a key characteristic of the genus is an ample subiculum surrounding fruiting bodies that are produced under the ascomata wall or in host tissues. Similarly, the intensive study of Jaklitsch and Voglmayr [41] pointed out that this feature is exclusive to delineate Thyridaria species from its phylogenetically closely related affiliates. We also observed that our new Thyridaria species was characterized with a pruinose layer of yellow to reddish- or orange-brown material forming around the apices of the ostiolar necks. The subiculum of Thyridaria species could be an adaptation for its terrestrial-based habitat, and their potential advantages should be further clarified with extensive sampling from both aquatic and non-aquatic habitats.

By contrast, wood-decaying Basidiomycota in tropical China are well studied, which has facilitated a better understanding of global Basidiomycota species diversity and systematics [62,63,64,65,66,67]. Nonetheless, the study of habitat properties and potential lifestyles of woody-based microfungal species is especially important to understanding their functional aspects. The trophic mode of many species can be highly variable, with the ability to switch between mutualistic, pathogenic, and saprotrophic strategies. Pathogenic taxa that cause leaf spots may begin as endophytes, but environmental stressors may necessitate becoming pathogenic or eventually saprotrophic after plant tissue [68,69]. Another example is Scedosporiosis, the human disease caused by Pseudallescheria/Scedosporium complex species (PSC), which can grow saprobically in wood [70,71]. Therefore, owing to their importance in all ecosystems, saprotrophic fungi simply cannot be overlooked in any region.

This study provides some insights into the saprotrophic taxa and contributes knowledge of microfungi associated with woody litter in Yunnan, China by introducing two novel species. Furthermore, these species were discovered by a 5-year-old boy, unassisted by any mycologists at the time of collection, thus highlighting how young enthusiasts can be involved in field studies and make a significant impact.

Acknowledgments

Austin G. Smith at World Agroforestry (ICRAF), Kunming Institute of Botany, China, is thanked for English editing. Shaun Pennycook is thanked for nomenclatural advice. Lu Wen Hua and Li Qin Xian are thanked for their invaluable assistance. We acknowledge Kunming Institute of Botany and the Chinese Academy of Sciences for providing the laboratories and instruments for molecular work.

Author Contributions

Conceptualization, D.N.W.; resources, P.E.M.; writing—original draft preparation, D.N.W.; writing—review and editing, P.E.M.; supervision, P.E.M.; funding acquisition, P.E.M. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets generated for this study can be found in the NCBI GenBank, MycoBank, and TreeBASE.

Conflicts of Interest

The authors declare no conflict of interest.

Funding Statement

Funding was provided by the High-End Foreign Experts in the High-Level Talent 318 Recruitment Plan of Yunnan Province, 2021; the CAS President’s International Fellowship Initiative (number 2021FYB0005); the National Science Foundation of China (NSFC) under the project code 32150410362; and the Postdoctoral Fund from Human Resources and Social Security Bureau of Yunnan Province.

Footnotes

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Bray J.R., Gorham E. Litter production in forests of the world. Adv. Ecol. Res. 1964;2:101–157. doi: 10.1016/S0065-2504(08)60331-1. [DOI] [Google Scholar]
  • 2.Lowman M.D. Litter fall and leaf decay in three Australian rainforest formations. J. Ecol. 1988;76:451–465. doi: 10.2307/2260605. [DOI] [Google Scholar]
  • 3.Rahman M.M., Tsukamoto J., Tokumoto Y., Shuvo M.A.R. The role of quantitative traits of leaf litter on decomposition and nutrient cycling of the forest ecosystems. J. For. Sci. 2013;29:38–48. doi: 10.7747/JFS.2013.29.1.38. [DOI] [Google Scholar]
  • 4.Jia B., Zhou G., Xu Z. Forest litter fall and its composition: A new data set of observational data from China. Ecology. 2016;97:1365. doi: 10.1890/15-1604.1. [DOI] [Google Scholar]
  • 5.Zhou J., Lang X., Du B., Zhang H., Liu H., Zhang Y., Shang L. Litter fall and nutrient return in moist evergreen broad-leaved primary forest and mixed subtropical secondary deciduous broad-leaved forest in China. Eur. J. For. Res. 2016;135:77–86. doi: 10.1007/s10342-015-0918-7. [DOI] [Google Scholar]
  • 6.Giweta M. Role of litter production and its decomposition, and factors affecting the processes in a tropical forest ecosystem: A review. J. Ecol. Environ. 2020;44:11. doi: 10.1186/s41610-020-0151-2. [DOI] [Google Scholar]
  • 7.Bao D.F., Luo Z.L., Liu J.K., Bhat D.J., Sarunya N., Li W.L., Su H.Y., Hyde K.D. Lignicolous freshwater fungi in China III: Three new species and a new record of Kirschsteiniothelia from northwestern Yunnan province. Mycosphere. 2018;9:755–768. doi: 10.5943/mycosphere/9/4/4. [DOI] [Google Scholar]
  • 8.Huang S.K., Maharachchikumbura S.S.N., Jeewon R., Bhat D.J., Chomnunti P., Hyde K.D., Lumyong S. Morphological and molecular taxonomy of Jahnula dianchia sp. nov. (Jahnulales) from submerged wood in Dianchi Lake, Yunnan China. Mycol. Prog. 2018;17:547–555. doi: 10.1007/s11557-018-1390-9. [DOI] [Google Scholar]
  • 9.Luo Z.L., Hyde K.D., Liu J.K., Bhat D.J., Bao D.F., Li W.L., Su H.W.Q. Lignicolous freshwater fungi from China II: Novel Distoseptispora (Distoseptisporaceae) species from northwestern Yunnan Province and a suggested unified method for studying lignicolous freshwater fungi. Mycosphere. 2018;9:444–461. doi: 10.5943/mycosphere/9/3/2. [DOI] [Google Scholar]
  • 10.Wanasinghe D.N., Wijayawardene N.N., Xu J., Cheewangkoon R., Mortimer P.E. Taxonomic novelties in Magnolia-associated pleosporalean fungi in the Kunming Botanical Gardens (Yunnan, China) PLoS ONE. 2020;15:e0235855. doi: 10.1371/journal.pone.0235855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Wanasinghe D.N., Mortimer P.E., Xu J. Insight into the systematics of microfungi colonizing dead woody twigs of Dodonaea viscosa in Honghe (China) J. Fungi. 2021;7:180. doi: 10.3390/jof7030180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Mortimer P.E., Jeewon R., Xu J.C., Lumyong S., Wanasinghe D.N. Morpho-phylo taxonomy of novel dothideomycetous fungi associated with dead woody twigs in Yunnan Province, China. Front. Microbiol. 2021;12:654683. doi: 10.3389/fmicb.2021.654683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Qiao M., Hua Z., Lv R., Yu Z. Neodactylariales, Neodactylariaceae (Dothideomycetes, Ascomycota): New order and family, with a new species from China. MycoKeys. 2020;73:69–85. doi: 10.3897/mycokeys.73.54054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Rathnayaka A.R., Dayarathne M.C., Maharachchikumbura S.S.N., Liu J.K., Tennakoon D.S., Hyde K.D. Introducing Seriascoma yunnanense sp. nov. (Occultibambusaceae, Pleosporales) based on evidence from morphology and phylogeny. AJOM. 2019;2:245–253. doi: 10.5943/ajom/2/1/15. [DOI] [Google Scholar]
  • 15.Thiyagaraja V., Wanasinghe D.N., Worthy F., Karunarathna S.C. Addition to Melanommataceae: A new geographical record of Alpinaria rhododendri from Shangri La, China. AJOM. 2020;3:335–344. doi: 10.5943/ajom/3/1/8. [DOI] [Google Scholar]
  • 16.Yasanthika E., Dissanayake L.S., Wanasinghe D.N., Karunarathna S.C., Mortimer P.E., Samarakoon B.C., Monkai J., Hyde K.D. Lonicericola fuyuanensis (Parabambusicolaceae) a new terrestrial pleosporalean ascomycete from Yunnan Province, China. Phytotaxa. 2020;446:103–113. doi: 10.11646/phytotaxa.446.2.3. [DOI] [Google Scholar]
  • 17.White T.J., Bruns T., Lee S.J.W.T., Taylor J.W. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols Appl. 1990;18:315–322. [Google Scholar]
  • 18.Rehner S.A., Samuels G.J. Taxonomy and phylogeny of Gliocladium analysed from nuclear large subunit ribosomal DNA sequences. Mycol. Res. 1994;98:625–634. doi: 10.1016/S0953-7562(09)80409-7. [DOI] [Google Scholar]
  • 19.Vilgalys R., Hester M. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J. Bacteriol. 1990;172:4238–4246. doi: 10.1128/jb.172.8.4238-4246.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Rehner S.A., Buckley E. A Beauveria phylogeny inferred from nuclear ITS and EF1-α sequences: Evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia. 2005;97:84–98. doi: 10.3852/mycologia.97.1.84. [DOI] [PubMed] [Google Scholar]
  • 21.Liu Y.J., Whelen S., Hall B.D. Phylogenetic relationships among ascomycetes evidence from an RNA polymerase II subunit. Mol. Biol. Evol. 1999;16:1799–1808. doi: 10.1093/oxfordjournals.molbev.a026092. [DOI] [PubMed] [Google Scholar]
  • 22.Sung G.H., Sung J.M., Hywel-Jones N.L., Spatafora J.W. A multi-gene phylogeny of Clavicipitaceae (Ascomycota, Fungi): Identification of localized incongruence using a combinational bootstrap approach. Mol. Phylogenet. Evol. 2007;44:1204–1223. doi: 10.1016/j.ympev.2007.03.011. [DOI] [PubMed] [Google Scholar]
  • 23.Hongsanan S., Hyde K.D., Phookamsak R., Wanasinghe D.N., McKenzie E.H.C., Sarma V.V., Boonmee S., Lücking R., Bhat D.J., Liu N.G., et al. Refined families of Dothideomycetes: Dothideomycetidae and Pleosporomycetidae. Mycosphere. 2020;11:1553–2107. doi: 10.5943/mycosphere/11/1/13. [DOI] [Google Scholar]
  • 24.Poli A., Bovio E., Ranieri L., Varese G.C., Prigione V. News from the Sea: A New Genus and Seven New Species in the Pleosporalean Families Roussoellaceae and Thyridariaceae. Diversity. 2020;12:144. doi: 10.3390/d12040144. [DOI] [Google Scholar]
  • 25.Samarakoon B.C., Wanasinghe D.N., Samarakoon M.C., Phookamsak R., McKenzie E.H.C., Chomnunti P., Hyde K.D., Lumyong S., Karunarathna S.C. Multi-gene phylogenetic evidence suggests Dictyoarthrinium belongs in Didymosphaeriaceae (Pleosporales, Dothideomycetes) and Dictyoarthrinium musae sp. nov. on Musa from Thailand. MycoKeys. 2020;71:101–118. doi: 10.3897/mycokeys.71.55493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Kuraku S., Zmasek C.M., Nishimura O., Katoh K. aLeaves facilitates on-demand exploration of metazoan gene family trees on MAFFT sequence alignment server with enhanced interactivity. Nucleic Acids Res. 2013;41:22–28. doi: 10.1093/nar/gkt389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Katoh K., Rozewicki J., Yamada K.D. MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Brief. Bioinform. 2019;20:1160–1166. doi: 10.1093/bib/bbx108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Hall T.A. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids Symp. Ser. 1999;41:95–98. [Google Scholar]
  • 29.Nylander J.A.A., Wilgenbusch J.C., Warren D.L., Swofford D.L. AWTY (are we there yet?): A system for graphical exploration of MCMC convergence in Bayesian phylogenetics. Bioinformatics. 2008;24:581–583. doi: 10.1093/bioinformatics/btm388. [DOI] [PubMed] [Google Scholar]
  • 30.Miller M.A., Pfeiffer W., Schwartz T. Creating the CIPRES Science Gateway for inference of large phylogenetic trees; Proceedings of the 2010 Gateway Computing Environments Workshop (GCE); New Orleans, LA, USA. 14 November 2010; pp. 1–8. [DOI] [Google Scholar]
  • 31.Stamatakis A. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30:1312–1313. doi: 10.1093/bioinformatics/btu033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Ronquist F., Huelsenbeck J.P. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003;19:1572–1574. doi: 10.1093/bioinformatics/btg180. [DOI] [PubMed] [Google Scholar]
  • 33.Rambaut A. FigTree Version 1.4.0. [(accessed on 23 January 2022)]. Available online: http://tree.bio.ed.ac.uk/software/figtree/
  • 34.Ariyawansa H.A., Tanaka K., Thambugala K.M., Phookamsak R., Tian Q., Camporesi E., Hongsanan S., Monkai J., Wanasinghe D.N., Mapook A., et al. A molecular phylogenetic reappraisal of the Didymosphaeriaceae (=Montagnulaceae) Fungal Divers. 2014;68:69–104. doi: 10.1007/s13225-014-0305-6. [DOI] [Google Scholar]
  • 35.Tanaka K., Hirayama K., Yonezawa H., Sato G., Toriyabe A., Kudo H., Hashimoto A., Matsumura M., Harada Y., Kurihara Y., et al. Revision of the Massarineae (Pleosporales, Dothideomycetes) Stud. Mycol. 2015;82:75–136. doi: 10.1016/j.simyco.2015.10.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Hyde K.D., Dong Y., Phookamsak R., Jeewon R., Bhat D.J., Jones E.B.G., Liu N.G., Abeywickrama P.D., Mapook A., Wei D.P., et al. Fungal diversity notes 1151–1276: Taxonomic and phylogenetic contributions on genera and species of fungal taxa. Fungal Divers. 2020;100:5–277. doi: 10.1007/s13225-020-00439-5. [DOI] [Google Scholar]
  • 37.Thambugala K.M., Wanasinghe D.N., Phillips A.J.L., Camporesi E., Bulgakov T.S., Phukhamsakda C., Ariyawansa H.A., Goonasekara I.D., Phookamsak R., Dissanayake A., et al. Mycosphere notes 1-50: Grass (Poaceae) inhabiting Dothideomycetes. Mycosphere. 2017;8:697–796. doi: 10.5943/mycosphere/8/4/13. [DOI] [Google Scholar]
  • 38.Dai D.Q., Bahkali A.H., Ariyawansa H.A., Li W.J., Bhat D.J., Hyde K.D. Neokalmusia didymospora (Didymosphaeriaceae), a new species from bamboo. Sydowia. 2016;68:17–25. doi: 10.1016/j.sjbs.2015.01.020. [DOI] [Google Scholar]
  • 39.Crous P.W., Wingfield M.J., Schumacher R.K., Summerell B.A., Giraldo A., Gené J., Guarro J., Wanasinghe D.N., Hyde K.D., Camporesi E., et al. Fungal Planet Description Sheets: 281–319. Persoonia. 2014;33:212–289. doi: 10.3767/003158514X685680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Boonmee S., Wanasinghe D.N., Calabon M.S., Huanraluek N., Chandrasiri S.K.U., Jones G.E.B., Rossi W., Leonardi M., Singh S.K., Rana S., et al. Fungal diversity notes 1387–1511: Taxonomic and phylogenetic contributions on genera and species of fungal taxa. Fungal Divers. 2021;111:1–335. doi: 10.1007/s13225-021-00489-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Jaklitsch W.M., Voglmayr H. Hidden diversity in Thyridaria and a new circumscription of the Thyridariaceae. Stud. Mycol. 2016;85:35–64. doi: 10.1016/j.simyco.2016.09.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Jeewon R., Hyde K.D. Establishing species boundaries and new taxa among fungi: Recommendations to resolve taxonomic ambiguities. Mycosphere. 2016;7:1669–1677. doi: 10.5943/mycosphere/7/11/4. [DOI] [Google Scholar]
  • 43.Rehm H. Ascomycetes philippinenses—VIII. Leafl. Philipp. Bot. 1916;8:2945. [Google Scholar]
  • 44.Petrak F. Ein Beitrag zur Pilzflora von Hawai. Sydowia. 1952;6:363–371. [Google Scholar]
  • 45.Saccardo P.A. Sylloge fungorum XXIV, sect. II. Abellini. Sylloge Fungorum. 1928;24:705–1438. [Google Scholar]
  • 46.Wehmeyer L.E. The genus Thyridaria (Pyrenomycetes) Lloydia. 1941;4:241–262. [Google Scholar]
  • 47.Barr M.E. Melanommatales (Loculoascomycetes) N. Am. Flora Ser. II. 1990;13:1–129. [Google Scholar]
  • 48.Munk A. The system of the Pyrenomycetes. Dansk botanisk Arkiv. 1953;15:1–163. [Google Scholar]
  • 49.Gonçalves M.F.M., Vicente T.F.L., Esteves A.C., Alves A. Neptunomyces aureus gen. et sp. nov. (Didymosphaeriaceae, Pleosporales) isolated from algae in Ria de Aveiro, Portugal. MycoKeys. 2019;60:31–44. doi: 10.3897/mycokeys.60.37931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Flakus A., Etayo J., Miadlikowska J., Lutzoni F., Kukwa M., Matura N., Rodriguez-Flakus P. Biodiversity assessment of ascomycetes inhabiting Lobariella lichens in Andean cloud forests led to one new family, three new genera and 13 new species of lichenicolous fungi. Plant Fungal Syst. 2019;64:283–344. doi: 10.2478/pfs-2019-0022. [DOI] [Google Scholar]
  • 51.Hyde K.D., Gareth J.E.B., Liu J.K., Ariyawansa H., Boehm E., Boonmee S., Braun U., Chomnunti P., Crous P.W., Dai D.Q., et al. Families of Dothideomycetes. Fungal Divers. 2013;63:1–313. doi: 10.1007/s13225-013-0263-4. [DOI] [Google Scholar]
  • 52.Species Fungorum. [(accessed on 23 January 2022)]. Available online: http://www.speciesfungorum.org/Names/Names.asp.
  • 53.Wehmeyer L.E. A revision of Melanconis, Pseudovalsa, Prosthecium and Titania. Univ. Mich. Stud. Sci. Ser. 1941;14:1–161. [Google Scholar]
  • 54.Wehmeyer L.E. The Pyrenomycetous Fungi. Mycologia Memoir No. 6. The New York Botanical Garden. J. Cramer Publishing; Lehre, Germany: 1975. pp. 1–250. [Google Scholar]
  • 55.Munk A. Danish pyrenomycetes: A preliminary flora. Dansk Botanisk Arkiv. 1957;17:491. [Google Scholar]
  • 56.Luttrell E.S. Loculoascomycetes. In: Ainsworth G.C., Sparrow F.K., Sussman A.S., editors. The Fungi. An Advanced Treatise, a Taxonomic Review with Keys: Ascomycetes and Fungi Imperfecti. Academic Press; New York, NY, USA: 1973. pp. 135–219. [Google Scholar]
  • 57.Müller E., von Arx J.A. Pyrenomycetes: Meliolales, Coronophorales, Sphaeriales. New Exot. Fungi. Grevillea. 1973;17:42–43. [Google Scholar]
  • 58.Dennis R.W.G. British Ascomycetes. 3rd ed. Cramer, J.; Vaduz, Liechtenstein: 1978. pp. 1–585. [Google Scholar]
  • 59.Barr M.E. A classification of Loculoascomycetes. Mycologia. 1979;71:935–995. doi: 10.1080/00275514.1979.12021099. [DOI] [Google Scholar]
  • 60.Barr M.E. On the Massariaceae in North America. Mycotaxon. 1979;9:17–37. [Google Scholar]
  • 61.Barr M.E. The affinities of Thyridaria. Mycotaxon. 2003;88:271–278. [Google Scholar]
  • 62.Dai Y.C., Cui B.K., Yuan H.S., He S.H., Wei Y.L., Qin W.M., Zhou L.W., Li H.J. Wood-inhabiting fungi in southern China 4. Polypores from Hainan province. Ann. Bot. Fenn. 2011;48:219–231. doi: 10.5735/085.048.0302. [DOI] [Google Scholar]
  • 63.Dai Y., Härkönen M., Niemelä T. Wood-inhabiting fungi in southern China 1. Polypores from Hunan province. Ann. Bot. Fenn. 2003;40:381–393. [Google Scholar]
  • 64.Dai Y., Wei Y., Wang Z. Wood-inhabiting fungi in southern China 2. Polypores from Sichuan province. Ann. Bot. Fenn. 2004;41:319–329. [Google Scholar]
  • 65.Cui B.K., Dai Y., Bao H.Y. Wood-inhabiting fungi in southern China 3. A new species of Phellinus (Hymenochaetales) from tropical China. Mycotaxon. 2009;110:125–130. doi: 10.5248/110.125. [DOI] [Google Scholar]
  • 66.Wang B., Cui B.K., Li H.J., Du P., Jia B.S. Wood-rotting fungi in eastern China 5. Polypore diversity in Jiangxi Province. Ann. Bot. Fenn. 2011;48:237–246. doi: 10.5735/085.048.0304. [DOI] [Google Scholar]
  • 67.Yuan H., Dai Y. Wood-inhabiting fungi in southern China. 6. Polypores from Guangxi autonomous region. Ann. Bot. Fenn. 2012;49:341–351. doi: 10.5735/085.049.0605. [DOI] [Google Scholar]
  • 68.Promputtha I., Lumyong S., Dhanasekaran V., McKenzie E.H.C., Hyde K.D., Jeewon R. A phylogenetic evaluation of whether endophytes become saprotrophs at host senescence. Microb. Ecol. 2007;53:579–590. doi: 10.1007/s00248-006-9117-x. [DOI] [PubMed] [Google Scholar]
  • 69.Photita W., Lumyong S., Lumyong P., McKenzie E.H.C., Hyde K.D. Are some endophytes from Musa acuminate latent pathogens? Fungal Divers. 2004;16:131–140. [Google Scholar]
  • 70.Luplertlop N. Pseudallescheria/Scedosporium complex species: From saprobic to pathogenic fungus. J. Mycol. Med. 2018;2:249–256. doi: 10.1016/j.mycmed.2018.02.015. [DOI] [PubMed] [Google Scholar]
  • 71.Põlme S., Abarenkov K., Nilsson R.H., Lindahl B.D., Clemmensen K.E., Kauserud H., Nguyen N., Kjøller R., Bates S.T., Baldrian P., et al. FungalTraits: A user-friendly traits database of fungi and fungus-like stramenopiles. Fungal Divers. 2020;105:1–16. doi: 10.1007/s13225-020-00466-2. [DOI] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets generated for this study can be found in the NCBI GenBank, MycoBank, and TreeBASE.

Articles from Biology are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI)

RESOURCES