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Citation: Du, T.-Y.; Dai, D.-Q.;
Mapook, A.; Lu, L.; Stephenson, S.L.;
Suwannarach, N.; Elgorban, A.M.;
Al-Rejaie, S.; Karunarathna, S.C.;
Tibpromma, S. Additions to
Rhytidhysteron (Hysteriales,
Dothideomycetes) in China. J. Fungi
2023,9, 148. https://doi.org/
10.3390/jof9020148
Academic Editor: Lei Cai
Received: 21 December 2022
Revised: 14 January 2023
Accepted: 18 January 2023
Published: 22 January 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Fungi
Journal of
Article
Additions to Rhytidhysteron (Hysteriales,Dothideomycetes)
in China
Tian-Ye Du 1,2,3 , Dong-Qin Dai 1, Ausana Mapook 2, Li Lu 1,2,3 , Steven L. Stephenson 4,
Nakarin Suwannarach 5, Abdallah M. Elgorban 6, Salim Al-Rejaie 7, Samantha C. Karunarathna 1, *
and Saowaluck Tibpromma 1,*
1
Center for Yunnan Plateau Biological Resources Protection and Utilization, College of Biological Resource and
Food Engineering, Qujing Normal University, Qujing 655011, China
2Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand
3School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
4Department of Biological Sciences, University of Arkansas, Fayetteville, AR 72701, USA
5Research Center of Microbial Diversity and Sustainable Utilization, Chiang Mai University,
Chiang Mai 50200, Thailand
6Department of Botany and Microbiology, College of Science, King Saud University,
Riyadh 11451, Saudi Arabia
7Department of Pharmacology & Toxicology, College of Pharmacy, King Saud University,
Riyadh 11451, Saudi Arabia
*Correspondence: samanthakarunarathna@gmail.com (S.C.K.); saowaluckfai@gmail.com (S.T.)
Abstract:
In this study, twelve terrestrial hysteriaceous saprobic fungi growing on different pieces of
dead wood were collected from Yunnan Province, China. All hysteriaceous strains isolated in this
study tallied with the general characteristics associated with Rhytidhysteron. Detailed morphological
characteristics and combined multigene phylogeny of LSU, ITS, SSU, and TEF showed that the twelve
hysteriaceous fungi strains represent four distinct new species, and seven new host or geographical
records of Rhytidhysteron. Based on morphological and phylogenetic evidence, the four new species
(Rhytidhysteron bannaense sp. nov., R. coffeae sp. nov., R. mengziense sp. nov., and R. yunnanense sp.
nov.) expand the number of species of Rhytidhysteron from thirty-three to thirty-seven, while seven
new geographical records expand the records of Rhytidhysteron in China from six to thirteen. In
addition, 10 new Rhytidhysteron host records are reported for the first time, thus expanding the known
hosts for Rhytidhysteron from 52 to 62. Full descriptions, images of the morphology, and phylogenetic
analyses to show the position of the Rhytidhysteron taxa are provided. In addition, the present study
summarizes the main morphological characteristics, host associations, and locations of this genus.
Keywords: Ascomycota; four new species; Hysteriaceae; hysteriaceous; saprobes; seven new records
1. Introduction
The Dothideomycetes O.E. Erikss. & Winka is the largest class in the Ascomycota Caval.-
Sm [
1
–
3
]. Currently, it is made up of the Dothideomycetidae P.M. Kirk, P.F. Cannon, J.C. David
& Stalpers (three orders with 25 families), and Pleosporomycetidae C.L. Schoch, Spatafora,
Crous & Shoemaker (four orders with 94 families) [
2
,
4
,
5
]. This highly diverse class is mainly
characterized by bitunicate asci (asci with two-wall layers), and often with fissitunicate
dehiscence [2,6].
The Hysteriales Lindau belong to the subclass Pleosporomycetidae [
4
,
5
]. This mono-
typic order is characterized by its thick-walled, navicular ascomata which dehisce by an
invaginated slit or sulcus [4,7].
The Hysteriaceae Chevall., the only family in Hysteriales, has been classified under the
Pseudosphaeriales E. Müll. & Arx, Dothiorales Luttrell, Dothideales Lindau, and the Pleosporales
Luttr. ex M.E. Barr, previously [
4
,
8
–
12
]. The Hysteriaceae include the hysteriaceous fungi,
J. Fungi 2023,9, 148. https://doi.org/10.3390/jof9020148 https://www.mdpi.com/journal/jof
J. Fungi 2023,9, 148 2 of 31
which currently contain 13 genera [
5
]. Hysteriaceous fungi are characterized by hysterithe-
cioid or apothecioid ascomata, semi-immersed to superficial, carbonaceous, thick-walled,
and distinctly navicular with a pronounced, longitudinal slit, ascospores that are hyaline to
pigmented, muriform, and one to multi-septate in bitunicate asci [2,12–18]
The genus Rhytidhysteron Speg. was introduced by Spegazzini [
19
] to accommodate
two species (R.brasiliense Speg. and R. viride Speg.), but no type species was designated. Sub-
sequently, Clements and Shear [
20
] designated R. brasiliense as the type species [
12
,
16
,
21
,
22
].
The genus was transferred by Boehm et al. [
13
] from the Patellariaceae Corda to Hysteriaceae,
based on molecular data. Currently, 33 records of Rhytidhysteron are listed in the Index
Fungorum [
23
]. The sexual morph is described as having large ascomata, conspicuous, nav-
icular, usually with a perpendicular striae margin, and pigmented, septate and muriform
to sub-muriform ascospores [
16
,
18
]. Currently, only four species (R. hysterinum [Dufour]
Samuels & E. Müll., R. rufulum [Spreng] Speg., R. thailandicum Thambug. & K.D. Hyde, and
R. xiaokongense G.C. Ren & K.D. Hyde) have been described with an asexual morph, and
conidia are classified into two types—“Aposphaeria-like” and “Diplodia-like” [
16
,
18
,
21
]. The
main characteristics used to distinguish some species in this genus are the shape and border
of the hysterothecium, the type of the exciple, the color and reaction of the epithecium,
and the size of the ascospores [
24
]. Species of Rhytidhysteron are widely distributed in
33 countries and on 52 hosts [
6
,
18
,
25
,
26
]. Members of Rhytidhysteron play an indispensable
role as saprobes, endophytes, and weak pathogens on woody plants in both terrestrial and
marine habitats, and some are rarely found as human pathogens [6,16,18,25,27].
In this study, we collected 12 strains of hysteriaceous fungi from Yunnan Province,
China, and based on molecular phylogenetic analyses (LSU, ITS, SSU, and TEF) and mor-
phological characteristic comparisons, they were identified as four distinct new species
(Rhytidhysteron bannaense sp. nov., R. coffeae sp. nov., R. mengziense sp. nov., and R. yunna-
nense sp. nov.) and seven new host records (R. bruguierae Dayar., R. camporesii Ekanayaka
& K.D. Hyde, R. hongheense Wanas., R. magnoliae N.I. de Silva & K.D. Hyde, R. neorufulum
Thambug. & K.D. Hyde, R. tectonae Doilom & K.D. Hyde, and R. thailandicum).
2. Materials and Methods
2.1. Sample Collection, Morphological Identification, and Single Spore Isolation
Dead plant specimens with fungal fruiting bodies were collected from Yunnan Province,
China, between 2020 and 2021. Specimens were placed in plastic bags, important informa-
tion such as collection date, location, and host name was recorded, and then the specimens
were brought them back to the lab for isolation and morphological observation.
Senanayake et al. [
28
] was followed for the morphological study and single spore
isolation. Morphological structures were examined under an OPTEC SZ650 dissecting
stereomicroscope. An OLYMPUS optical microscope (Tokyo, Japan) was used to observe
microscopic fungal structures and an OLYMPUS DP74 (Tokyo, Japan) digital camera fitted
to the microscope was used to take photographs. All micro-morphological structures were
measured with the Tarosoft
®
Image Framework program and photo plates were made
using Adobe Photoshop CS3 Extended version 10.0 software (Adobe Systems, San Jose,
CA, USA).
Single spore isolation was carried out for all the specimens and the pure cultures were
grown on potato dextrose agar (PDA). Germinated spores were transferred to new PDA
plates under sterile conditions and incubated at 28
◦
C. Culture characteristics (mycelia
color, shape, and edge feature) were observed after one week.
The specimens were deposited in the Kunming Institute of Botany, Academia Sinica
(HKAS), Kunming, China. Living cultures were deposited in the Kunming Institute of
Botany Culture Collection (KUMCC), China. Facesoffungi (FoF) numbers were registered
as described in Jayasiri et al. [
29
], and MycoBank number (MB) was registered as outlined
in MycoBank (http://www.MycoBank.org, accessed on 24 October 2022).
J. Fungi 2023,9, 148 3 of 31
2.2. DNA Extraction, PCR Amplification, and Sequencing
Dissanayake et al. [
30
] was followed for molecular studies. Fresh mycelia which
grew on PDA plates for two weeks were scraped off the plates, and then DNA was
extracted using DNA Extraction Kit-BSC14S1 (BioFlux, Hangzhou, P.R. China), following
the manufacturer’s protocol. Polymerase chain reaction (PCR) was used to amplify four
gene regions and the primers and protocols were used for the amplification following
Wanasinghe et al. [
25
]. The LSU gene was amplified by using the primers LR0R and
LR5 [
31
], the ITS gene was amplified by using the primers ITS5 and ITS4 [
32
], the SSU
gene was amplified using the primers NS1 and NS4 [
32
], and the TEF gene was amplified
using the primers EF1-983F and EF1-2218R [
33
]. The total volume of the PCR mixture
for amplifications was 25
µ
L, which consisted of 12.5
µ
L 2xMaster Mix (mixture of Easy
Taq TM DNA Polymerase, dNTPs, and optimized buffer (Beijing Trans Gen Biotech Co.,
Chaoyang District, Beijing, China)), 8.5
µ
L ddH
2
O, 2
µ
L of DNA template, and 1
µ
L of each
forward and reverse primer (10 pM) [
34
]. Purification and sequencing of PCR products
were carried out by Qinke Biotech Co., Kunming, China.
2.3. Phylogenetic Analyses
The sequences of all strains obtained in this study were checked in BioEdit v.7.2.6.1 [
35
]
for quality. Geneious 9.1.8 was used to splice forward and reverse sequences. The combined
sequences were searched for similar taxa via Blast in NCBI (http://blast.ncbi.nlm.nih.gov/
(accessed on 10 November 2022)), and the most closely related taxa were put together for the
phylogenetic analyses. Phylogenetic analyses were carried out with 65 sequences (Table 1)
and two outgroup taxa—Gloniopsis calami S. Konta & K.D. Hyde (MFLUCC 15-0739) and
G. praelonga (Schwein.) Underw. & Earle (CBS 112415). All four gene sequences were down-
loaded from NCBI (http://www.ncbi.nlm.nih.gov/ (accessed on 10 November 2022)) and
aligned by MAFFT v.7 (http://mafft.cbrc.jp/alignment/server/ (accessed on 10 Novem-
ber 2022)) [
36
]. TrimAl.v1.2rev59 was used to optimize the alignment of sequences [
37
],
sequences were combined in BioEdit v.7.2.6.1. FASTA alignment formats were converted to
PHYLIP and NEXUS formats in ALTER (http://www.sing-group.org/ALTER/ (accessed
on 10 November 2022)) [38].
In phylogenetic analyses, Randomized Accelerated Maximum Likelihood (RAxML)
and Bayesian inference analyses (BI) were carried out in the CIPRES Science Gateway
(https://www.phylo.org/portal2/login!input.action (accessed on 10 November 2022)) [
39
].
The RAxML trees are performed using RAxML-HPC2 on XSEDE (8.2.12) [
40
,
41
] with the
GTR + I + G model of evolution. Additionally, Bayesian analyses were conducted using the
Markov Chain Monte Carlo (MCMC) method in MrBayes on XSEDE (3.2.7a) [
42
] to evaluate
posterior probabilities [
43
,
44
]: the best model of LSU and ITS is GTR + I+G, the best model
of SSU is HKY + I, and the best model of TEF is GTR + G. Six simultaneous Markov chains
were run for 1,000,000 generations, and trees were sampled at every 100th generation. Max-
trees was set to 5000 and clade robustness was assessed using a bootstrap (BT) analysis
of 1000 replicates. Phylogenetic trees were visualized with FigTree v.1.4.2 [
45
], bootstrap
values showing at the nodes, and edited by Microsoft Office PowerPoint 2010. The newly
obtained alignments and phylogenetic trees were deposited in TreeBASE, submission
ID: 30049 (https://treebase.org/treebase-web/user/submissionList.html, accessed on 28
November 2022).
J. Fungi 2023,9, 148 4 of 31
Table 1. Taxa name, strain numbers, and GenBank accession numbers included in the phylogenetic analyses carried out in the present study.
Taxa Name Strain Number
GenBank Accession Numbers
References
LSU ITS SSU TEF
Gloniopsis calami MFLUCC 15-0739 NG059715 KX669036 KX669034 KX671965 [46]
G. praelonga CBS 112415 FJ161173 — FJ161134 FJ161090 [13]
Rhytidhysteron bannaense KUMCC 21-0482 TOP526408 OP526398 OP526395 OP572199 This study
R. bannaense KUMCC 21-0483 OP526409 OP526399 OP526396 OP572200 This study
R. bruguierae KUMCC 21-0484 OP442285 OP494090 OP482277 OP572207 This study
R. bruguierae MFLU 18-0571 TMN017833 — MN017901 MN077056 [47]
R. bruguierae MFLUCC 17-1515 MN632452 MN632457 MN632463 MN635661 [48]
R. bruguierae MFLUCC 17-1511 MN632454 MN632459 MN632465 — [48]
R. bruguierae MFLUCC 17-1502 MN632453 MN632458 MN632464 MN635662 [48]
R. bruguierae MFLUCC 17-1509 MN632455 MN632460 MN632466 — [48]
R. camporesii KUMCC 21-0488 OP482286 OP494091 OP482278 OP572208 This study
R. camporesii KUN-HKAS 104277 TMN429072 MN429069 — MN442087 [49]
R. chromolaenae MFLUCC 17-1516 TMN632456 MN632461 MN632467 MN635663 [48]
R. coffeae KUMCC 21-0489 TOP526406 OP605963 OP526412 OP572201 This study
R. coffeae KUMCC 21-0492 OP526407 OP605964 OP526413 OP572202 This study
R. cozumelense A. Cobos-Villagrán 951 MW939459 MZ056797 — MZ457338 [24]
R. cozumelense T. Raymundo 7321 MW939460 MZ056798 — MZ457339 [24]
R. erioi MFLU 16-0584 TMN429071 MN429068 — MN442086 [49]
R. esperanzae T. Raymundo 6579 MZ477203 MZ056795 — MZ457336 [24]
R. esperanzae R. Valenzuela 17206 MZ477204 MZ056796 — MZ457337 [24]
R. hongheense KUMCC 21-0487 OP482287 OP494092 OP482279 OP572209 This study
R. hongheense KUMCC 20-0222 MW264193 MW264214 MW264223 MW256815 [25]
R. hongheense HKAS112348 MW541820 MW541824 MW541831 MW556132 [25]
R. hongheense HKAS112349 MW541821 MW541825 MW541832 MW556133 [25]
R. hysterinum EB 0351 GU397350 — — GU397340 [13]
R. hysterinum CBS 316.71 MH871912 MH860141 — — [50]
R. magnoliae KUMCC 21-0478 OP482288 OP494093 OP482280 OP572210 This study
R. magnoliae MFLUCC 18-0719 TMN989384 MN989383 MN989382 MN997309 [6]
R. mangrovei MFLU 18-1894 TMK357777 MK425188 — MK450030 [12]
R. mengziense KUMCC 21-0490 TOP526396 OP526402 OP526414 OP572203 This study
R. mengziense KUMCC 21-0491 OP526397 OP526403 OP526415 OP572204 This study
R. mesophilum A. Trejo 74 MW939461 MZ056799 — MZ457340 [24]
R. mesophilum A. Cobos-Villagrán 1800 MW939462 MZ056800 — MZ457341 [24]
J. Fungi 2023,9, 148 5 of 31
Table 1. Cont.
Taxa Name Strain Number
GenBank Accession Numbers
References
LSU ITS SSU TEF
R. mexicanum RV17107.1 TMT626028 MT626026 — — [51]
R. mexicanum RV17107.2 MT626029 MT626027 — — [51]
R. neorufulum KUMCC 21-0480 OP482290 OP494095 OP482282 OP572212 This study
R. neorufulum MFLUCC 21-0035 MZ346015 MZ346020 MZ346025 MZ356249 [18]
R. neorufulum MFLUCC 13-0216 TKU377566 KU377561 KU377571 KU510400 [16]
R. neorufulum GKM 361A GQ221893 — GU296192 — [13]
R. neorufulum HUEFS 192194 KF914915 — — — [15]
R. neorufulum MFLUCC 12-0528 KJ418117 KJ418118 KJ418119 — [16]
R. neorufulum CBS 306.38 FJ469672 — GU296191 GU349031 [15]
R. neorufulum MFLUCC 12-0011 KJ418109 KJ206287 KJ418110 — [16]
R. neorufulum MFLUCC 12-0567 KJ526126 KJ546124 KJ546129 — [16]
R. neorufulum MFLUCC 12-0569 KJ526128 KJ546126 KJ546131 — [16]
R. neorufulum EB 0381 GU397351 — GU397366 — [13]
R. opuntiae GKM 1190 GQ221892 — — GU397341 [52]
R. rufulum MFLUCC 14-0577 TKU377565 KU377560 KU377570 KU510399 [16]
R. rufulum EB 0384 GU397354 — GU397368 — [13]
R. rufulum EB 0382 GU397352 — — — [13]
R. rufulum EB 0383 GU397353 — GU397367 — [13]
R. rufulum MFLUCC 12-0013 KJ418111 KJ418112 KJ418113 — [6]
R. tectonae KUMCC 21-0479 OP482291 OP494096 OP482283 OP572213 This study
R. tectonae MFLUCC 21-0037 MZ346013 MZ346018 MZ346023 MZ356247 [18]
R. tectonae MFLUCC 21-0034 MZ346014 MZ346019 MZ346024 MZ356248 [18]
R. tectonae MFLUCC 13-0710 TKU764698 KU144936 KU712457 KU872760 [53]
R. thailandicum KUMCC 21-0493 OP482292 OP494097 OP482284 OP572214 This study
R. thailandicum MFLUCC 14-0503 TKU377564 KU377559 KU377569 KU497490 [16]
R. thailandicum MFLUCC 12-0530 KJ526125 KJ546123 KJ546128 — [16]
R. thailandicum MFLU17-0788 MT093472 MT093733 MT093495 — [6]
R. thailandicum MFLUCC 13-0051 MN509434 MN509433 — MN509435 [54]
R. xiaokongense KUMCC 20-0158 MZ346011 MZ346016 MZ346021 MZ356245 [18]
R. xiaokongense KUMCC 20-0160 TMZ346012 MZ346017 MZ346022 MZ356246 [18]
R. yunnanense KUMCC 21-0485 TOP526404 OP526410 OP526400 OP572205 This study
R. yunnanense KUMCC 21-0486 OP526405 OP526411 OP526401 OP572206 This study
Remarks: The newly generated sequences are indicated in bold, the superscript Tindicates ex-type, and “—” indicates information unavailable.
J. Fungi 2023,9, 148 6 of 31
3. Results
3.1. Phylogenetic Analyses
The phylogenetic trees obtained from RAxML and BI analyses provided essentially
similar topologies. The RAxML analyses of the combined dataset yielded the best scoring
tree (Figure 1), with a final ML optimization likelihood value of
−
12081.382479. The
matrix had 761 distinct alignment patterns, with 23.87% being undetermined characters
or gaps. Parameters for the GTR + I + G model of the combined LSU, ITS, SSU, and
TEF were as follows: estimated base frequencies A = 0.239683, C = 0.247793, G = 0.276186,
T = 0.236338
; substitution rates AC = 1.225201, AG = 2.493063, AT = 1.186723, CG = 0.732384,
CT = 4.977839
, GT = 1.0; proportion of invariable sites I = 0.675821; and gamma distribution
shape parameter α= 0.554013. The final RAxML tree is shown in Figure 1.
J. Fungi 2023, 9, x FOR PEER REVIEW 7 of 36
3. Results
3.1. Phylogenetic Analyses
The phylogenetic trees obtained from RAxML and BI analyses provided essentially
similar topologies. The RAxML analyses of the combined dataset yielded the best scoring
tree (Figure 1), with a final ML optimization likelihood value of −12081.382479. The matrix
had 761 distinct alignment patterns, with 23.87% being undetermined characters or gaps.
Parameters for the GTR + I + G model of the combined LSU, ITS, SSU, and TEF were as
follows: estimated base frequencies A = 0.239683, C = 0.247793, G = 0.276186, T = 0.236338;
substitution rates AC = 1.225201, AG = 2.493063, AT = 1.186723, CG = 0.732384, CT =
4.977839, GT = 1.0; proportion of invariable sites I = 0.675821; and gamma distribution
shape parameter α = 0.554013. The final RAxML tree is shown in Figure 1.
Figure 1. Phylogenetic tree generated from RAxML analyses based on combined LSU, ITS, SSU, and
TEF sequence data for Rhytidhysteron. The tree was rooted with Gloniopsis calami (MFLUCC 15-0739)
and G. praelonga (CBS 112415). Bootstrap support values equal to or higher than 60% ML and poste-
rior probability values equal to or higher than 0.90 Bayesian PP are indicated on the nodes. New
species are in red, new records are in blue, and ex-type strains are in bold.
Figure 1.
Phylogenetic tree generated from RAxML analyses based on combined LSU, ITS, SSU,
and TEF sequence data for Rhytidhysteron. The tree was rooted with Gloniopsis calami (MFLUCC
15-0739) and G. praelonga (CBS 112415). Bootstrap support values equal to or higher than 60% ML
and posterior probability values equal to or higher than 0.90 Bayesian PP are indicated on the nodes.
New species are in red, new records are in blue, and ex-type strains are in bold.
J. Fungi 2023,9, 148 7 of 31
The final RAxML tree was divided into two clades of Rhytidhysteron and the results
are similar to those reported by Wanasinghe et al. [
25
]. In this study, two new species
R. mengziense (KUMCC 21-0490, KUMCC 21-0491) and R. yunnanense (KUMCC 21-0485,
KUMCC 21-0486), and five new records for R. camporesii (KUMCC 21-0488), R. hongheense
(KUMCC 21-0487), R. magnoliae (KUMCC 21-0478), R. neorufulum (KUMCC 21-0480), and
R. tectonae (KUMCC 21-0479) were clustered within clade A. Two new species R. bannaense
(KUMCC 21-0482, KUMCC 21-0483) and R. coffeae (KUMCC 21-0489, KUMCC 21-0492),
along with two new records, R. bruguierae (KUMCC 21-0484) and R. thailandicum (KUMCC
21-0493), were included in clade B.
The four new species formed separate branches in the phylogenetic tree. Rhytidhysteron
bannaense was well separated from R. thailandicum in an independent lineage with relatively
good statistical support (100% ML/1.00 PP). Rhytidhysteron coffeae was separated from
R. mangrovei Vinit & K.D. Hyde with good statistical support (99% ML/1.00 PP). Rhytidhys-
teron mengziense was well separated from R. camporesii with low statistical support. Rhytid-
hysteron yunnanense was separated from R. mesophilum Cobos-Villagrán, R. Valenz, Hern.-
Rodr., Calvillo-Medina & Raymundo, with good statistical support (90% ML/0.92 PP).
The seven newly recorded strains and the known species in Rhytidhysteron clustered to-
gether in the phylogenetic tree with significant statistical support. Rhytidhysteron bruguierae
(KUMCC 21-0484) grouped within five strains of R. bruguierae, with moderate statisti-
cal support (65% ML). Rhytidhysteron camporesii (KUMCC 21-0488) grouped with R. cam-
poresii (KUN-HKAS 104277) with good statistical support (100% ML/1.00 PP). Rhytid-
hysteron hongheense (KUMCC 21-0487) grouped with R. hongheense (KUMCC 20-0222, H
KAS112348, HKAS112349) with moderate statistical support (79% ML). Rhytidhysteron mag-
noliae (KUMCC 21-0478) grouped with R. magnoliae (MFLUCC 18-0719) with good statistical
support (100% ML/1.00 PP). Rhytidhysteron neorufulum (KUMCC 21-0480) grouped with
ten strains of R. neorufulum with low statistical support. Rhytidhysteron tectonae (KUMCC
21-0479) grouped with R. tectonae (MFLUCC 13-0710, MFLUCC 21-0037, MFLUCC 21-0034)
with good statistical support (89% ML/0.98 PP). Rhytidhysteron thailandicum (KUMCC
21-0493) grouped within four strains of R. thailandicum with moderate statistical support
(72% ML).
3.2. Taxonomy
Rhytidhysteron bannaense T.Y. Du and Tibpromma sp. nov. (Figure 2)
MycoBank number: MB 845999. Facesoffungi number: FoF 12957
Etymology: Named after the region Xishuangbanna where the type specimen of the
new species was collected.
Holotype: HKAS 122695
Saprobic on decaying wood of Buddleja officinalis (Loganiaceae). Sexual morph: As-
comata. 1000–1500
µ
m long
×
550–1100
µ
m wide
×
350–850
µ
m high (
x
= 1350
×
750
×
670
µ
m, n = 5), hysterothecial, solitary to aggregated, semi-immersed to superficial,
black, apothecioid, navicular, rough, perpendicular striae, elongate and depressed, com-
pressed at apex, opening through a longitudinal slit, green at the center. Exciple: 40–150
µ
m
wide, composed of dark brown, thick-walled cells of textura angularis, outer layer brown
to dark brown, inner layer pale brown to hyaline. Hamathecium: 1–2
µ
m wide, dense,
hyaline, septate, branched, cellular pseudoparaphyses, forming an orange epithecium
above asci when mounted in water, becoming a purple epithecium above the asci when
mounted in 10% KOH and turns hyaline after 5 s. Asci: 140–189(–199)
×
12–15(–16)
µ
m
(
x = 166 ×14 µm
,
n = 20
), 8-spored, bitunicate, cylindrical, with short pedicel, rounded
at the apex, with an ocular chamber, J- apical ring. Ascospores: 22–27
×
10–12.8
µ
m
(
x = 25 ×11.5 µm
, n = 30), uni-seriate, slightly overlapping, hyaline, 1-septate when im-
mature, becoming brown to dark brown, 3-septate when mature, ellipsoidal, rounded to
slightly pointed at both ends, smooth-walled, without guttules or mucilaginous sheath.
Asexual morph: Undetermined.
J. Fungi 2023,9, 148 8 of 31
Culture characteristics: Ascospores germinated on PDA within 24 h and germ tubes
produced from one or both ends. Colonies on PDA reached a 6 cm diameter after two
weeks at 28
◦
C. The colony is soft, circular, irregularly raised, with an undulated edge,
white to gray on the forward, and grayish yellow in reverse.
Material examined: China, Yunnan Province, Xishuangbanna Prefecture, Jinghong
City, Manlie Village, 101
◦
0
0
1
00
E, 21
◦
55
0
15
00
N, on a decaying wood piece of Buddleja officinalis
Maxim. (Loganiaceae), 12 September 2021, T.Y. Du, BND72 (holotype, HKAS 122695, ex-
type living culture, KUMCC 21-0482 = KUMCC 21-0483).
Notes: In phylogenetic analyses, Rhytidhysteron bannaense was well separated from
R. thailandicum with relatively good statistical support (100% ML/1.00 PP). In morphology,
R. bannaense is distinct from R. thailandicum, having 8-spored asci, and ascospores 3-septate
when mature, without guttules, while R. thailandicum has (3–)6–8-spored asci, (1–)3-septate
ascospores, and guttulate [
6
,
16
,
54
]. In addition, the asci and ascospore size in R. bannaense
are larger than those in R. thailandicum (asci: 166
×
14
µ
m vs. 145
×
12.8
µ
m; ascospores:
25
×
11.5
µ
m vs. 24.5
×
9.5
µ
m) [
16
]. Moreover, according to the comparison results of
different gene fragments (Table 2), R. bannaense is different from R.thailandicum in ITS and
TEF genes (
≥
1.5%, without gaps). Therefore, in this study, R. bannaense is introduced as a
new species.
Table 2.
Comparison of LSU, ITS, LSU, and TEF gene fragments of R. bannaense and four strains of
R. thailandicum (without gaps).
Closest Known
Species Strain Number LSU Gene ITS Gene SSU Gene TEF Gene References
R. thailandicum
MFLUCC 14-0503 T0.00% 1.79%
(9/503 bp) 0.00% 2.26%
(21/929 bp) [16]
MFLUCC 12-0530 0.00% 1.89%
(10/527 bp) 0.00% — [16]
MFLU 17-0788 0.00% 1.42%
(7/494 bp) 0.00% — [6]
MFLUCC 13-0051 0.00% 1.81%
(9/496 bp) —2.28%
(21/923 bp) [54]
Remarks: The “—” represents unavailable data for this gene, superscript Tindicates the ex-type.
J. Fungi 2023, 9, x FOR PEER REVIEW 10 of 36
Figure 2. Rhytidhysteron bannaense (HKAS 122695, holotype). (a,b) Appearance of hysterothecia
on the host; (c,d) Vertical section through hysterothecium; (e–h) Asci; (i) Epithecium mounted in
water; (j) Pseudoparaphyses; (k) Exciple; (l–o) Ascospores; (p) A germinating ascospore; (q,r) Col-
ony on PDA medium (after four weeks). Scale bars: (c,d) = 500 μm; (e–h) = 100 μm; (i,j,l–p) = 20 μm;
(k) = 50 μm.
Figure 2. Cont.
J. Fungi 2023,9, 148 9 of 31
J. Fungi 2023, 9, x FOR PEER REVIEW 10 of 36
Figure 2. Rhytidhysteron bannaense (HKAS 122695, holotype). (a,b) Appearance of hysterothecia
on the host; (c,d) Vertical section through hysterothecium; (e–h) Asci; (i) Epithecium mounted in
water; (j) Pseudoparaphyses; (k) Exciple; (l–o) Ascospores; (p) A germinating ascospore; (q,r) Col-
ony on PDA medium (after four weeks). Scale bars: (c,d) = 500 μm; (e–h) = 100 μm; (i,j,l–p) = 20 μm;
(k) = 50 μm.
Figure 2. Rhytidhysteron bannaense
(HKAS 122695,
holotype
). (
a
,
b
) Appearance of hysterothecia
on the host; (
c
,
d
) Vertical section through hysterothecium; (
e
–
h
) Asci; (
i
) Epithecium mounted in
water; (
j
) Pseudoparaphyses; (
k
) Exciple; (
l
–
o
) Ascospores; (
p
) A germinating ascospore; (
q
,
r
) Colony
on PDA medium (after four weeks). Scale bars: (
c
,
d
) = 500
µ
m; (
e
–
h
) = 100
µ
m; (
i
,
j
,
l
–
p
) = 20
µ
m;
(k) = 50 µm.
Rhytidhysteron bruguierae Dayarathne, Mycosphere 11(1): 20 (2020) (Figure S1)
MycoBank number: MB 556574. Facesoffungi number: FoF 06154
Description: See Dayarathne et al. [47], Supplementary Notes 1
Distribution: China (this study), Thailand [47,48].
Host: Alnus nepalensis (this study), Bruguiera sp. [47], Chromolaena odorata [48].
Material examined: China, Yunnan Province, Xishuangbanna Prefecture, Jinghong
City, Manlie Village, 101
◦
1
0
1
00
E, 21
◦
54
0
0
00
N, on decaying wood of Alnus nepalensis D. Don
(Betulaceae), 12 September 2021, T.Y. Du, BND77 (HKAS 122690, living culture, KUMCC
21-0484).
Notes: Rhytidhysteron bruguierae was introduced by Dayarathne et al. (2020) based
on both morphology and phylogenetic analyses. According to phylogenetic analyses
based on combined multi-gene (LSU, ITS, SSU, and TEF), our collection grouped together
with R. bruguierae (MFLUCC 18-0398, MFLUCC 17-1515, MFLUCC 17 1511, MFLUCC
17-1502, MFLUCC 17-1509). The morphological characteristics of our collection resemble
R. bruguierae in having superficial, perpendicular striae, orange at the center hysterothecia,
hamathecium comprising septate, branched pseudoparaphyses, forming a red epithecium
above the asci, cylindrical, short pedicellate asci, and ellipsoidal to fusiform, brown as-
cospores [
47
,
48
]. Our collection and R. bruguierae are extremely similar in molecular data
analyses and morphological characteristics. Previously, this species was only recorded in
Thailand from Bruguiera sp. and Chromolaena odorata [
47
,
48
]. Therefore, our collection was
introduced as a new geographical and host record of R. bruguierae from the decaying wood
of Alnus nepalensis (Betulaceae) in the Yunnan Province of China. This is the first record of
R. bruguierae on Alnus nepalensis.
Rhytidhysteron camporesii
Ekanayaka & K.D. Hyde, Fungal Diversity 100: 5–277
(2020) (Figure S2)
J. Fungi 2023,9, 148 10 of 31
MycoBank number: MB 556783. Facesoffungi number: FoF 06459
Description: See Hyde et al. [49], Supplementary Notes 2
Distribution: China [49] (this study).
Host: Cotoneaster franchetii (this study), unidentified wood [49].
Material examined: China, Yunnan Province, Kunming City, Panlong District, Chang-
chong Mountain, on decaying wood of Cotoneaster franchetii Bois (Rosaceae), 5 September
2021, T.Y. Du, KMD93 (HKAS 122698, living culture, KUMCC 21-0488).
Notes: Rhytidhysteron camporesii was introduced by Hyde et al. [
49
] based on both
morphology and phylogenetic analyses. According to phylogenetic analyses based on
combined multi-gene (LSU, ITS, SSU, and TEF), our collection grouped together with
R. camporesii (KUN-HKAS 104277). In addition, our collection shows similar morphological
characteristics with R. camporesii, having black, striated hysterothecia, branched pseudopa-
raphyses, 8-spored, cylindrical, short pedicellate asci, and 3-septate, uni-seriate, ellipsoidal
to fusiform, brown ascospores [
49
]. Therefore, we report our collection as a new host record
of R. camporesii from decaying wood of Cotoneaster franchetii (Rosaceae) in the Yunnan
Province of China. This is the first record of R. camporesii on Cotoneaster franchetii.
Rhytidhysteron coffeae T.Y. Du and Tibpromma sp. nov. (Figure 3)
MycoBank number: MB 846000. Facesoffungi number: FoF 12958
Etymology: Named after the host name, Coffea sp.
Holotype: HKAS 122700
Saprobic on decaying wood of Coffea sp. (Rubiaceae). Sexual morph: Ascomata
1000–1700 µm
long
×
1000–1200
µ
m wide
×
300–600
µ
m high (
x = 1520 ×1120 ×450 µm
,
n = 5), hysterothecial, solitary to aggregated, mostly solitary, superficial, base is embedded
in the plant tissue, navicular, black, apothecioid, rough, perpendicular striae, elongate and
depressed, compressed at apex, and opening through a nearly circular longitudinal slit,
reddish brown at the center. Exciple: 70–160
µ
m wide (
x
= 95
µ
m, n = 10), composed of
dark brown, thick-walled cells of textura angularis, outer layer brown to dark brown, inner
layer pale brown to hyaline. Hamathecium: 2–3
µ
m wide, dense, hyaline, septate, branched,
cellular pseudoparaphyses, forming a red to purple epithecium above asci when mounted
in water, becoming dark purple epithecium above the asci when mounted in 10% KOH, and
turns hyaline after 5 s. Asci: (162–)170–197
µ
m
×
(9–)10–14(–16)
µ
m (
x = 179.5 ×13 µm
,
n = 20
), 8-spored, bitunicate, cylindrical, with short pedicel, rounded at the apex, with an
ocular chamber, and J- apical ring. Ascospores: 23–28.5
µ
m
×
8.5–11.5
µ
m (
x = 26 ×10 µm
,
n = 30
), uni-seriate, slightly overlapping, hyaline, 1-septate when immature, becoming
reddish brown to brown, 3-septate when mature, ellipsoidal to fusoid, straight or curved,
rounded to slightly pointed at both ends, guttulate, smooth-walled, without a mucilaginous
sheath. Asexual morph: Undetermined.
Culture characteristics: Ascospores germinated on PDA within 24 h and germ tubes
produced from one or both ends. Colonies on PDA reached a 6 cm diameter after two
weeks at 28
◦
C. The colony is flossy, velvety, circular, slightly raised, with an entire edge,
reddish brown on the forward and in reverse, with a green circle in the middle.
Material examined: China, Yunnan Province, Pu’er City, Mojiang County, Jinggong
coffee plantation, 101
◦
44
0
20
00
E, 23
◦
15
0
15
00
N, decaying wood of Coffea sp. L. (Rubiaceae),
23 December 2020, L. Lu, MJC2 (holotype, HKAS 122700, ex-type living culture, KUMCC
21-0489); Yunnan Province, Pu’er City, Qixiang coffee plantation, 101
◦
20
0
47
00
E, 22
◦
42
0
15
00
N, on decaying wood of Coffea sp. (Rubiaceae), 25 December 2020, L. Lu, QXC8 (HKAS
122701 paratype, ex-paratype culture, KUMCC 21-0492).
Notes: In the phylogenetic analyses, Rhytidhysteron coffeae clearly separated from
R. mangrovei with good statistical support (99% ML/1.00 PP). With respect to morphology,
R. coffeae is distinct from R. mangrovei in having branched pseudoparaphyses,
8-spored
asci, and 3-septate ascospores when mature, while R. mangrovei has unbranched pseu-
doparaphyses, (2–6–)8-spored asci, and (1–)3-septate ascospores [
12
]. In addition, the
ascomata, asci, and ascospore size of R. coffeae are larger than those of R. mangrovei
(ascomata:
1520 ×1120 ×450 µm
vs. 940
×
800
×
500
µ
m, asci: 179.5
×
13
µ
m vs.
J. Fungi 2023,9, 148 11 of 31
146 ×9.5 µm
, ascospores:
26 ×10 µm
vs. 23
×
8.3
µ
m) [
12
]. Moreover, according to
the comparison results of different gene fragments, R. coffeae is different from R. mangrovei
in ITS (77/651 bp, 11.83%, without gaps) and TEF (21/960 bp, 2.19%, without gaps) genes
(≥1.5%). Therefore, in this study, R. coffeae is introduced as a new species.
J. Fungi 2023, 9, x FOR PEER REVIEW 13 of 36
Figure 3. Rhytidhysteron coffeae (HKAS 122701, holotype). (a,b) Appearance of hysterothecia on
the host; (c,d) Vertical section through hysterothecium; (e–g) Asci; (h) Epithecium mounted in water;
(i,j) Pseudoparaphyses; (k) Pedicel of asci; (l) Exciple; (m–r) Ascospores; (s) A germinating asco-
spore; (t) Colony on PDA medium (after four weeks). Scale bars: (c,d) = 500 μm; (e–g) = 100 μm; (h,l)
= 50 μm; (i–k,m–s) = 20 μm.
Figure 3. Rhytidhysteron coffeae
(HKAS 122701,
holotype
). (
a
,
b
) Appearance of hysterothecia
on the host; (
c
,
d
) Vertical section through hysterothecium; (
e
–
g
) Asci; (
h
) Epithecium mounted in
water; (
i
,
j
) Pseudoparaphyses; (
k
) Pedicel of asci; (
l
) Exciple; (
m
–
r
) Ascospores; (
s
) A germinating
ascospore; (
t
) Colony on PDA medium (after four weeks). Scale bars: (
c
,
d
) = 500
µ
m; (
e
–
g
) = 100
µ
m;
(h,l) = 50 µm; (i–k,m–s) = 20 µm.
J. Fungi 2023,9, 148 12 of 31
Rhytidhysteron hongheense Wanas. J. Fungi 7, 180 (2021) (Figure S3)
MycoBank number: MB 837992
Description: See Wanasinghe et al. [25], Supplementary Notes 3
Distribution: China [25] (this study).
Host: Dodonaea sp. [25], Phyllanthus emblica (this study).
Material examined: China, Yunnan Province, Kunming City, Panlong District, Kunming
Institute of Botany, 102
◦
45
0
5
00
E, 25
◦
8
0
30
00
N, on decaying wood of Phyllanthus emblica (Euphor-
biaceae), 1 February 2021, T.Y. Du, KMD24 (HKAS 122697, living culture, KUMCC 21-0487).
Notes: Rhytidhysteron hongheense was introduced by Wanasinghe et al. [
25
] based
on both morphology and phylogenetic analyses. According to phylogenetic analyses
based on combined multi-gene (LSU, ITS, SSU, and TEF), our collection grouped together
with R. hongheense (KUMCC 20-0222, HKAS112348, and HKAS112349). In addition, our
collection shows similar morphological characteristics with R. hongheense in having solitary
to aggregated, slightly striated hysterothecia, branched pseudoparaphyses forming a
red epithecium above asci, 8-spored, cylindrical, short pedicellate asci, and uni-seriate
ascospores, brown when mature [
25
]. Therefore, we report our collection as a new host
record of R. hongheense from decaying wood of Phyllanthus emblica (Euphorbiaceae) in the
Yunnan Province of China. This is the first record of R. hongheense on Phyllanthus emblica.
Rhytidhysteron magnoliae
N.I. de Silva, Lumyong S & K.D. Hyde, Asian Journal of
Mycology 3(1): 295–306 (2019) (Figure S4)
MycoBank number: MB 557220. Facesoffungi number: FoF 07369
Description: See de Silva et al. [6], Supplementary Notes 4
Distribution: China [6] (this study).
Host: Hevea brasiliensis (this study), Magnolia grandiflora [6].
Material examined: China, Yunnan Province, Xishuangbanna Prefecture, Mengla
County, Xishuangbanna Tropical Botanical Garden, 101
◦
15
0
40
00
E, 21
◦
55
0
57
00
N, on decaying
wood of Hevea brasiliensis (Willd. ex A. Juss.) Muell. Arg. (Euphorbiaceae), 24 November
2020, T.Y. Du, BND10 (HKAS 122693, living culture, KUMCC 21-0478).
Notes: Rhytidhysteron magnoliae was introduced by de Silva et al. [
6
] based on both
morphology and phylogenetic analyses. According to phylogenetic analyses based on
combined multi-gene (LSU, ITS, SSU, and TEF), our collection grouped together with
R. magnoliae (MFLUCC 18-0719). In addition, our collection shows similar morphological
characteristics to R. magnoliae in having solitary to aggregated, semi-immersed to superficial,
coriaceous, striated hysterothecia, septate pseudoparaphyses slightly swollen at the apex
and enclosed in a gelatinous matrix, 8-spored, cylindrical, short pedicellate asci, and
ellipsoidal to fusoid, 1–3-septate, guttulate, brown to dark brown ascospores [
6
]. There is a
small difference between our collection and R. magnoliae in that the perpendicular striae
of ascomata in our collection is not as obvious as that in R. magnoliae. However, in the
phylogenetic tree, our collection (KUMCC 21-0478) grouped with R. magnoliae (MFLUCC
18-0719) (100% ML/1.00 PP). Therefore, we report our collection as a new host record
of R. magnoliae from decaying wood of Hevea brasiliensis (Euphorbiaceae) in the Yunnan
Province of China. This is the first record of R. magnoliae on Hevea brasiliensis.
Rhytidhysteron mengziense T.Y. Du and Tibpromma sp. nov. (Figure 4)
MycoBank number: MB 846001. Facesoffungi number: FoF 12959
Etymology: Named after the region Mengzi where the type specimen was collected.
Holotype: HKAS 122699
Saprobic on decaying twigs of Crataegus scabrifolia (Rosaceae). Sexual morph: Asco-
mata: 1000–1600
µ
m long
×
800–1000
µ
m wide
×
400–700
µ
m high (
x
= 1400
×
910
×
640
µ
m,
n=5
), hysterothecial, solitary to aggregated, mostly solitary, semi-immersed to
superficial, navicular, black, apothecioid, smooth, perpendicular striae, elongate and de-
pressed, compressed at apex, opening through a longitudinal slit, reddish brown at the
center. Exciple: 60–135
µ
m wide, composed of outer layer brown to black, thick-walled
cells of textura angularis, and inner layer light brown, thin-walled cells of textura prismatica.
Hamathecium: 1–2.5
µ
m wide, dense, hyaline, septate, branched, cellular pseudoparaphy-
J. Fungi 2023,9, 148 13 of 31
ses, forming a reddish brown to brown epithecium above asci when mounted in water,
becoming purple epithecium above the asci when mounted in 10% KOH, and turns hyaline
after 30 s, while appendages turn dark brown. Asci: 150–176(–182)
µ
m
×
10–14(–16.3)
µ
m
(
x = 164.5 ×13 µm
, n = 20), 8-spored, bitunicate, cylindrical, with short pedicel, rounded at
the apex, with an ocular chamber, J- apical ring, always fused with hamathecium. Ascospores:
(22.5–)24.5–27.5(–29) µm×
10.5–12.5
µ
m (
x
= 27
×
12
µ
m, n = 30), slightly overlapping, uni-
seriate, slightly overlapping, hyaline, 1-septate when immature, becoming reddish brown
to brown, (1–)3-septate when mature, ellipsoidal to fusoid, straight or curved, rounded to
slightly pointed at both ends, guttulate, rough-walled, without the mucilaginous sheath.
Asexual morph: Undetermined.
Culture characteristics: Ascospores germinated on PDA within 24 h and germ tubes
produced from one or both ends. Colonies on PDA reached a 6 cm diameter after one week
at 28
◦
C. The colony is flossy, velvety, circular, slightly raised, with an undulated edge,
white aerial hyphae on the forward and cream white in reverse.
Material examined: China, Yunnan Province, Honghe Prefecture, Mengzi City, on a de-
caying piece of wood of Crataegus scabrifolia (Rosaceae), 21 May 2020, S. Tibpromma, MZD5
(holotype, HKAS 122699, ex-type living culture, KUMCC 21-0490 = KUMCC 21-0491).
Notes: In phylogenetic analyses, Rhytidhysteron mengziense was well separated from
R. camporesii with low statistical support. However, R. mengziense is distinct from R. cam-
poresii in having exciple cells of textura angularis to prismatica, and rough-walled ascospores,
while R. camporesii has exciple cells of textura globulosa to angularis, and smooth-walled of
ascospores [
49
]. In addition, the ascomata and ascospore size of R. mengziense are larger
than those of R. camporesii (ascomata: 1400
×
640
µ
m vs. 1002.4
×
570.1
µ
m, ascospores:
27
×
12
µ
m vs. 26.1
×
10.4
µ
m) [
49
]. Moreover, according to the comparison results of
different gene fragments, R. mengziense is different from R. camporesii in ITS gene (10/651 bp,
1.54%, without gaps). Therefore, in this study, R. mengziense is introduced as a new species.
Rhytidhysteron neorufulum
Thambug. & K.D. Hyde, Cryptog. Mycol. 37(1): 110
(2016) (Figure S5)
MycoBank number: MB 551865. Facesoffungi number: FoF 01840
Description: See Thambugala et al. [16], Supplementary Notes 5
Distribution: China (this study), Mexico [55], Thailand [16,18].
Host: Bursera sp. [
55
], Elaeagnus sarmentosa (this study), Hevea brasiliensis [
16
], Tectona
grandis [18].
Material examined: China, Yunnan Province, Xishuangbanna Prefecture, Mengla
County, Xishuangbanna Tropical Botanical Garden, 101
◦
15
0
45” E, 21
◦
55
0
50” N, on decaying
wood of Elaeagnus sarmentosa Rehd. (Elaeagnaceae), 24 November 2020, T.Y. Du, BND49
(HKAS 122691, living culture, KUMCC 21-0480).
Notes: Rhytidhysteron neorufulum was introduced by Thambugala et al. [
16
] based on
both morphology and phylogenetic analyses. According to phylogenetic analyses based on
combined multi-gene (LSU, ITS, SSU, and TEF), our collection grouped together with R. ne-
orufulum (MFLUCC 13-0216, GKM 361A, HUEFS 192194, MFLUCC 12-0528, CBS 306.38,
MFLUCC 12-0011, MFLUCC 12-0567, MFLUCC 12-0569, EB 0381, MFLUCC 21-0035). In ad-
dition, our collection shows similar morphological characteristics to R. neorufulum, having
solitary to aggregated, superficial, non-striated hysterothecia, septate pseudoparaphy-
ses, 8-spored, cylindrical, short pedicellate asci, and uni-seriate, ellipsoidal to fusiform,
ascospores, brown when mature [
16
,
18
]. This species was previously only recorded in Thai-
land and Mexico. Therefore, we report our collection as new geographical and host record
of R. neorufulum from the decaying wood of Elaeagnus sarmentosa (Elaeagnaceae) in the
Yunnan Province of China. This is the first record of R. neorufulum on Elaeagnus sarmentosa.
Rhytidhysteron tectonae
Doilom & K.D. Hyde, Fungal Diversity. 82: 107–182 (2017)
(Figure S6)
MycoBank number: MB 551964. Facesoffungi number: FoF 01849
Description: See Doilom et al. [53], Supplementary Notes 6
Distribution: China (this study), Thailand [18,53].
J. Fungi 2023,9, 148 14 of 31
J. Fungi 2023, 9, x FOR PEER REVIEW 16 of 36
Figure 4. Rhytidhysteron mengziense (HKAS 122699, holotype). (a,b) Appearance of hysterothecia
on the host; (c,d) Vertical section through hysterothecium; (e) Epithecium mounted in water; (f–h)
Asci; (i) Exciple; (j) Pseudoparaphyses; (k–m) Ascospores; (n) A germinating ascospore; (o,p) Col-
ony on PDA medium (after one week). Scale bars: (c,d) = 500 μm; (e,k–n) = 20 μm; (f–i) = 100 μm; (j)
= 50 μm.
Figure 4. Rhytidhysteron mengziense
(HKAS 122699,
holotype
). (
a
,
b
) Appearance of hysterothecia on
the host; (
c
,
d
) Vertical section through hysterothecium; (
e
) Epithecium mounted in water; (
f
–
h
) Asci;
(
i
) Exciple; (
j
) Pseudoparaphyses; (
k
–
m
) Ascospores; (
n
) A germinating ascospore; (
o
,
p
) Colony on
PDA medium (after one week). Scale bars: (
c
,
d
) = 500
µ
m; (
e
,
k
–
n
) = 20
µ
m; (
f
–
i
) = 100
µ
m; (
j
) = 50
µ
m.
Host: Betula sp., an unidentified member of the Fabaceae [
18
], Magnolia delavayi (this
study), and Tectona grandis [53].
Material examined: China, Yunnan Province, Xishuangbanna Prefecture, Mengla
County, Xishuangbanna Tropical Botanical Garden, 101
◦
15
0
25
00
E, 21
◦
55
0
37
00
N, on decaying
wood of Magnolia delavayi Franch. (Magnoliaceae), 24 November 2020, T.Y. Du, BND33
(HKAS 122692, living culture, KUMCC 21-0479).
J. Fungi 2023,9, 148 15 of 31
Notes: Rhytidhysteron tectonae was introduced by Doilom et al. [
53
] based on both
morphology and phylogenetic analyses. According to phylogenetic analyses based on
combined multi-gene (LSU, ITS, SSU, and TEF), our collection grouped together with
R. tectonae (MFLUCC 13-0710, MFLUCC 21-0034, and MFLUCC 21-0037). In addition, our
collection shows similar morphological characteristics to R. tectonae, having solitary to
aggregated, semi-immersed to superficial, non-striated, yellow at the center hysterothecia,
septate, branched pseudoparaphyses, 8-spored, cylindrical, short pedicellate asci, and uni-
seriate, slightly overlapping, 1–3-septate ascospores, dark brown when mature [
18
,
53
]. This
species was previously only recorded in Thailand. Therefore, we report our collection as a
new geographical and host record of R. tectonae from decaying wood of Magnolia delavayi
(Magnoliaceae) in the Yunnan Province of China. This is the first record of R. tectonae on
Magnolia delavayi.
Rhytidhysteron thailandicum
Thambug. & K.D. Hyde, Cryptog. Mycol. 37(1): 110
(2016) (Figure S7)
MycoBank number: MB 551866. Facesoffungi number: FoF 01841
Description: See Thambugala et al. [16], Supplementary Notes 7
Distribution: China [6] (this study), Mexico [55], Thailand [16,54].
Host: Acacia sp. [
55
], Afzelia xylocarpa [
54
], Aquilaria sinensis (this study), Morus aus-
tralis [6], and unidentified wood [16].
Material examined: China, Yunnan Province, Xishuangbanna Prefecture, Menghai
County, agarwood plantation, on decaying wood of Aquilaria sinensis (Lour.) Spreng.
(Thymelaeaceae), 15 September 2021, T.Y. Du, YNA62 (HKAS 122689, living culture,
KUMCC 21-0493).
Notes: Rhytidhysteron thailandicum was introduced by Thambugala et al. [
16
] based on
both morphology and phylogenetic analyses. According to phylogenetic analyses based on
combined multi-gene (LSU, ITS, SSU, and TEF), our collection grouped together with R. thai-
landicum (MFLUCC 14-0503, MFLUCC 12-0530, MFLU17-0788, and MFLUCC 13-0051).
In addition, our collection shows similar morphological characteristics to R. thailandicum,
having solitary to aggregated, globose to subglobose, striated hysterothecia, exciple cells
of textura angularis, septate, branched pseudoparaphyses, cylindrical, short pedicellate
asci, and uni-seriate, brown ascospores [
6
,
16
,
54
]. There is a small difference between our
collection and R. thailandicum in that the surface of the ascomata of our collection is covered
with green. However, in the phylogenetic tree, our collection (KUMCC 21-0493) grouped
with the four strains of R. thailandicum. Therefore, we report our collection as a new host
record of R. thailandicum from decaying wood of Aquilaria sinensis (Thymelaeaceae) in the
Yunnan Province of China. This is the first record of R. thailandicum on Aquilaria sinensis.
Rhytidhysteron yunnanense T.Y. Du and Tibpromma, sp. nov. (Figure 5)
MycoBank number: MB 846002. Facesoffungi number: FoF 12960
Etymology: Named after the region Yunnan where the type specimen was collected.
Holotype: HKAS 122696
Saprobic on decaying wood of Rhus chinensis (Anacardiaceae). Sexual morph: Ascomata:
1900–3000
µ
m long
×
400–800
µ
m wide
×
300–600
µ
m high (
x = 2510 ×625 ×455 µm
,
n=5
), hysterothecial, solitary to aggregated, mostly aggregated, semi-immersed, navicular
to irregular, black, apothecioid, rough, each hysterothecia has two parallel striae parallel to
the longitudinal slit, and slight perpendicular striae, elongate and depressed, compressed
at apex, longitudinal slit, no opening. Exciple: 60–180
µ
m wide, composed of dark brown,
thick-walled cells of textura globulosa, outer layer brown to dark brown, inner layer pale
brown to hyaline. Hamathecium: 1–2.5
µ
m wide, dense, hyaline, septate, branched, cellular
pseudoparaphyses, forming a yellow epithecium above asci when mounted in water, and
becoming hyaline epithecium above the asci when mounted in 10% KOH, while appendages
turn dark. Asci: (205–)215–250(–265)
µ
m
×
12–16(–17)
µ
m (
x = 230 ×14 µm
,
n = 20
),
8-spored
, bitunicate, cylindrical, short with club-like perdicel, rounded at the apex, with
an ocular chamber, J- apical ring. Ascospores:
28.5–36 µm×11–14.5 µm
(
x = 32.5 ×13 µm
,
n = 30
), uni-seriate when mature, hyaline, 1-septate when immature, becoming reddish
J. Fungi 2023,9, 148 16 of 31
brown to brown, 3-septate when mature, ellipsoidal to fusoid, straight or curved, rounded
to slightly pointed at both ends, guttulate, without the mucilaginous sheath. Asexual
morph: Undetermined.
Culture characteristics: Ascospores germinated on PDA within 24 h and germ tubes
produced from one or both ends. Colonies on PDA reached a 6 cm diameter after two
weeks at 28
◦
C. The colony is velvety, circular, slightly raised, with a filiform edge, white
on the forward and white in reverse.
Material examined: China, Yunnan Province, Honghe Prefecture, Honghe County,
102
◦
14
0
24
00
E, 23
◦
25
0
30
00
N, on a decaying wood piece of Rhus chinensis Mill. (Anacardiaceae),
8 December 2020, T.Y. Du, HHD5 (holotype, HKAS 122696, ex-type living culture, KUMCC
21-048 = KUMCC 21-0486).
Notes: In phylogenetic analyses, Rhytidhysteron yunnanense was well separated from
R. mesophilum with good statistical support (90% ML/0.92 PP). In morphology, R. yun-
nanense is distinct from R. mesophilum, having navicular to irregular ascomata, each hys-
terothecia has two parallel striae parallel to the longitudinal slit, and slight perpendicular
striae, and a longitudinal slit with no opening, while R. mesophilum has boat-shaped
ascomata, with perpendicular striae with a perpendicular to longitudinal slit, and a lon-
gitudinal slit opening [
24
]. In addition, the asci and ascospore size of R. yunnanense are
smaller than those of R. mesophilum (asci: 230
×
14
µ
m vs. 267–282
×
15.5–16
µ
m, as-
cospores:
32.5 ×13 µm vs. 44.2 ×13.6 µm
) [
24
]. Moreover, according to the comparison
results of different gene fragments, R. yunnanense is different from R. mesophilum in the ITS
(28/651 bp, 4.30%, without gaps) gene. Therefore, in this study, R. yunnanense is introduced
as a new species.
Moreover, in the previous study of the genus Rhytidhysteron, ascomata have a transverse-
striae, perpendicular to the longitudinal slit, and this study is the first to find parallel striae
that are parallel to the longitudinal slit.
J. Fungi 2023, 9, x FOR PEER REVIEW 20 of 36
Figure 5. Rhytidhysteron yunnanense (HKAS 122696, holotype). (a–c) Appearance of hysterothecia
on the host; (d,e) Vertical section through hysterothecium; (f–j) Asci; (k) Epithecium mounted in
water; (l) Pseudoparaphyses; (m) Exciple; (n–q) Ascospores; (r) A germinating ascospore; (s,t) Col-
ony on PDA medium (after one week); Scale bars: (d,e) = 500 μm; (f–j) = 100 μm; (k,l,n–r) = 20 μm;
(m) = 50 μm.
Figure 5. Cont.
J. Fungi 2023,9, 148 17 of 31
J. Fungi 2023, 9, x FOR PEER REVIEW 20 of 36
Figure 5. Rhytidhysteron yunnanense (HKAS 122696, holotype). (a–c) Appearance of hysterothecia
on the host; (d,e) Vertical section through hysterothecium; (f–j) Asci; (k) Epithecium mounted in
water; (l) Pseudoparaphyses; (m) Exciple; (n–q) Ascospores; (r) A germinating ascospore; (s,t) Col-
ony on PDA medium (after one week); Scale bars: (d,e) = 500 μm; (f–j) = 100 μm; (k,l,n–r) = 20 μm;
(m) = 50 μm.
Figure 5. Rhytidhysteron yunnanense
(HKAS 122696,
holotype
). (
a
–
c
) Appearance of hysterothecia
on the host; (
d
,
e
) Vertical section through hysterothecium; (
f
–
j
) Asci; (
k
) Epithecium mounted in
water; (
l
) Pseudoparaphyses; (
m
) Exciple; (
n
–
q
) Ascospores; (
r
) A germinating ascospore; (
s
,
t
) Colony
on PDA medium (after one week); Scale bars: (
d
,
e
) = 500
µ
m; (
f
–
j
) = 100
µ
m; (
k
,
l
,
n
–
r
) = 20
µ
m;
(m) = 50 µm.
4. Discussion
Based on the morphological study and phylogenetic analyses, four new species and
seven new records of Rhytidhysteron are introduced in this paper. Rhytidhysteron bannaense
sp. nov., R. coffeae sp. nov., R. mengziense sp. nov., and R. yunnanense sp. nov. are proposed
as new to science based on their unique morphological characteristics and moderate to
good statistical support. Seven collections of R. bruguierae,R. camporesii,R. hongheense,
R. magnoliae,R. neorufulum,R. tectonae, and R. thailandicum are identified as new records
because of their identical morphological characteristics with the type species of the same
species and high statistical support.
In this study, we found that the pseudoparaphyses of all 11 species are branched
and septate. Interestingly, the epithecia of ten species all turn purple in 10% KOH, but
the purple color of the epithecium fades and becomes hyaline in a short period of time
(5–30 s). On the contrary, the epithecium of R. yunnanense sp. nov. (HKAS 122696) turns
hyaline in 10% KOH, and the appendages become dark. In addition, R. yunnanense is
unique in Rhytidhysteron because it has both parallel and perpendicular striae, relative
to the longitudinal slit. This is also the first discovery of parallel striae in this genus,
while other species of Rhytidhysteron have perpendicular striae or are non-striated (Table 3).
The presence or absence of striae on the margin of ascomata is one of the important
characteristics of this genus and is used to identify different species [16].
J. Fungi 2023,9, 148 18 of 31
The results of the phylogenetic tree generated in this study are consistent with those
reported by Ren et al. [
18
], and R. erioi Ekanayaka & K.D. Hyde is grouped as a sister to R.
bruguierae. Therefore, to find out the correct taxonomic placement of R. erioi, it is necessary
to recollect more samples and confirm their placement. Boehm et al. [
13
] suggested that R.
opuntiae (J.G. Br.) M.E. Barr should be removed from Rhytidhysteron based on morphological
and molecular data. Subsequently, Almeida et al. [
15
] suggested that R. opuntiae should
be accommodated by a new genus in future studies. The main reason is that R. opuntiae
grouped with Hysterodifractum partisporum D.A.C. Almeida, Gusmão & A.N. Mill. [
15
,
56
],
but morpho-molecular differences exist between Hysterodifractum D.A.C. Almeida, Gusmão
& A.N. Mill. and R. opuntiae [
12
]. This study agrees with Boehm et al. [
13
] and Almeida
et al. [
15
] in that R. opuntiae should be included in a new genus, due to the fact that morpho-
molecular data of R. opuntiae are different from those of Rhytidhysteron and Hysterodifractum.
Therefore, more studies on R. opuntiae are needed.
Rhytidhysteron neorufulum and R. rufulum are the most common and most reported
species in this genus [
51
]. Rhytidhysteron rufulum is considered a complex species based on
studies on molecular and chemical data [
16
,
22
,
27
,
51
,
57
]. Thambugala et al. [
16
] indicated
that some fungal specimens were incorrectly classified as R. rufulum, which needs to be
reviewed exhaustively, as they might represent new species [
51
]. This action is meaningful,
and can clarify the taxonomic placement of unclear species and enrich the diversity of
Rhytidhysteron. Unfortunately, in this study, no strains with very similar morpho-molecular
data to R. rufulum were found.
In previous studies, six species were reported in China. These are R. camporesii [
49
],
R. hongheense [
25
], R. magnoliae [
6
], R. rufulum [
15
], R. thailandicum [
6
], and R. xiaokon-
gense [
18
]. This study provides seven additional species from China—R. bannaense sp. nov.,
R. bruguierae,R. coffeae sp. nov., R. mengziense sp. nov., R. neorufulum,R. tectonae, and R. yun-
nanense sp. nov. With these additions, the number of species of Rhytidhysteron in China
increases from six to thirteen. At the same time, the four new species added in this study
expand the species of Rhytidhysteron from thirty-three to thirty-seven, and the known hosts
for Rhytidhysteron expand from 52 to 62 records. However, only 22 species of Rhytidhysteron
have sequence data (including this study), so more research needs to be carried out, and
more samples need to be collected, isolated, and sequenced. This study also summarizes
the morphological characteristics, hosts, and countries of the species of this genus for the
first time (Table 3), which provides references for future research on Rhytidhysteron.
J. Fungi 2023,9, 148 19 of 31
Table 3.
Morphological characteristics, hosts, and location information of species of Rhytidhysterons. Host and location information of taxa are from the following
references and Farr and Rossman [26].
Species Ascomata/
Conidiomata
Exciples/Conidio-
mata Wall
Hamathecium/
Paraphysoids
Asci/
Conidiogenous Cells
Ascospores/
Conidia Sequences Hosts Countries References
Holomorph
R. hysterinum
Sexual: ascomata
1000–3000 µm long ×
500 µm wide ×
500–1000 µm high,
smooth-striated,
erumpent, solitary or
aggregated, sessile, deep
longitudinal slit
extending the entire
length of the ascoma and
with irregularly spaced,
pseudoepithecium,
orange or black when
fresh and when dry
Exciples tightly
compact, hyaline
to light brown,
becoming red in
Melzer’s reagent,
cells toward the
interior less
heavily pigmented
Paraphyses exceeding the
ascus by ca. 25 µm,
branching dichotomously
just below the tip, tip cells
globose to clavate,
disintegrating and
embedded in an
amorphous substance to
form the
pseudoepithecium,
becoming blue-green in
Melzer’s reagent
Asci 185–220 µm×
15–17 µm, 4–8-spored,
cylindrical, pedicellate
Ascospores 21–32 µm×
8–12 µm, 1-septate,
septum median, fusiform
with rounded to acute
ends, slightly constricted
at the septum, brown and
translucent to nearly black,
and opaque with septum
obscured
ITS, LSU,
TEF
Buxus spp.,
Diospyros
sp., Ilex sp.,
Prosopis sp.
Australia,
France,
India, USA
[13,21]
Asexual
(Aposphaeria-like):
pycnidia produced
abundantly in the
aerialmycelium and on
the surface of the agar
on both MEA and PDA,
non-stromatic, globose
with a short papilla,
250–330 µm high ×
220–250 µm wide, black
— —
Phialides forming in a
single layer over the
entire inner surface of
the pycnidial wall,
hyaline, ampulliform to
cylindrical, 7–8 µm long,
×2µm wide basally,
and 1.5 µm wide at the
opening
Conidia globose, 2–3.5 µm
diam., smooth, held in
hyaline slime at the
pycnidial opening
Asexual (Diplodia-like):
pycnidia form on MEA
within one month,
immersed,
non-stromatic,
subglobose, 350 µm
high ×400 µm wide,
non-papillate, black,
smooth
Pycnidial wall
25–35 µm wide,
consisting of
pseudoparenchy-
matous cells,
5–7 µm×3–4 µm,
thin-walled,
brown
Paraphyses arisingfrom
among conidiogenous
cells, up to 50 µm long ×
3µm wide, septate,
unbranched, with rounded
ends, hyaline
Conidiogenous cells
forming in a single layer
over the entire inner
surface of the pycnidial
wall, barely
distinguishable from
cells of the wall;
consisting of a basal cell
6–7 µm across, and a
5–10
µ
m long elongation
Conidia arising
holoblastically from the tip
of the conidiogenous cell;
at first hyaline and
unicellular, becoming dark
brown, opaque, minutely
punctate, and 1-septate
with a pore in the middle
of the septum, oblong,
with a truncate,
non-cicatrized base,
22–26 µm×9–11 µm
J. Fungi 2023,9, 148 20 of 31
Table 3. Cont.
Species Ascomata/
Conidiomata
Exciples/Conidio-
mata Wall
Hamathecium/
Paraphysoids
Asci/
Conidiogenous Cells
Ascospores/
Conidia Sequences Hosts Countries References
R. rufulum
Sexual: ascomata
900–2350 µm long ×
1134–1450 µm wide ×
461–820 µm high,
superficial,
rough-striated, black
or red at the center
Exciples
75–228 µm wide,
two layers. Outer
layer dark brown
to black, cells of
textura angularis or
textura globosa.
Inner layer
hyaline cells of
textura angularis to
textura prismatica
Septate, branched
pseudoparaphyses
Asci 150–250 µm×
11–16 µm, 8-spored
Ascospores 28–36 µm×
9–13 µm, 1–3-septate,
reddish brown to brown
when mature
ITS, LSU,
SSU, TEF
Abrus precatorius,
Abrus pulchellus,
Acacia
auriculiformis,
Acacia
cochliacantha,
Acacia farnesiana,
Acacia macracantha,
Acacia spp.,
Adhatoda vasica,
Albizia lebbeck,
Albizia
odoratissima,
Alphitonia excelsa,
Annona muricata,
Bignonia unguis,
Bougainvillea
glabra,
Capparis sepiaria,
Casuarina sp.,
Celtis pallida,
Citrus aurantifolia,
Citrus aurantium,
Codiaeum
variegatum,
Euterpe oleracea,
Grevillea robusta,
Guaiacum officinale,
Helietta parvifolia,
Juniperus lucayana,
Nothofagus sp.,
Pisonia aculeata,
Pithecellobium
dulce,
Prosopis juliflora,
Torresia cearensis
Argentina,
Australia,
Brazil,
China,
Cook
Islands,
Costa Rica,
Cuba,
Dominica,
France,
Ghana,
India,
Jamaica,
Japan,
Kenya,
Malaysia,
Mexico,
Micronesia,
New
Guinea,
New
Zealand,
Philippine,
Puerto, Rico,
Spain,
Tanzania,
Thailand,
Tonga,
United
States,
Venezuela,
West Indies
[15,16,21,
55,58–70]
Asexual
(Aposphaeria-like):
pycnidia form
abundantly in aerial
mycelium, often
associated with small
tufts of red-brown
hyphae, non-stromatic,
globose to oblong,
100–150 µm high ×
70–150 µm wide,
non-papillate, black
— —
Phialides forming in a
single layer over the
entire inner surface of
the pycnidial wall,
hyaline, ampulliform to
cylindrical, 4.5–9.0 µm
long ×1.5–3.0 µm diam.
basally, and 1.5
µ
m wide
at the opening
Conidia globose to elliptic,
2–3 µm diam. or 3.0 µm×
2.5 µm, smooth, held in a
drop of hyaline slime at
the pycnidial opening
Asexual (Diplodia-like):
pycnidia abundant to
rare, immersed,
non-stromatic,
subglobose, 460 µm
high ×400 µm wide,
papillate or
non-papillate, or
seated on the surface
of the agar, pyriform,
270 µm high ×
130–180 µm wide and
with a papilla
130–180 µm long ×
70 µm wide, black,
smooth
Pycnidial wall
45 µm wide,
consisting of
pseudoparenchy-
matous cells
8–20 µm×
8–10 µm,
thin-walled,
brown
Paraphyses arising from
among conidiogenous
cells, up to 50 µm long ×
3µm wide, septate,
unbranched, with rounded
ends, hyaline
Conidiogenous cells
forming in a single layer
over the entire inner
surface of the pycnidial
wall, consisting of a
hyaline, globose cell
4–5 µm in diam. basally,
and with a 5 µm long
elongation
Conidia arising
holoblastically from the tip
of the elongation of the
conidiogenous cell, at first
hyaline and unicellular,
becoming dark brown to
opaque and 1-septate with
a pore in the middle of the
septum following
discharge, oblong with a
truncate, non-cicatrized
base, 19.5–23.5 (–29.5) µm
×(6.5–) 8–10 (–12) µm,
smooth
J. Fungi 2023,9, 148 21 of 31
Table 3. Cont.
Species Ascomata/
Conidiomata
Exciples/Conidio-
mata Wall
Hamathecium/
Paraphysoids
Asci/
Conidiogenous Cells
Ascospores/
Conidia Sequences Hosts Countries References
R. thailandicum
Sexual: ascomata
700–1200 µm long×
530–750 µm wide ×
360–640 µm high,
semi-immersed to
superficial, rough
without striations
Exciples
72–130 µm wide,
brown to dark
brown,
thick-walled cells
of textura angularis,
becoming hyaline
towards the inner
layers and base
Septate, branched
pseudoparaphyses,
forming a yellow
epithecium above asci
when mounted in water
Asci 135–160 µm×
10.5–15 µm,
(3–)6–8-spored
Ascospores 20–31 µm×
7.5–12 µm, (1–)3-septate,
yellowish to brown when
mature
ITS, LSU,
SSU, TEF
Acacia sp.,
Aquilaria
sinensis,
Morus
australis
China,
Mexico,
Thailand
[6,16,55],
this study
Asexual
(Aposphaeria-like):
conidiomata 70–108 µm
long ×63–110 µm wide,
superficial on PDA,
globose, black,
appearing in a mycelium
mass
Conidiomata wall
thin, arranged in
textura angularis
—
Conidiophores reduced
to conidiogenous cells.
Conidiogenous cells
5.9 µm×3µm,
cylindrical to
subcylindrical, truncate
apex, short, smooth,
hyaline
Conidia 2.9 µm×2.2 µm,
globose to subglobose,
hyaline, smooth
Asexual morph
R.
xiaokongense
Asexual (Diplodia-like):
conidiomata 448–464
µ
m
long ×324–422 µm
wide, solitary, scattered,
semi-immersed in the
host, black, unilocular,
subglobose to
ampulliform. Ostioles
178–227 µm long ×
166–234 µm wide,
central, short papillate
Conidiomata wall
30–40 µm thick,
4–6 layers, reddish
brown to dark
brown cells of
textura angularis
—
Conidiogenous cells
5–8 µm×3–6 µm,
subglobose or
ellipsoidal, hyaline,
smooth, discrete,
producing a single
conidium at the apex
Conidia 20–25 µm×
8–10 µm, 1-septate and
brown to dark brown at
maturity, oblong to
ellipsoidal, straight to
slightly curved, with
granular appearance
ITS, LSU,
SSU, TEF Prunus sp. China [18]
Sexual morph
R. bannaense
1350 µm long ×750 µm
wide ×670 µm high,
rough, solitary to
aggregated,
semi-immersed to
superficial,
perpendicular striae,
green at the center
40–150 µm wide,
composed of dark
brown,
thick-walled cells
of textura angularis,
outer layer brown
to dark brown,
inner layer pale
brown to hyaline
Septate, branched, cellular
pseudoparaphyses,
forming an orange
epithecium above asci
when mounted in water
166 µm×14 µm,
8-spored, J- apical ring
25 µm×11.5 µm,
3-septate, brown to dark
brown when mature
ITS, LSU,
SSU, TEF
Buddleja
officinalis China This study
J. Fungi 2023,9, 148 22 of 31
Table 3. Cont.
Species Ascomata/
Conidiomata
Exciples/Conidio-
mata Wall
Hamathecium/
Paraphysoids
Asci/
Conidiogenous Cells
Ascospores/
Conidia Sequences Hosts Countries References
R. beccarianum
1000
µ
m long, erumpent,
solitary, dark brown — — 60 µm×6µm, 8-spored
12–15 µm×5–6 µm,
3-septate, constriction at
the septa
— — Sri Lanka [71]
R. brasiliense Erumpent to nearly
superficial —Septate, branched
pseudoparaphyses
230–250
µ
m
×
20–30
µ
m,
8-spored
40–45 µm×15–20 µm,
3-septate —On rotten
branches
Brazil,
Thailand [16,19]
R. bruguierae
400–950 µm long ×
548–570 µm wide ×
410–520 µm high,
superficial, striated
148–162 µm wide,
dark brown to
black,
thick-walled cells
of textura angularis
Septate, branched
pseudoparaphyses,
forming a red epithecium
above asci when mounted
in water
128–148
µ
m
×
10–14
µ
m,
6–8-spored, J- apical ring
14–26 µm×6.2–9 µm,
1–3-septate,
yellowish-brown to
reddish brown when
mature
ITS, LSU,
SSU, TEF
Alnus
nepalensis,
Bruguiera
sp., Chro-
molaena
odorata
China,
Thailand
[47,48], this
study
R. camporesii
800–1100 µm long ×
500–650 µm high,
erumpent, slightly
dentate
Ectal excipulum
65–95 µm wide,
blackish cells of
textura globulosa to
angularis.
Medullary
excipulum
19–22 µm wide,
thin-walled,
hyaline to brown
cells of textura
porrecta
Paraphyses septate,
branched at the base,
forming an orange-red
epithecium above asci
when mounted in water
165–175
µ
m
×
13–15
µ
m,
8-spored, J- apical ring
25–28 µm×9–11 µm,
3-septate, dark brown
when mature
ITS, LSU,
SSU, TEF
Cotoneaster
franchetii China [49], this
study
R.
chromolaenae
500–1000 µm long ×
250–500 µm high,
superficial, not
perpendicular striae,
scattered, dark brown to
black with dark orange
at the center
45–60(–110) µm
wide, hyaline or
pale brown to
brown cells
arranged in textura
globulosa to textura
angularis
Septate, branched
pseudoparaphyses
120–140
µ
m
×
10–15
µ
m,
8-spored
18–22 µm×7–9 µm,
3-septate, pale brown to
brown when mature
ITS, LSU,
SSU, TEF
Chromolaena
odorata Thailand [48]
R. coffeae
1520
µ
m long
×
1120
µ
m
wide ×450 µm high,
rough, solitary to
aggregated, mostly
solitary, superficial,
perpendicular striae,
reddish brown at the
center
70–160 µm wide,
composed of dark
brown,
thick-walled cells
of textura angularis,
outer layer brown
to dark brown,
inner layer pale
brown to hyaline
Septate, branched, cellular
pseudoparaphyses,
forming a red to purple
epithecium above asci
when mounted in water
179.5 µm×13 µm,
8-spored, J- apical ring
26 µm×10 µm, 3-septate,
reddish brown to brown
when mature
ITS, LSU,
SSU, TEF Coffea sp. China This study
J. Fungi 2023,9, 148 23 of 31
Table 3. Cont.
Species Ascomata/
Conidiomata
Exciples/Conidio-
mata Wall
Hamathecium/
Paraphysoids
Asci/
Conidiogenous Cells
Ascospores/
Conidia Sequences Hosts Countries References
R. columbiense
1500–3000 µm long ×
1200–1800 µm wide ×
600–700 µm high,
superficial, striated,
yellowish green on the
margins
60–90 µm wide,
dark brown to
black,
thick-walled cells
of textura angularis
Septate, branched
pseudoparaphyses
175–190
µ
m
×
14–18
µ
m,
6–8-spored
38–52 µm×13–18 µm,
(1–)3-septate, reddish
brown when mature
—
Unidentified
woody Colombia [27]
R. cozumelense
2500–3500 µm long ×
1100–1500 µm wide ×
800–1900 µm high,
erumpent, solitary,
smooth to slightly
striated, dark at the
center
Two layers, the
first carbonaceous,
45–100 µm wide
thick cells of
textura prismatica.
The second cells
hyaline,
thin-walled
Septate pseudoparaphyses
182–191
µ
m
×
12–13
µ
m,
8-spored
26–29 µm×9–11 µm,
3-septate, dark brown
when mature
ITS, LSU,
TEF
Tabebuia
rosea Mexico [24]
R. discolor 1000–2000 µm long,
cracking after maturity Carbonaceous Paraphyses filiform
180–220
µ
m
×
12–15
µ
m,
8-spored
28–30 µm×10–12 µm,
3-septate, elongated
ellipse, guttules
—Celtis tala Argentina [72]
R. erioi
600–1200 µm long ×
270–360 µm high,
superficial or slightly
erumpent, dentate
Ectal excipulum
55–75 µm wide,
thin-walled, dark
brown cells of
textura angularis to
textura globulosa.
Medullary
excipulum
14–20 µm wide,
hyaline cells of
textura porrecta
Paraphyses septate,
slightly branched at the
base
140–200 µm×9–16 µm,
8-spored, J- apical ring
22–28 µm×9–11 µm,
3-septate, dark brown
when mature
ITS, LSU,
TEF
Unidentified
wood Thailand [49]
R. esperanzae
2000–4500 µm long ×
1200–3000 µm wide ×
1000–2400 µm high,
superficial, solitary,
rarely gregarious,
margin greyish green,
striated, dark green to
black at the center
Exciple in two
layers, the first
carbonaceous,
60–220 µm wide
cells of textura
globulosa-angularis.
The second
slightly
pigmented to
hyaline,
thin-walled
Septate pseudoparaphyses
265–270
µ
m
×
19–20
µ
m,
8-spored
45–47 µm×17–19 µm,
3-septate, reddish brown
to brown when mature
ITS, LSU,
TEF
Oreomunnea
mexicana Mexico [24]
J. Fungi 2023,9, 148 24 of 31
Table 3. Cont.
Species Ascomata/
Conidiomata
Exciples/Conidio-
mata Wall
Hamathecium/
Paraphysoids
Asci/
Conidiogenous Cells
Ascospores/
Conidia Sequences Hosts Countries References
R.
guaraniticum
1000–4000 µm long ×
700–100 µm wide,
superficial
— — 200 µm×12–14 µm30–31 µm×10–12 µm,
3-septate —
On bark,
rotten
branches
Jawa,
Paraguay [73]
R. hongheense
1200–2000 µm long ×
600–1000 µm wide ×
350–500 µm high,
slightly erumpent,
slightly dentate
Ectal excipulum
70–100 µm wide,
thick-walled, with
black cells of
textura globulosa to
textura angularis.
Medullary
excipulum is
composed of
narrow, long,
thin-walled,
hyaline to brown
cells of textura
angularis
Septate, branched
pseudoparaphyses,
forming a red epithecium
above asci when mounted
in water
140–180
µ
m
×
12–16
µ
m,
8-spored
20–33 µm×9–13 µm,
3-septate, dark brown
when mature, rarely
muriform, with one
longitudinal septum
ITS, LSU,
SSU, TEF,
RPB2
Dodonaea
sp.,
Phyllanthus
emblica
China [25], this
study
R. indicum
1800–3000 µm long,
black, carbonaceous,
scattered, erumpent,
uniloculate, discoid to
elongated
—Paraphyses filiform,
septate, clavate expansion
200–220
µ
m
×
18–20
µ
m,
8-spored
30–32 µm×12–14 µm,
dark brown, 3-septate, end
cells slightly tapering,
constricted at septa,
uniseriate
—Scutia indica India [74]
R. magnoliae
1200–2300 µm long ×
540–600 µm wide ×
430–550 µm high µm
semi-immersed to
superficial, striated, dark
brown at the center
80–100 µm wide,
two layers. Outer
layer black to dark
brown,
thick-walled cells
of textura angularis.
Inner layer
hyaline,
thin-walled cells
of textura angularis
to textura
prismatica
Septate, branched
pseudoparaphyses,
forming an orange
epithecium above asci
when mounted in water
160–200 µm×14 µm,
8-spored
25–32 µm×8–12 µm,
1–3-septate, pale brown to
dark brown when mature
ITS, LSU,
SSU, TEF
Hevea
brasilien-
sis,Magnolia
grandiflora
China [6], this
study
J. Fungi 2023,9, 148 25 of 31
Table 3. Cont.
Species Ascomata/
Conidiomata
Exciples/Conidio-
mata Wall
Hamathecium/
Paraphysoids
Asci/
Conidiogenous Cells
Ascospores/
Conidia Sequences Hosts Countries References
R. mangrovei
930–1980 µm long ×
780–910 µm wide ×
500–520 µm high,
crowded to aggregate,
semi-immersed to
superficial,
rough-striated
65–90 µm wide,
dark brown to
black, thin-walled
cells of textura
angularis
Septate, unbranched
pseudoparaphyses
110–150 µm×9.4–10
µm, (2–6–) 8-spored
21–28 µm×7.5–8.5 µm,
1–3-septate, reddish brown
when mature
ITS, LSU,
TEF
Mangrove
sp. Thailand [12]
R. mengziense
1400 µm long ×910 µm
wide ×640 µm high,
smooth, solitary to
aggregated, mostly
solitary, semi-immersed
to superficial,
perpendicular striae,
reddish brown at the
center
60–135 µm wide,
composed of outer
layer brown to
black,
thick-walled cells
of textura angularis
and inner layer
light brown,
thin-walled cells
of textura
prismatica
Septate, branched, cellular
pseudoparaphyses,
forming a reddish brown
to brown epithecium
above asci when mounted
in water
164.5 µm×13 µm,
8-spored, J- apical ring
27 µm×12 µm, 3-septate,
reddish brown to brown
when mature
ITS, LSU,
SSU, TEF
Crataegus
scabrifolia China This study
R. mesophilum
2500–4000 µm long ×
1000–1500 µm wide ×
1400–1700 µm high,
superficial or erumpent,
gregarious, rarely
solitary, margin
yellowish green, striated,
orange at the center
Two layers, the
first carbonaceous,
62.5–75 µm wide,
green yellowish
cells of textura
prismatica. The
second hyaline,
thin-walled
Aseptate, branched
pseudoparaphyses
267–282 µm×15.5–16
µm, 8-spored
40–44 µm×12–14 µm,
3-septate, light brown
when mature
ITS, LSU,
TEF
On decayed
wood Mexico [24]
R. mexicanum
2000–4000 µm long ×
1500–2500 µm wide ×
1500
µ
m high, superficial
or erumpent, gregarious,
rarely solitary, striated
Two layers, the
first carbonaceous,
104.5–114 µm
wide in the
medium cells of
textura globulosa to
textura angularis,
thick-walled. The
second composed
of cells of textura
prismatica, hyaline,
thin-walled
Aseptate, bifurcated to
branched
pseudoparaphyses
285–297
µ
m
×
16–17
µ
m,
8-spored
34–40 µm×10–12 µm,
3-septate, reddish brown
when mature
ITS, LSU — Mexico [51]
J. Fungi 2023,9, 148 26 of 31
Table 3. Cont.
Species Ascomata/
Conidiomata
Exciples/Conidio-
mata Wall
Hamathecium/
Paraphysoids
Asci/
Conidiogenous Cells
Ascospores/
Conidia Sequences Hosts Countries References
R.
neohysterinum
1500–2500 µm long ×
700–2200 µm wide ×
700–1100 µm high,
superficial, solitary,
rarely gregarious,
striated, orange at the
center
52–68 µm wide,
dark brown to
black,
thick-walled cells
of textura
prismatica
Septate pseudoparaphyses
160–185
µ
m
×
12–13
µ
m,
8-spored
24.8–29 µm×8.8–10 µm,
1-septate, brown when
mature
—Acacia sp. Mexico [55]
R. neorufulum
835–2100 µm long ×
350–1320 µm wide ×
430–1000 µm high,
superficial, elliptic or
irregular, without
striations, black or
yellow at the center
64–160 µm wide,
dark brown to
black,
thick-walled cells
of textura angularis
Septate, branched
pseudoparaphyses,
forming a yellow
epithecium above asci
when mounted in water
185–260 µm×9.5–18
µm, 8-spored
27–44 µm×6.5–17 µm,
1–3-septate, reddish brown
to brown when mature
ITS, LSU,
SSU, TEF
Bursera sp.,
Elaeagnus
sarmentosa,
Hevea
brasiliensis,
Tectona
grandis
China,
Mexico,
Thailand
[16,18,55],
this study
R. opuntiae 640–1700 µm long — — 85–160 µm×12.5–16
µm, 3–8-spored
17–33 µm×13 µm,
3–5-septate LSU, TEF Opuntia
fulgida USA [52]
R. prosopidis
Superficial, very hard
when dry, elliptical or
triangular, black, with
very obtuse, thick,
yellowish green disc
— — —
3-septate, unineriate,
oblong, sometimes slightly
curved
—Prosopis
juliflora USA [75]
R. quercinum
1000–3000 µm in diam.,
leathery apothecia,
scattered, superficial,
erumpent, pedicellate
(short pedicel)
Excipulum black
with low seated,
reddish
—
Asci cylindrical, slender,
stalked hyaline with
inconspicuous wall
19.0–24.7 µm×
7.6–11.4 µm, 1–3-septate
more commonly 3
—Quercus sp. India [76]
R. tectonae
550–3365 µm long ×
325–728 µm wide ×
370−835 µm high,
semi-immersed to
superficial, smooth
without striation, yellow
at the center
80–135 µm wide,
two layers. Outer
layer black to dark
reddish,
thick-walled cells
of textura angularis.
Inner layer
hyaline,
thin-walled cells
of textura angularis
Septate, branched
pseudoparaphyses,
forming an orange
epithecium above asci
when mounted in water
150–200
µ
m
×
10–15
µ
m,
8-spored
19–31 µm×8–13 µm,
1–3-septate, pale brown to
dark brown when mature
ITS, LSU,
SSU, TEF
Betula sp.,
Fabaceae
sp.,
Magnolia
delavayi,
Tectona
grandis
China,
Thailand
[18,53], this
study
J. Fungi 2023,9, 148 27 of 31
Table 3. Cont.
Species Ascomata/
Conidiomata
Exciples/Conidio-
mata Wall
Hamathecium/
Paraphysoids
Asci/
Conidiogenous Cells
Ascospores/
Conidia Sequences Hosts Countries References
R. viride
1000–1500 µm long ×
500–600 µm wide,
erumpent
— Filiform, hyaline 200–250 µm×10–12 µm20–30 µm×7–9 µm,
3-septate —
On bark,
associated
with lichens
Brazil [77]
R. yunnanense
2510 µm×625 µm×
455 µm, solitary to
aggregated, mostly
aggregated,
semi-immersed, each
hysterothecia has two
parallel striae parallel to
the longitudinal slit and
slight perpendicular
striae, longitudinal slit,
no opening
60–180 µm wide,
composed of dark
brown,
thick-walled cells
of textura globulosa,
outer layer brown
to dark brown,
inner layer pale
brown to hyaline
Septate, branched, cellular
pseudoparaphyses,
forming a yellow
epithecium above asci
when mounted in water
230 µm×14 µm,
8-spored, J- apical ring
32.5 µm×13 µm,
3-septate, reddish brown
to brown when mature
ITS, LSU,
SSU, TEF
Rhus
chinensis China This study
Remarks: the symbol “—” denotes no information available.
J. Fungi 2023,9, 148 28 of 31
Supplementary Materials:
The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/jof9020148/s1, Supplementary Notes S1–S7: Description of seven
new records; Supplementary Figures S1–S7: Photo plates of seven new records.
Author Contributions:
Conceptualization, S.C.K. and S.T.; Data curation, T.-Y.D. and L.L.; Formal
analysis, T.-Y.D.; Funding acquisition, S.C.K., D.-Q.D., N.S., A.M.E., S.A.-R. and S.T.; Methodology,
T.-Y.D. and L.L.; Software, T.-Y.D.; Supervision, S.C.K. and S.T.; Writing—original draft, T.-Y.D.;
Writing—review & editing, S.C.K., D.-Q.D., A.M., L.L., N.S., S.L.S., A.M.E., S.A.-R. and S.T. All
authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by the National Natural Science Foundation of China, grant num-
ber NSFC 31760013, 31950410558, 32260004, High-Level Talent Recruitment Plan of Yunnan Provinces
(“Young Talents” Program), and the Researchers Supporting Project, number (RSP2023R120), King
Saud University, Riyadh, Saudi Arabia. The authors extend their appreciation to Chiang Mai Univer-
sity for financial support.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
We are grateful to Gui-Qing Zhang for her help. Tian-Ye Du thanks Mae Fah
Luang University for the award of fee-less scholarship. Nakarin Suwannarach thanks Chiang Mai
University, Thailand for financial support. Shaun Pennycook is thanked for his assistance in selecting
a species epithet for the new species.
Conflicts of Interest: The authors declare no conflict of interest.
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