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Pseudodidymellaceae fam. nov.: Phylogenetic affiliations of
mycopappus-like genera in Dothideomycetes
A. Hashimoto
1
,
2
, M. Matsumura
1
,
3
, K. Hirayama
4
, R. Fujimoto
1
, and K. Tanaka
1
,
3*
1
Faculty of Agriculture and Life Sciences, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori, 036-8561, Japan;
2
Research Fellow of the Japan Society for the
Promotion of Science, 5-3-1 Kojimachi, Chiyoda-ku, Tokyo, 102-0083, Japan;
3
The United Graduate School of Agricultural Sciences, Iwate University, 18–8 Ueda 3
chome, Morioka, 020-8550, Japan;
4
Apple Experiment Station, Aomori Prefectural Agriculture and Forestry Research Centre, 24 Fukutami, Botandaira, Kuroishi,
Aomori, 036-0332, Japan
*Correspondence: K. Tanaka,
k-tanaka@hirosaki-u.ac.jp
Abstract: The familial placement of four genera, Mycodidymella,Petrakia,Pseudodidymella, and Xenostigmina, was taxonomically revised based on morphological
observations and phylogenetic analyses of nuclear rDNA SSU, LSU, tef1, and rpb2 sequences. ITS sequences were also provided as barcode markers. A total of 130
sequences were newly obtained from 28 isolates which are phylogenetically related to Melanommataceae (Pleosporales,Dothideomycetes) and its relatives. Phylo-
genetic analyses and morphological observation of sexual and asexual morphs led to the conclusion that Melanommataceae should be restricted to its type genus
Melanomma, which is characterised by ascomata composed of a well-developed, carbonaceous peridium, and an aposphaeria-like coelomycetous asexual morph.
Although Mycodidymella,Petrakia,Pseudodidymella, and Xenostigmina are phylogenetically related to Melanommataceae, these genera are characterised by epi-
phyllous, lenticular ascomata with well-developed basal stroma in their sexual morphs, and mycopappus-like propagules in their asexual morphs, which are clearly
different from those of Melanomma.Pseudodidymellaceae is proposed to accommodate these four genera. Although Mycodidymella and Xenostigmina have been
considered synonyms of Petrakia based on sexual morphology, we show that they are distinct genera. Based on morphological observations, these genera in
Pseudodidymellaceae are easily distinguished by their synasexual morphs: sigmoid, multi-septate, thin-walled, hyaline conidia (Mycodidymella); globose to ovoid,
dictyosporus, thick-walled, brown conidia with cellular appendages (Petrakia); and clavate with a short rostrum, dictyosporus, thick-walled, brown conidia (Xenostigmina).
A synasexual morph of Pseudodidymella was not observed. Although Alpinaria was treated as member of Melanommataceae in a previous study, it has hyaline cells at
the base of ascomata and pseudopycnidial, confluent conidiomata which is atypical features in Melanommataceae, and is treated as incertae sedis.
Key words: Foliar pathogen, Synasexual morph, Systematics.
Taxonomic novelties: New family: Pseudodidymellaceae A. Hashim. & Kaz. Tanaka; New species: Melanomma japonicum A. Hashim. & Kaz. Tanaka,
Pseudodidymella minima A. Hashim. & Kaz. Tanaka; New combination: Xenostigmina aceris (Dearn. & Barthol.) A. Hashim. & Kaz. Tanaka.
Available online 13 July 2017; http://dx.doi.org/10.1016/j.simyco.2017.07.002.
INTRODUCTION
The family Melanommataceae (Pleosporales) was proposed for
its type genus, Melanomma (Winter 1887). Currently, more than
20 genera with diverse ecological and morphological features are
recognised in this family (Tian et al. 2015). Petrakia and Xeno-
stigmina have epiphyllous, lenticular ascomata with well-
developed basal stroma, mycopappus-like propagules, and pet-
rakia- or stigmina-like synasexual morphs, and were also
accepted in Melanommataceae (Funk 1986, Funk & Dorworth
1988, Crous 1998, Crous et al. 2009, Butin et al. 2013, Tian
et al. 2015). Subsequently, two additional genera, Mycodidy-
mella and Pseudodidymella, were reported to be phylogenetically
related to this family (Gross et al. 2017), although their morpho-
logical features were clearly different from those of Melanomma,
which is characterised by carbonaceous ascomata, trabecular
pseudoparaphyses, and aposphaeria-like coelomycetous
asexual morphs (Barr 1987, 1990, Lumbsch & Huhndorf 2007,
Kirk et al. 2008, Tian et al. 2015, Jaklitsch & Voglmayr 2017).
The genus Petrakia was originally characterised by spor-
odochial conidiomata and muriform, brown conidia with cellular,
hyaline appendages (Sydow & Sydow 1913, Butin et al. 2013).
Recently, the complete life cycle of Pe. echinata, which is the
type species and a known causal agent of leaf blotch disease of
Acer spp., was revealed (Butin et al. 2013). Subsequently,
phylogenetic analysis using large subunit nrDNA sequences
indicated that this genus is related to Melanommataceae or
Pleomassariaceae (Dothideomycetes;Butin et al. 2013).
Xenostigmina zilleri, the type species of the genus, is a known
pathogen that causes brown spot disease in Acer macrophyllum
in Canada (Funk 1986). This species was originally described as
Cercosporella aceris (Dearness 1917). Redhead & White (1985)
introduced Mycopappus, and transferred two species to this
genus, i.e. C. aceris and C. alni. The type species of Myco-
pappus,Mycop. alni, was suggested to be a member of Scle-
rotiniaceae (Helotiales,Leotiomycetes) based on its sclerotial
morph and phylogenetic analyses using ITS sequences
(Takahashi et al. 2006). Mycopappus aceris was excluded from
the genus, because the synasexual morph of this species is the
dothideomycetous taxon X. zilleri (Funk & Dorworth 1988, Crous
1998, Wei et al. 1998, Crous et al. 2009). According to phylo-
genetic analysis, this genus was accepted as Melanommataceae
(Phookamsak et al. 2014, Tian et al. 2015).
The genera Mycodidymella and Pseudodidymella are also
members of Melanommataceae that produce mycopappus-like
propagules in their asexual morphs (Wei et al. 1997, 1998,
Gross et al. 2017). The genus Mycodidymella, which is based
on the type species Mycod. aesculi, is known as a pathogen of
Peer review under responsibility of Westerdijk Fungal Biodiversity Institute.
© 2017 Westerdijk Fungal Biodiversity Institute. Production and hosting by ELSEVIER B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
available online at www.studiesinmycology.org STUDIES IN MYCOLOGY 87: 187–206 (2017).
187
Studies in Mycology
concentric ring spot disease in Aesculus turbinata (Wei et al.
1998). The life cycle of Mycod.aesculi is similar to those of
Petrakia and Xenostigmina, except it has sigmoid and hyaline
conidia in its synasexual morph. Although the synasexual morph
of Petrakia seems to be clearly different from that of Mycodi-
dymella and Xenostigmina, the latter two genera were synony-
mised with an older name, Petrakia (Jaklitsch & Voglmayr 2017).
The monotypic genus Pseudodidymella was established for
Pseudod. fagi (Wei et al. 1997). The species was found to be
associated with brown leaf spots of Fagus crenata in Japan, and
was originally characterised by lenticular ascomata with a well-
developed basal stroma and a pycnopleiospora-like asexual
morph, which is characterised by sporodochial conidiomata and
conidia with appendages (Wei et al. 1997). Mycodidymella is
morphologically similar to this genus, but can be distinguished by
its pycnopleiospora-like asexual morph (Wei et al. 1998). Gross
et al. (2017) discovered Pseudod. fagi on F. sylvatica in
Switzerland and suggested that the pycnopleiospora-like asexual
morph has mycopappus-like propagules rather than individual
conidia. Thus, morphological delimitation of these two genera is
problematic and requires further research. According to a phylo-
genetic study using ITS sequences (Gross et al. 2017), four genera
with mycopappus-like propagules (Mycodidymella,Petrakia,
Pseudodidymella, and Xenostigmina) formed a strongly supported
clade within Melanommataceae sensu lato; however, familial
placement and generic validity of each genus remain unresolved.
During our ongoing studies of ascomycetous fungi in Japan
(Tanaka et al. 2010, 2011, 2015, Hashimoto et al. 2015a, b, 2016,
2017), we collected strains which are morphologically similar or
phylogenetically related to Melanommataceae sensu lato. The
main objectives of the present study were to clarify familial
placement of genera in this family, and establish a taxonomic
framework of Melanommataceae sensu lato based on morpho-
logical observations and molecular phylogenetic analyses of
small subunit nrDNA (18S; SSU), large subunit nrDNA (28S;
LSU), translation elongation factor 1-α(tef1), and DNA-directed
RNA polymerase II second largest subunit (rpb2) sequences.
ITS sequences were also obtained as DNA barcode markers.
MATERIALS AND METHODS
Isolates
All fungal structures were studied in preparations mounted in
distilled water. Morphological characters were observed by differ-
ential interference and phase contrast microscopy (Olympus BX53,
Japan), and images captured with an Olympus digital camera
(DP21, Japan). A total of 28 single-spore isolates were used for
morphological observation and phylogenetic analyses (Tab l e 1 ).
DNA isolation, amplification and phylogenetic
analysis
DNA extraction was carried out with an ISOPLANT II kit (Nippon
Gene, Japan) based on the manufacturer's protocol. Sequences of
SSU, ITS, LSU, and tef1 and rpb2 were amplified by PCR with the
primer pairs NS1/NS4, ITS1/ITS4 (White et al. 1990), LR0R/LR7
(Rehner & Samuels 1994, Vilgalys & Hester 1990), EF1-983F/
EF1-2218R (Rehner & Buckley 2005), and fRPB2-5F/fRPB2-
7cR (Liu et al. 1999), respectively. Amplifications were per-
formed in 25 μL volumes that consisted of 2 μL DNA extract, 2.5 μL
of 10 × TEMPase Buffer I, 10 mM dNTP mix, 1 μL of each 20-pM
primer, 25 mL MgCl
2
, 14.5 μL MilliQ water, and 0.5 μL TEMPase
Hot Start DNA polymerase (Ampliqon, Denmark). PCRs were
carried out on a PC 320 thermo-cycler (ASTEC, Japan) as follows:
95 °C for 15 min; followed by 35 cycles of 1 min at 94 °C, 1 min at
the designated annealing temperature (42.2 °C for SSU, 61.5 °C
for ITS, 46 °C for LSU, 60 °C for tef1, and 58 °C for rpb2), and
1 min at 72 °C; and a final denaturation of 7 min at 72 °C. The PCR
products were directly sequenced at SolGent (South Korea).
Newly generated sequences were deposited in GenBank
(Table 1). Sequences of 73 taxa of Pleosporales and Hysteriales
were also phylogenetically analysed (Table 1). Hysterium puli-
care and Hysterobrevium mori (Hysteriaceae,Hysteriales) were
used as outgroups. All sequences were aligned using the
MUSCLE algorithm as implemented in the program MEGA v. 5
(Tamura et al. 2011). Phylogenetic analyses were conducted
using maximum likelihood (ML) and Bayesian methods. The
optimal substitution models for each dataset were estimated by
Kakusan4 (Tanabe 2011) based on the Akaike information Cri-
terion (AIC; Akaike 1974) for ML analysis and Bayesian infor-
mation Criterion (BIC; Schwarz 1978) for the Bayesian analysis.
The ML analysis was performed using TreeFinder Mar 2011
(Jobb 2011) based on the models selected with the AICc4
parameter (a proportional model among genes and codons):
J2+G for SSU; GTR+G for LSU; F81+G for the tef1 first codon
position, J1ef+G for the tef1 second codon position, and J2+G for
the tef1 third codon position; and J2+G for the rpb2 first codon
position, J1+G for the rpb2 second codon position, and J2+G for
the rpb2 third codon position. Bootstrap percentages (BPs) were
obtained by 1 000 bootstrap replications.
Bayesian analysis was performed with MrBayes v. 3.2.2
(Ronquist et al. 2012) with substitution models for different re-
gions selected with the BIC4 parameter (proportional model
among loci and codons): K80+G for SSU; SYM+G for LSU;
GTR+G for the tef1 first codon position, JC69+G for the tef1
second codon position, and GTR+G for the tef1 third codon
position; and GTR+G for the rpb2 first codon position, GTR+G
for the rpb2 second codon position, and GTR+G for the rpb2 third
codon position. Two simultaneous, independent runs of
Metropolis-coupled Markov chain Monte Carlo (MCMC) were
performed for 2 M generations with trees sampled every 1 000
generations. Convergence of the MCMC runs assessed from the
average standard deviation of split frequencies (<0.01) and
effective sample size scores (all >100) using MrBayes v. 3.2.2
and Tracer v. 1.6 (Rambaut et al. 2014), respectively. The first
25 % of trees were discarded as burn-in, and the remaining trees
were used to calculate 50 % majority rule trees and determine
posterior probabilities (PPs) for individual branches. The align-
ment was submitted to TreeBase under study number S20165.
Morphology
Colony characteristics of cultures grown on 2 % potato dextrose
agar (PDA; Difco, France) were observed after 3 wk incubation at
20 °C in the dark. Colours were noted based on those described
by Rayner (1970).
To induce sexual or asexual fructification in culture, 5 mm
square mycelial agar discs were placed on water agar that
included sterilised natural substrate, such as Aesculus turbinata
HASHIMOTO ET AL.
188
and Fagus crenata leaves and rice straw, and the plates were
incubated at 20 °C for 2 wk in the dark. When the substrate was
colonised, the plates were incubated at 20 °C under blacklight
blue illumination for 2 mo to observe sporulation. Cultures were
deposited in the Westerdijk Fungal Biodiversity Institute (CBS),
the Japan Collection of Microorganisms (JCM), and the Gene-
bank Project of NARO, Japan (MAFF). Specimens were
deposited in the Herbarium of Hirosaki University, Fungi (HHUF).
RESULTS
Phylogeny
The ML and Bayesian phylogenetic analyses were conducted
using an aligned sequence dataset composed of 941 nucleotides
from SSU, 1 276 from LSU, 886 from tef1, and 1 021 from rpb2.
The alignment contained a total of 73 taxa, which consisted of 59
taxa (80.8 %) in SSU, 73 (100 %) in LSU, 63 (86.3 %) in tef1,51
(69.9 %) in rpb2 (Table 1 and 2). No significant conflict was
observed among individual gene phylogenies, but the familial
and generic nodes mostly lacked significant support in SSU and
LSU phylogenetic trees generated (data not shown). However,
this combined dataset provided higher confidence values for the
familial level than did those of the individual gene trees (data not
shown). Of the 3 824 characters included in the alignment, 1 205
were variable and 2 844 were conserved. The ML tree with the
highest log likelihood (−26580.8637) is shown in Fig. 1. The
Bayesian likelihood score was −26638.727. The topology
recovered by the Bayesian analysis was almost identical to that
of the ML tree, except for the position of Aposphaeria cor-
allinolutea,Bertiella macrospora,Herpotrichia macrotricha,
Table 1. Specimens, isolates and new sequences used in this study.
Species Original no. Specimen no.
1
Strain no. Host/substrate GenBank accession no.
2
SSU LSU tef1 rpb2 ITS
Alpinaria rhododendri KT 2520 HHUF 30554 CBS 142901 Rhododendron
brachycarpum
LC203314 LC203360 LC203388 LC203416 LC203335
Melanomma japonicum KT 2076 HHUF 30539
P
CBS 142902 dead wood LC203290 LC203336 LC203364 LC203392 LC203318
KT 3028 HHUF 30540
P
CBS 142903 Fagus crenata LC203291 LC203337 LC203365 LC203393 LC203319
KT 3425 HHUF 30541
P
CBS 142904 F. crenata LC203292 LC203338 LC203366 LC203394 LC203320
–HHUF 26520
H
CBS 142905 Dead wood LC203293 LC203339 LC203367 LC203395 LC203321
= JCM 13124
= MAFF 239634
Me. pulvis-pyrius KT 2110 HHUF 30542 CBS 142906 Acer sp. LC203294 LC203340 LC203368 LC203396 LC203322
KT 2113 HHUF 30543 CBS 142907 Dead wood LC203295 LC203341 LC203369 LC203397 LC203323
AH 375 HHUF 30544 CBS 142908 F. crenata LC203296 LC203342 LC203370 LC203398 LC203324
KH 27 HHUF 30545 CBS 142909 Dead wood LC203297 LC203343 LC203371 LC203399 LC203325
KH 77 HHUF 30546 CBS 142910 Dead wood LC203298 LC203344 LC203372 LC203400 LC203326
KH 86 HHUF 30547 CBS 142911 Dead wood LC203299 LC203345 LC203373 LC203401 LC203327
KH 197 HHUF 30548 CBS 142912 Dead wood LC203300 LC203346 LC203374 LC203402 LC203328
Mycodidymella aesculi KT 3060 HHUF 30549 CBS 142913 Aesculus turbinata LC203301 LC203347 LC203375 LC203403 LC203329
H 2610 HHUF 22892
H
CBS 142914 A. turbinata LC203302 LC203348 LC203376 LC203404 LC194192
H 2620 –CBS 142915 A. turbinata LC203303 LC203349 LC203377 LC203405 LC203330
AH 560 HHUF 30550 CBS 142916 A. turbinata LC203304 LC203350 LC203378 LC203406 LC203331
Petrakia echinata –– CBS 133072 Acer pseudoplatanus LC203305 LC203351 LC203379 LC203407 –
–– CBS 133070 A. pseudoplatanus LC203306 LC203352 LC203380 LC203408 –
Pseudodidymella fagi KT 3058 HHUF 30515 CBS 142917 F. crenata LC203307 LC203353 LC203381 LC203409 LC150785
= MAFF 245738
KT 3074-3 HHUF 30516 CBS 142918 F. crenata LC203308 LC203354 LC203382 LC203410 LC150786
= MAFF 245739
RF 5 HHUF 30517 CBS 142919 F. crenata LC203309 LC203355 LC203383 LC203411 LC150788
= MAFF 245741
H 2579 HHUF 22903
H
MAFF 245740 F. crenata LC203310 LC203356 LC203384 LC203412 LC150787
AH 561 HHUF 30553 CBS 142920 F. crenata LC203311 LC203357 LC203385 LC203413 LC203332
Pseudod. minima KT 2918 HHUF 30551
H
CBS 142921 Fagus japonica LC203312 LC203358 LC203386 LC203414 LC203333
= MAFF 246249
AH 556 HHUF 30552
P
CBS 142922 F. japonica LC203313 LC203359 LC203387 LC203415 LC203334
Xenostigmina aceris –– CBS 124109 Acer macrophyllum LC203315 LC203361 LC203389 LC203417 –
–– CBS 115685 Acer sp. LC203316 LC203362 LC203390 LC203418 –
–– CBS 115686 Acer sp. LC203317 LC203363 LC203391 LC203419 –
1
“H”: holotype, “P”: paratype.
2
Sequences generated in this study are shown in bold.
PSEUDODIDYMELLACEAE AND ALLIED GENERA
www.studiesinmycology.org 189
Table 2. GenBank accession numbers of species used in the phylogenetic study.
Species name Family Strain no.
1
GenBank accession no.
SSU LSU tef1 rpb2
Alpinaria rhododendri incertae sedis ANM 73 –GU385198 ––
A. rhododendri incertae sedis CBS 141994
E
KY190004 KY189973 KY190009 KY189989
Alternaria alternata Pleosporaceae CBS 916.96
E
DQ678031 DQ678082 DQ677927 DQ677980
Aposphaeria corallinolutea incertae sedis CBS 131287
H
–JF740330 ––
Bertiella macrospora incertae sedis IL 5005 –GU385150 ––
Beverwykella pulmonaria incertae sedis CBS 283.53
H
KY190005 GU301804 –GU371768
Byssosphaeria jamaicana incertae sedis SMH 1403 –GU385152 GU327746 –
B. rhodomphala incertae sedis GKM L153N –GU385157 GU327747 –
B. salebrosa incertae sedis SMH 2387 –GU385162 GU327748 –
B. schiedermayeriana incertae sedis SMH 3157 –GU385163 GU327745 –
B. siamensis incertae sedis MFLUCC 10-0099
H
KT289897 KT289895 KT962059 KT962061
B. villosa incertae sedis GKM 204N –GU385151 GU327751 –
Corynespora cassiicola Corynesporascaceae CBS 100822 GU296144 GU301808 GU349052 GU371742
Cyclothyriella rubronotata Cyclothyriellaceae CBS 141486
E
KX650507 KX650544 KX650519 KX650574
Gemmamyces piceae incertae sedis CBS 141555 KY190006 KY189976 KY190011 KY189992
Herpotrichia diffusa incertae sedis CBS 250.62 DQ678019 DQ678071 DQ677915 DQ677968
H. juniperi incertae sedis CBS 200.31 DQ678029 DQ678080 DQ677925 DQ677978
H. macrotricha incertae sedis GKM 196N –GU385176 GU327755 –
H. vaginatispora incertae sedis MFLUCC 13-0865
H
KT934256 KT934252 KT934260 –
Hysterium pulicare Hysteriaceae CBS 123377 FJ161161 FJ161201 FJ161109 FJ161127
Hysterobrevium mori Hysteriaceae CBS 123563 FJ161155 FJ161196 FJ161104 –
Leptosphaeria doliolum Leptosphaeriaceae CBS 505.75 GU296159 GU301827 GU349069 KT389640
Lophiostoma arundinis Lophiostomataceae CBS 621.86 DQ782383 DQ782384 DQ782387 DQ782386
Massaria inquinans Massariaceae CBS 125591
E
HQ599442 HQ599400 HQ599340 –
Massarina eburnea Massarinaceae CBS 473.64 GU296170 GU301840 GU349040 GU371732
Melanomma populina Melanommataceae CBS 543.70
E
EU754031 EU754130 ––
M. populina Melanommataceae CBS 350.82 –JF740265 ––
M. pulvis-pyrius Melanommataceae CBS 124080
E
GU456302 GU456323 GU456265 GU456350
M. pulvis-pyrius Melanommataceae CBS 109.77 FJ201987 FJ201986 GU456274 GU456359
M. pulvis-pyrius Melanommataceae CBS 371.75 FJ201989 FJ201988 GU349019 GU371798
Muriformistrickeria rubi incertae sedis MFLUCC 15-0681
H
KT934257 KT934253 KT934261 –
Neoophiosphaerella sasicola Lentitheciaceae MAFF 239644
E
AB524458 AB524599 AB539111 AB539098
Nigrograna obliqua Nigrogranaceae CBS 141475
P
KX650512 KX650558 KX650530 KX650579
Phragmocephala atra incertae sedis MFLUCC 15-0021 KP698729 KP698725 ––
Praetumpfia obducens incertae sedis CBS 141474
E
KY190008 KY189984 KY190019 KY190000
Prosthemium betulinum Pleomassariaceae CBS 279.74 DQ678027 DQ678078 DQ677923 KT216532
Prosthemium canba Pleomassariaceae KT 2083-1 AB553646 AB553760 ––
Pseudostrickeria incertae sedis MFLUCC 13-0764
H
KT934258 KT934254 KT934262 –
muriformis
Pseudotrichia mutabilis incertae sedis PM 1 –KY189988 KY190022 KY190003
Roussoella verrucispora Thyridariaceae CBS 125434
H
AB524481 AB524622 AB539115 AB539102
Sarimanas shirakamiense incertae sedis KT 3000
H
LC001712 LC001715 ––
Seifertia azaleae incertae sedis DAOM 239136 –EU030276 ––
S. shangrilaensis incertae sedis MFLUCC 16-0238
H
KU954102 KU954100 KU954101 –
Teichospora trabicola Teichosporaceae CBS 140730
E
–KU601591 KU601601 KU601600
Tumularia tuberculata incertae sedis CBS 256.84 –GU301851 GU349006 –
1
“H”: ex-holotype, “P”: ex-paratype, “E”: ex-epitype.
HASHIMOTO ET AL.
190
Fig. 1. Maximum-likelihood (ML) tree of Melanommataceae sensu stricto and Pseudodidymellaceae with its relatives. ML bootstrap percentages (BP) greater than 60 % and
Bayesian posterior probabilities (PP) above 0.95 are presented at the nodes as ML BP/ Bayesian PP. A hyphen (“-”) indicates values lower than 60 % BP or 0.95 PP, and a
node not present in the Bayesian analysis is shown with “x”. Ex-holotype, paratype, epitype, strains are indicated in with a superscript
H
,
P
and
E
, respectively. The newly
obtained sequences are shown in bold. The scale bar represents nucleotide substitution per site.
PSEUDODIDYMELLACEAE AND ALLIED GENERA
www.studiesinmycology.org 191
Phragmocephala atra,Pseudostrickeria murigormis and Sar-
imanas shirakamiense.
Monophyly of the genera with mycopappus-like propagules
(Mycodidymella,Petrakia,Pseudodidymella, and Xenostigmina)
was well-supported (91 % ML BP/ 1.00 Bayesian PP). Although
these four genera are phylogenetically related to Melanomma-
taceae sensu lato, their morphological and ecological features
are clearly distinct from those of the type genus Melanomma.
Therefore, we establish a new family, Pseudodidymellaceae,to
accommodate these genera with mycopappus-like propagules.
Results from phylogenetic analyses of this study indicate that
Alpinaria, formerly classified in Melanommataceae sensu lato
(Jaklitsch & Voglmayr 2017), is phylogenetically distant from
Melanommataceae sensu stricto (Fig. 1), but its familial place-
ment is unresolved.
Taxonomy
Two families, including a new family (Pseudodidymellaceae),
four genera, and seven species, including two new species and
one new combination (Melanomma japonicum,Pseudodidymella
minima, and Xenostimgmina aceris) are described below.
Melanommataceae G. Winter [as ‘Melanommeae’], Rabenh.
Krypt.-Fl., Edn 2 (Leipzig) 1.2: 220. 1887.
Saprobic on dead twigs of woody plants. Sexual morph: Asco-
mata globose to ovoid, immersed to superficial, gregarious,
ostiolate. Peridium composed of thick-walled, pseudoparenchy-
matous, hyaline to brown cells. Pseudoparaphyses trabeculate,
septate, branched and anastomosed. Asci bitunicate, fissituni-
cate, cylindrical, 8-spored. Ascospores olive brown, multi-
septate, smooth. Asexual morph: Conidiomata pycnidial,
globose to subglobose, superficial, black, ostiolate. Peridium
composed of elongate, brown cells. Conidiophores absent.
Conidiogenous cells holoblastic, ampliform to cylindrical, hyaline.
Conidia ellipsoidal, hyaline, smooth, aseptate.
Type genus:Melanomma Nitschke ex Fuckel.
Notes:Melanommataceae was established by Winter (1887).
Byssosphaeria,Keissleriella,Melanomma,Ostropella, and
Strickeria have been referred to as members of Melanomma-
taceae, and this family was characterised by gregarious asco-
mata composed of well-developed, carbonaceous or coriaceous
peridium, trabecular pseudoparaphyses, and aposphaeria-like
coelomycetous asexual morphs (Barr 1987). This familial
concept was supported in “Outline of Ascomycota –2007”for 18
genera (Lumbsch & Huhndorf 2007).
A study by Mugambi & Huhndorf (2009) on LSU and tef1
sequences showed that Melanommataceae is composed of
Byssosphaeria,Herpotrichia,Melanomma,andPseudotrichia,
and previous familial concepts did not reflect natural relation-
ships. Several genera, such as Keissleriella and Ostropella,
were phylogenetically scattered in other Pleosporales
(Mugambi & Huhndorf 2009, Zhang et al. 2012, Tanaka et al.
2015), and Strickeria was placed in Sporocadaceae (Xylar-
iales,Sordariomycetes)(Jaklitsch et al. 2016a). It was clear
that the traditional concept of Melanommataceae is polyphyletic
and needed revision (Kirk et al. 2008, Mugambi & Huhndorf
2009, Hyde et al. 2013). Later, two genera, Tumularia (as
Monotosporella) and Phragmocephala, which have mono-
nematous or synnematous conidiophores in their asexual
morphs, were reported in Melanommataceae (Schoch et al.
2009, Su et al. 2015). Wijayawardene et al. (2012, 2014)
also listed additional dematiaceous genera, Exosporiella and
Nigrolentilocus, as members of this family without molecular
evidence. A broad concept of Melanommataceae was pro-
posed by Tian et al. (2015) and Jaklitsch & Voglmayr (2017),
and Mycodidymella,Petrakia and Xenostigmina were treated
as members of this family. However, the results of our phylo-
genetic analyses and morphological observations indicate that
Melanommataceae should be restricted to its type genus,
Melanomma.
Melanomma Nitschke ex Fuckel, Jb. nassau. Ver. Naturk.
23–24: 159. 1870 (1869–1870).
Synonym:Moriolopis Norman ex Keissl., Nytt Mag. Natur. 66: 88.
1927.
Saprobic on dead twigs of woody plants. Sexual morph: Asco-
mata globose to ovoid, immersed or erumpent to superficial,
gregarious, with a short ostiolar neck. Peridium composed of
thick-walled, pseudoparenchymatous, hyaline to brown cells.
Pseudoparaphyses trabecular, septate, branched and anasto-
mosed. Asci bitunicate, fissitunicate, cylindrical, 8-spored. As-
cospores olive brown, sometimes with paler ends, strongly or
slightly curved, multi-septate, smooth. Asexual morph: Con-
idiomata pycnidial, globose to subglobose, superficial, black, with
a papillate ostiole. Peridium composed of elongate, brown cells.
Conidiophores absent. Conidiogenous cells holoblastic, ampli-
form to cylindrical, hyaline, smooth. Conidia ellipsoidal, hyaline,
smooth, aseptate.
Type species:Melanomma pulvis-pyrius (Pers.) Fuckel.
Notes: The genus Melanomma was established by Fuckel
(1870). Species in this genus are known to be saprobes on
decaying plant material or weak plant pathogens (Chesters 1938,
Holm 1957, Zhang et al. 2008). Melanomma pulvis-pyrius is a
well-studied, widespread species in this genus. However, other
species have rarely been reported or have not been recorded
since their initial description. Only a few species have received
modern taxonomic treatment (Holm 1957, Mathiassen 1989,
1993, Barr 1990), although approximately 300 epithets are lis-
ted in Index Fungorum (http://indexfungorum.org). Asexual
morphs of this genus were reported to be aposphaeria-like
coelomycetes or Nigrolentilocus (Ichinoe 1970, Sivanesan
1984, Casta~
neda-Ruiz et al. 2001, S
anchez & Bianchinotti
2015, Tian et al. 2015).
Melanomma japonicum A. Hashim. & Kaz. Tanaka, sp. nov.
MycoBank MB819613; Fig. 2.
Etymology: Referring to its country of origin, Japan.
Saprobic on dead twigs of woody plants. Sexual morph: Asco-
mata globose to ovoid, superficial, gregarious, 190–320 μm
diam, 200–340 μm high. Ostiolar neck short papillate, composed
of carbonaceous, thick-walled, black cells. Peridium 40–60 μm
thick of two layers at side; outer layer 25–40 μm thick of elon-
gate, thin-walled, 12–20 × 3–4μm, brown cells; inner layer
HASHIMOTO ET AL.
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12.5–30 μm thick of globose to rectangular, 10–17.5 × 5–7μm,
hyaline cells; base of ascomata 40–53 μm thick, of two layers;
outer layer 15–30 μm thick of elongate, thin-walled,
3.5–7.5 × 3.5–5μm, brown cells; inner layer 10–30 μm thick
of globose to rectangular, 7–10.5 × 6 –9μm, brown cells.
Pseudoparaphyses trabeculate, 0.5 μm wide, septate, branched
and anastomosed. Asci bitunicate, fissitunicate, cylindrical,
73–105 × 5.5–9μm(
x= 89.9 × 7 μm, n = 26), with a short stipe
(7–16 μm long, x= 10.3 μm, n = 20), apically rounded with an
ocular chamber, 8-spored. Ascospores fusiform, with broad
rounded ends, straight to slightly curved, 12 –19 × 3 –7μm
(x= 15.1 × 4.6 μm, n = 151), l/w 2.5–4.9 (x= 3.4, n = 151), 3-
septate, with a primary septum nearly median (0.44–0.57,
x= 0.51, n = 75), olive brown, sometimes with paler ends,
constricted at the septa, smooth. Asexual morph: Conidiomata
pycnidial, globose to subglobose, up to 230 μm high in section,
150–250 μm diam, semi-immersed, solitary. Ostiolar neck short
papillate, composed of thick-walled, black cells. Peridium
12–33.5 μm wide, composed of 8.5–16.5 × 3.5–7.5 μm, rect-
angular, brown cells. Conidiophores reduced to conidiogenous
cells. Conidiogenous cells holoblastic, 8–13.5 × 2–3μm, cy-
lindrical, hyaline, smooth. Conidia cylindrical with rounded ends,
3–4×2–2.5 μm(
x= 3.3 × 2.2 μm, n = 50), l/w 1.1 –2.1 (x= 1.5,
n = 50), hyaline, smooth, aseptate, guttulate when young.
Culture characteristics: Colonies on PDA attaining 25–27 mm
diam within 21 d in the dark, floccose, centrally raised, smoke
grey (Rayner 1970), grey olivaceous at centre; reverse smoke
grey, grey olivaceous at margin (Fig. 8A); asexual morph formed.
Specimens examined:Japan, Aomori, Hakkoda, Okiagetai, on dead twigs of
woody plant, 15 Apr. 2006, K. Tanaka, KT 2076 (HHUF 30539 paratype, ex-
Fig. 2. Melanomma japonicum. A, B. Ascomata on substrate. C. Ascoma in longitudinal section. D. Lateral peridium of ascoma. E. Basal peridium of ascoma. F. Ascus. G.
Apex of ascus. H. Stipe of ascus. I. Pseudoparaphyses. J–L. Ascospores. M–O. Conidiomata in culture. P. Conidioma in longitudinal section. Q. Peridium of conidioma. R, S.
Conidiogenous cells. T. Conidia. U. Germinating conidia. A, C–Jfrom HHUF 26520; B, K, L from HHUF 30540; M–Ofrom culture CBS 142903; P–Ufrom culture CBS
142905 = JCM 13124 = MAFF 239634. Scale bars: A, M = 500 μm; B = 200 μm; C, N –P = 100 μm; D, E, G –L, R–U=5μm; F, Q = 10 μm.
PSEUDODIDYMELLACEAE AND ALLIED GENERA
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paratype living culture CBS 142902); Akita, Kazuno, Hachimantai, Yakeyama,
Mousen pass, on dead twigs of Fagus crenata, 24 Jun. 2012, K. Tanaka, KT 3028
(HHUF 30540 paratype, ex-paratype living culture CBS 142903); Kagoshima,
Tarumizu, Mt. Oonogara, on dead twigs of Fagus crenata, 25 Oct. 2013, K.
Tanaka, KT 3425 (HHUF 30541 paratype, ex-paratype living culture CBS
142904); Aomori, Hakkoda, near Yunotai, on dead twigs of woody plant, 21 Jul.
2001, Y. Harada (HHUF 26520 holotype designated here, ex-holotype living
culture CBS 142905 = JCM 13124 = MAFF 239634).
Notes: This species is morphologically closest to Me. pulvis-
pyrius in ascospore size, but the size of conidia of this species is
slightly longer and slenderer (3–4μm vs. (2–)2.5–3.5 μm long;
1.1–2.1 vs. 1.0–1.7 length/width). ITS sequences of these two
species differed by 13 positions with one gap.
Melanomma pulvis-pyrius (Pers.) Fuckel, Jb. nassau. Ver.
Naturk. 23–24: 160. 1870 (1869–1870). Fig. 3.
Saprobic on dead twigs of woody plants. Sexual morph: Ascomata
globose to ovoid, 210–310(–410) μm diam. Ostiolar neck short
papillate, composed of carbonaceous cells. Peridium 75–88 μm
thick of two layers at side; outer layer 35–45 μm thick; inner layer
30–40 μm thick, 65 –75 μm thick at base. Pseudoparaphyses
trabeculate, 1–1.5 μm wide. Asci 71–92 × 5–8.5 μm
(x= 82.1 × 6.3 μm, n = 14), with a short stipe (5–8μmlong,
x= 5.7 μm, n = 12). Ascospores 11.5–15.5 × 4–5μm
(x= 13 × 4.2 μm, n = 75), l/w 2.5–3.6 (x= 3.1, n = 75), 3-septate,
with a primary septum nearly median (0.45–0.58, x= 0.50, n = 75).
Asexual morph: Conidiomata pycnidial, globose to subglobose,
160–300 μm diam, with a papillate ostiolar neck. Peridium
18.5–22 μm wide, composed of 4–16.5 × 2.5–5μm, rectangular,
brown cells. Conidiophores reduced to conidiogenous cells.
Conidiogenous cells holoblastic, 8–17.5 × 1.5 –4μm, cylindrical,
hyaline, smooth. Conidia cylindrical with rounded ends, (2–)
2.5–3.5 × 2 –2.5( –3) μm(
x= 2.9 × 2.3 μm, n = 50), l/w 1.0–1.7
(x= 1.3, n = 50), hyaline, smooth, aseptate, guttulate when young.
Culture characteristics: Colonies on PDA attaining 22 –24 mm
diam within 21 d, floccose, fasciculate, centrally raised, pale
olivaceous grey; reverse greyish sepia, olivaceous buff at margin
(Fig. 8B); asexual morph formed.
Specimens examined:Japan, Aomori, Minamitsugaru, Owani, on dead twigs of
Acer mono var. mayrii, 1 Jul. 2006, K. Tanaka, KT 2110 (HHUF 30542, culture
CBS 142906); Hirosaki, Zatoishi, on dead twigs of woody plant, 8 Jul. 2006, H.
Yonezawa, KT 2113 (HHUF 30543, culture CBS 142907); Noheji, near Mt.
Eboshi, on dead twigs of Fagus crenata, 2 Sep. 2015, A. Hashimoto et al.,AH
375 (HHUF 30544, culture CBS 142908); Nishimeya, Ooshirosawa stream, on
dead twigs of woody plant, 25 Jun. 2007, K. Hirayama et al., KH 27 (HHUF
30545, culture CBS 142909); on dead twigs of woody plant, 21 Jul. 2007, K.
Hirayama et al., KH 77 (HHUF 30546, culture CBS 142910); Kawaratai, Ooka-
wazoe, on dead twigs of woody plant, 28 Aug. 2007, K. Hirayama et al.,KH86
(HHUF 30547, culture CBS 142911); on dead twigs of woody plant, 30 Aug. 2008,
K. Hirayama et al., KH 197 (HHUF 30548, culture CBS 142912).
Notes: The above specimens were identified as Me. pulvis-pyrius,
the type species of Melanomma. The size of ascospores in our
materials was almost identical to that of Me. pulvis-pyrius reported
by Holm (1957), who observed the neotype of this species. The
rpb2 sequences of our isolates were identical or had one or two
differences compared with those of Me. pulvis-pyrius (GU456350)
obtained from the ex-epitype culture (CBS 124080).
Melanomma pulvis-pyrius is a well-studied species in Mela-
nomma; its taxonomy and ontogeny of sexual morphs have been
described (Chesters 1938), and it has been reported worldwide
(Holm 1957, Sivanesan 1984, Vassilieva 1987, Vasyagina et al.
1987, Romero 1998, Mathiassen 1989, 1993, Zhang et al. 2008,
Mugambi & Huhndorf 2009, Jaklitsch & Voglmayr 2017). How-
ever, this is the first report of Me. pulvis-pyrius from Japan. This
species was epitypified by Zhang et al. (2008) based on a
specimen collected from Salix caprea in France.
In the phylogenetic tree, Me. pulvis-pyrius clustered with Me.
populina (CBS 543.70 and CBS 350.82) with moderate to strong
support (93 % ML BP/ 1.00 Bayesian PP). Because we could not
compare the characters of these two species, further study is
needed in the future to confirm whether these two species are
conspecific.
Pseudodidymellaceae A. Hashim. & Kaz. Tanaka, fam. nov.
MycoBank MB819614.
Parasitic on living leaves of woody plants. Sexual morph:
Ascomata subglobose to lenticular, immersed, ostiolate.
Peridium pale brown to brown, distinctly thickened at base.
Pseudoparaphyses septate, branched and anastomosed. Asci
bitunicate, fissitunicate, cylindrical, 8-spored. Ascospores fusi-
form with rounded ends, straight, 1-septate, hyaline, smooth.
Spermatia cylindrical, hyaline. Asexual morph: Propagules epi-
phyllous, white to yellowish, globose to subglobose, multicellular,
with numerous, flexuous, cylindrical, multi-septate hyphal ap-
pendages, detached at stroma-like base composed of sub-
globose to oblong, hyaline to yellow cells. Synasexual morph:
Conidiomata sporodochial, superficial. Stromata composed of
globose to subglobose cells. Conidiophores reduced. Con-
idiogenous cells annellidic or holoblastic. Conidia clavate, sig-
moid or rounded to oval or broadly ellipsoidal, phragmosporous
to muriform, hyaline to brown, falcate to sigmoid.
Type genus:Pseudodidymella C.Z. Wei et al.
Notes:Mycodidymella,Petrakia,Pseudodidymella, and Xeno-
stigmina have mycopappus-like propagules in their life cycles.
Although sexual morphs of these genera were reported, and
several molecular studies were performed, the phylogenetic
placement of these genera remains unresolved (Crous et al.
2009, Butin et al. 2013, Li et al. 2016, Gross et al. 2017).
According to the multi-locus phylogenies, these genera are
closely related to each other (Li et al. 2016, Gross et al. 2017,
Jaklitsch & Voglmayr 2017). Based on phylogenetic study,
Phookamsak et al. (2014) proposed to include Petrakia and
Xenostigmina in Melanommataceae.Tian et al. (2015)
accepted these two genera in Melanommataceae in a sub-
sequent study. In our study, the monophyly of these four
genera with mycopappus-like propagules was strongly sup-
ported (91 % ML BP/ 1.00 Bayesian PP; Fig. 1). Therefore, we
introduce a new family, Pseudodidymellaceae, to accommo-
date the above four genera. Species in this family bear several
common features, including sexual morphs with lenticular and
subcuticular ascomata erumpent from host tissue, asexual
morphs with mycopappus-like propagules, and with or without
a synasexual morph that has sporodochial conidiomata.
Pseudodidymellaceae can be distinguished from Mela-
nommataceae sensu stricto based on the presence of
mycopappus-like propagules.
HASHIMOTO ET AL.
194
Mycodidymella C.Z. Wei et al., Mycologia 90: 336. 1998.
Synonym:Blastostroma C.Z. Wei et al., Mycologia 90: 337. 1998.
Parasitic on living leaves of woody plant. Sexual morph: Asco-
mata subglobose to lenticular, immersed, ostiolate. Peridium with
rim-like side wall, composed of rectangular, thin-walled, pale
brown cells, well-developed at base. Pseudoparaphyses septate,
branched and anastomosed. Asci bitunicate, fissitunicate, cy-
lindrical, 8-spored. Ascospores fusiform, 1-septate, hyaline,
smooth. Spermatia cylindrical, hyaline. Asexual morph: Propa-
gules epiphyllous, white to yellowish, globose to subglobose,
multicellular; main bodies subglobose to oblong, bearing
numerous, unbranched, flexuous, cylindrical, multi-septate
hyphal appendages. Synasexual morph: Conidiomata sporodo-
chial, white to yellowish. Stromata composed of globose to
subglobose cells. Conidiophores absent. Conidiogenous cells
holoblastic, hyaline. Conidia falcate to sigmoid, hyaline, multi-
septate, obtuse at the apex, truncate at the base.
Type species:Mycodidymella aesculi C.Z. Wei et al.
Mycodidymella aesculi C.Z. Wei et al., Mycologia 90: 336.
1998. Fig. 4.
Synonyms:Blastostroma aesculi C.Z. Wei et al., Mycologia 90:
338. 1998.
Mycopappus aesculi C.Z. Wei et al., Mycologia 90: 336. 1998.
Fig. 3. Melanomma pulvis-pyrius. A, B. Ascomata on substrate. C. Ascoma in longitudinal section. D. Lateral peridium of ascoma. E. Basal peridium of ascoma. F. Pseu-
doparaphyses. G. Ascus. H. Apex of ascus. I. Stipe of ascus. J, K. Ascospores. L. Germinating ascospore. M, N. Conidiomata in culture. O. Conidioma in longitudinal section. P.
Peridium of conidioma. Q, R. Conidiogenous cells. S. Conidia. T. Germinating conidia. A–F, J –Lfrom HHUF 30544; G–Ifrom HHUF 30543; M–Q, T from culture CBS 142912;
R, S from culture CBS 142908. Scale bars: A, M = 500 μm; B, N = 200 μm; C, O = 100 μm; D, E, P = 10 μm; F –L, Q –T=5μm.
PSEUDODIDYMELLACEAE AND ALLIED GENERA
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HASHIMOTO ET AL.
196
Petrakia aesculi (C.Z. Wei et al.) Jaklitsch & Voglmayr, Sydowia
69: 91. 2017.
Parasitic on living leaves of Aesculus turbinata. Sexual morph:
Ascomata subglobose to lenticular, solitary to 3–5 grouped,
immersed, up to 210 μm high, 260–380 μm diam. Ostiolar neck
short papillate, composed of thick-walled, black cells. Peridium
17.5–27.5 μm thick at side, with rim-like side wall, composed of
rectangular, thin-walled, 10–13.5 × 6 –9μm, pale brown cells, at
base 105–140 μm thick, composed of 8.5–11.5 × 6.5–8.5 μm,
hyaline to pale brown cells. Pseudoparaphyses numerous,
trabeculate, 0.8–1.3 μm wide, septate, branched and anasto-
mosed. Asci bitunicate, fissitunicate, cylindrical,
45.5–60 × 7–12.5 μm(
x= 53.3 × 10 μm, n = 20), with or without
a short stipe, apically rounded with an ocular chamber, 8-spored.
Ascospores fusiform with rounded ends, straight,
16–21.5 × 3–4.5 μm(
x= 18.6 × 3.9 μm, n = 21), l/w 4.3–5.3
(x= 4.7, n = 21), with a septum nearly median (0.44 –0.55,
x= 0.51, n = 21), constricted at the septum, hyaline, smooth,
guttulate when young. Spermatia 3–5×1–2μm
(x= 3.6 × 1.5 μm, n = 50), l/w 1.7–3.7 (x= 2.5, n = 50), cy-
lindrical, hyaline. Asexual morph: Propagules epiphyllous, white
to yellowish, globose to subglobose, 200–565 μm diam
(x= 331.9 μm, n = 30); main bodies subglobose to oblong,
85–193 × 116–228 μm(
x= 127.6 × 152.4, n = 30), composed of
7.5–10 μm diam cells; hyphal appendages 19 to 37, unbranched
flexuous, cylindrical, 3–7-septate, 72–150 × 3.5–5.5 μm
(x= 111.5 × 4.6, n = 30). Synasexual morph: Conidiomata
sporodochial, white to yellowish. Stromata 15–20 μm thick,
composed of hyaline, globose to subglobose cells. Co-
nidiophores reduced to conidiogenous cells. Conidiogenous cells
holoblastic, hyaline, smooth, 9–12 × 4–5.5 μm. Conidia falcate
to sigmoid, 57–94 × 5.5–8.5 μm(
x= 75.8 × 6.8, n = 50), hyaline,
8–13-septate, obtuse at the apex, truncate at the base.
Culture characteristics: Colonies on PDA attaining 31 –40 mm
diam within 21 d, velvety, floccose, centrally raised, buff,
grey olivaceous at centre; reverse buff; grey olivaceous at
centre (Fig. 8C); spermatial, asexual and synasexual morphs
formed.
Specimens examined:Japan, Aomori, Minamitsugaru, Owani, on living leaves of
Aesculus turbinata, 12 Aug. 2012, K. Tanaka et al., KT 3060 (HHUF 30549,
culture CBS 142913); Nishimeya, Kawaratai, Ookawazoe, near Annmon waterfall
trail, on living leaves of Aesculus turbinata, 4 Oct. 1995, C. Z. Wei & Y. Harada
(HHUF 23078 holotype of Blastostroma aesculi); 10 Sep. 2016, A. Hashimoto,
AH 560 (HHUF 30550, culture CBS 142916); Hirakawa, Ikarigaseki, on living
leaves of Aesculus turbinata, 18 Apr. 1995, C. Z. Wei & Y. Harada, H 2610 (HHUF
22892 holotype of Mycodidymella aesculi, ex-holotype living culture CBS
142914); on living leaves of Aesculus turbinata, 18 Apr. 1995, C. Z. Wei & Y.
Harada, H 2620 (culture CBS 142915).
Notes: The genus Mycodidymella was established to accom-
modate a single species, Mycod. aesculi, and this species
causes large concentric leaf spots on Aesculus turbinata in
Japan (Wei et al. 1998). This species is morphologically
characterised by lenticular ascomata and 1-septate, hyaline
ascospores in the sexual morph, mycopappus-like propagules in
the asexual morph, and blastostroma-like sigmoid conidia in the
synasexual morph. The sexual morph of this species morpho-
logically resembles those of Didymella or Pseudodidymella.Wei
et al. (1998) assigned this genus to Phaeosphaeriaceae based
on morphology. Later, familial placement of this genus was
treated as incertae sedis in Dothideomycetes (Lumbsch &
Huhndorf 2007). Recently, Butin et al. (2013) described the
sexual morph of Pe. echinata, which is the type species of
Petrakia; they found that the sexual morphology of Petrakia
matches that of Mycodidymella and thus synonymised Mycodi-
dymella with Petrakia (Butin et al. 2013). This proposal was
accepted by subsequent studies (Tian et al. 2015, Li et al. 2016,
Jaklitsch & Voglmayr 2017). However, Mycod. aesculi was not
included in their analyses. Our phylogenetic study revealed that
their monophyletic status was not supported in any analyses
(below 60 % ML BP/ 0.95 Bayesian PP, Fig. 1). We retained
Mycodidymella as a natural genus in Pseudodidymellaceae
(discussed below).
Pseudodidymella C.Z. Wei et al., Mycologia 89: 496. 1997.
Synonym:Pycnopleiospora C.Z. Wei et al., Mycologia 89: 496.
1997.
Parasitic on living leaves of Fagus spp. Sexual morph: Ascomata
subglobose to lenticular, solitary to grouped, immersed, ostiolate.
Peridium composed of rectangular, thin-walled, pale brown cells,
well-developed at base. Pseudoparaphyses septate, branched
and anastomosed. Asci bitunicate, fissitunicate, cylindrical, 8-
spored. Ascospores fusiform with rounded ends, 1-septate, hy-
aline, smooth. Spermatia cylindrical, hyaline. Asexual morph:
Propagules epiphyllous, white to yellowish, globose, multicel-
lular; main bodies globose, subglobose, hyaline to yellow,
bearing numerous, unbranched, flexuous, multi-septate hyphal
appendages.
Type species:Pseudodidymella fagi C.Z. Wei et al.
Notes: The genus Pseudodidymella, based on the type species
Pseudod. fagi, has lenticular ascomata and a pycnopleiospora-
like asexual morph which is characterised by sporodochial
conidiomata and appendage-bearing conidia (Wei et al. 1997).
Because its sexual morph morphologically resembles that of
Didymella, this genus was considered a member of Phaeos-
phaeriaceae (Wei et al. 1997). The sexual morph of this genus
superficially resembles that of Mycodidymella, but it can be
distinguished based on its pycnopleiospora-like asexual morph
(Wei et al. 1998). Since then, this genus has been treated as
incertae sedis in Dothideomycetes (Lumbsch & Huhndorf 2007).
Gross et al. (2017) discovered Pseudod. fagi on Fagus sylvatica
in Switzerland; they noted that the asexual morph of this species
was previously recorded as Pycnopleiospora, but actually has
mycopappus-like propagules rather than individual conidia, and
Fig. 4. Mycodidymella aesculi. A, B. Ascomata on substrate. C. Ascoma in longitudinal section. D. Peridium of ascoma. E. Ascus. F. Apex of ascus. G. Stipe of ascus. H, I.
Ascospores. J–L. Spermatogonia in culture. M. Spermatogonium in longitudinal section. N. Perdium of spermatogonium. O, P. Spermatogenous cells. Q, R. Spermatia. S, T.
Leaves of Aesculus turninata with necrotic brown spots. U, V. Propagules on the leaf surface. W, X. Propagules. Y. Appendage of propagule. Z, AA. Sporodochia on the leaf
surface. AB. Sporodochium in longitudinal section. AC. Stroma of sporodochium. AD. Conidiogenous cells. AE, AF. Conidia. A–Ifrom HHUF 22892. J–Rfrom culture CBS
142913. S, T from HHUF 30550. U–Yfrom HHUF 30549. Z–AF from HHUF 23078. Scale bars: A, J, T, Z = 500 μm; B, K, L, U, V = 250 μm; C, M, W, X, AA, AB = 50 μm; D, E,
N, Y, AC, AE, AF = 10 μm; F–I, O–R, AD = 5 μm.
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the original description (Wei et al. 1997) seemed to misinterpret
over-mature propagules. They also confirmed that Pseudodidy-
mella is phylogenetically related to other mycopappus-forming
genera, such as Mycodidymella,Petrakia, and Xenostigmina,
based on the ITS phylogeny. Thus, morphological delimitation of
Pseudodidymella and Mycodidymella is problematic and requires
further research. In the present study, we recollected Pseudod.
fagi from its type locality, and compared the fresh materials to the
holotype of Py. fagi. Based on morphological and phylogenetic
comparisons of these specimens, we also conclude that Wei
et al. (1997) misinterpreted the pieces of broken overmatured
mycopappus-like propagules (Fig. 5AA and AB) as conidia of
Pseudodidymella, but Pseudodidymella actually has
mycopappus-like propagules in its asexual morph.
Species in this genus bear common features, with more than
60 hyphal appendages in mycopappus-like propagules. Although
other related genera have sporodochial synasexual morphs, no
synasexual morph is known from Pseudodidymella (Wei et al.
1997, Gross et al. 2017, present study). Morphologically,
Pseudodidymella resembles Mycodidymella, but can be distin-
guished based on the rim-like walls of the ascomata, and
numerous hyphal appendages in the asexual morph.
Pseudodidymella fagi C.Z. Wei et al., Mycologia 89: 496. 1997.
Fig. 5.
Synonym:Pycnopleiospora fagi C.Z. Wei et al., Mycologia 89:
496. 1997.
Parasitic on living leaves of Fagus crenata. Sexual morph:
Ascomata subglobose to lenticular, solitary to 3–5 grouped,
immersed, up to 175 μm high, 200–300 μm diam. Ostiolar neck
short papillate, composed of thick-walled, black cells. Peridium
20–22 μm thick at side, composed of rectangular, thin-walled,
7.5–10.5 × 6.5–8.5 μm, pale brown cells, at base 58–67 μm
thick, composed of 10–13.5 × 5–11.5 μm, hyaline to pale brown
cells. Pseudoparaphyses numerous, 1–2μm wide, septate,
branched and anastomosed. Asci bitunicate, fissitunicate, cy-
lindrical, 49–76.5 × 10–14 μm(
x= 60.3 × 11.5 μm, n = 20), with
a short stipe (3.5–8μm long, x= 6.1 μm, n = 20), apically
rounded with an ocular chamber, 8-spored. Ascospores fusiform
with rounded ends, straight, 18.5–24 × 4–5μm
(x= 20.5 × 4.3 μm, n = 20), l/w 4.3–5.6 (x= 4.8, n = 20), with a
septum nearly median (0.47–0.58, x= 0.52, n = 20), constricted
at the septum, hyaline, smooth, guttulate when young. Spermatia
3–5×1–1.5 μm(
x= 3.9 × 1.2 μm, n = 50), l/w 2.1–4.8 (x= 3.3,
n = 50), cylindrical, hyaline. Asexual morph: Propagules epi-
phyllous, white to yellowish, globose, 290–500 μm diam
(x= 387.2 μm, n = 30); main bodies globose, 160–315 μm diam
(x= 227.4 μm, n = 30), composed of subglobose, hyaline to
yellow, 11.5–15 × 7.5–11.5 μm cells; hyphal appendages 63 to
138, unbranched, flexuous, cylindrical, 1–4-septate,
67–133 × 3–5μm(
x= 97.1 × 3.7 μm, n = 52).
Culture characteristics: Colonies on PDA attaining 27 –37 mm
diam within 21 d, velvety, plane, buff to olivaceous black at
centre; reverse buff to olivaceous black at centre (Fig. 8D);
spermatial and asexual morphs formed.
Specimens examined:Japan, Aomori, Nakatsugaru, Onikawabe, on living
leaves of Fagus crenata, 12 Aug. 2012, K. Tanaka et al., KT 3058 (HHUF 30515,
culture CBS 142917 = MAFF 245738); Nishimeya, Ookawazoe, near Annmon
waterfall trail, on living leaves of Fagus crenata, 2 Sep. 2012, K. Tanaka et al.,
KT 3074-3 (HHUF 30516, culture CBS 142918 = MAFF 245739); 2 Sep. 2012,
R. Fujimoto et al., RF 5 (HHUF 30517, culture CBS 142919 = MAFF 245741); 10
Sep. 2016, A. Hashimoto, AH 561 (HHUF 30553, culture CBS 142920); Hir-
akawa, Ikarigaseki, on living leaves of Fagus crenata, 28 Apr. 1995, C. Z. Wei &
Y. Harada, H 2579 (HHUF 22903, holotype of Pseudodidymella fagi, ex-
holotype living culture MAFF 245740); artificial inoculation on leaves of Fagus
crenata, 30 Sep. 1996, C. Z. Wei (HHUF 23672, holotype of Pycnopleiospora
fagi).
Notes: This species was originally reported to cause brown leaf
spots on Fagus crenata in Japan. More recently, it was reported
from a new host, F. sylvatica (Gross et al. 2017). To elucidate its
host spectrum, further surveys for this fungus and other species
on Fagus is needed.
Pseudodidymella minima A. Hashim. & Kaz. Tanaka, sp. nov.
MycoBank MB819615. Fig. 6.
Etymology: Referring to the smaller-sized propagules observed
in this species.
Parasitic on living leaves of Fagus japonica. Sexual morph:
Unknown. Asexual morph: Propagules epiphyllous, white to
yellowish, globose, 110–220( –240) μm diam (x= 164.4 μm,
n = 60); main bodies globose, multicellular, 78–168 μm diam
(x=115μm, n = 60), composed of subglobose, 7.5–10 μm diam,
hyaline to yellow cells; hyphal appendages 65 to 135, un-
branched, flexuous, cylindrical, 1–2-septate or rarely aseptate,
27–44 × 3–6μm(
x= 35.5 × 4.4 μm, n = 59).
Culture characteristics: Colonies on PDA attaining 32–38 mm
diam within 21 d, floccose, plane, smoke grey; reverse honey to
isabelline (Fig. 8E); asexual morph formed.
Specimens examined:Japan, Iwate, Hanamaki, near Dai spa, on living leaves of
Fagus japonica, 9 Oct. 2011, K. Tanaka, KT 2918 (HHUF 30551 holotype
designated here; ex-holotype living culture CBS 142921 = MAFF 246249); 3
Sept. 2016, A. Hashimoto, AH 556 (HHUF 30552 paratype, ex-paratype living
culture CBS 142922).
Notes: This species on Fagus japonica is easily distinguished
from Pseudod. fagi on F. crenata by its much smaller propagules.
Sequence differences between these two species were found at
six nucleotide positions with one gap in the ITS sequences.
We did not observe the sexual or synasexual morph of
Pseudod. minima. Further surveys are therefore needed to
reveal the ecological features of this species.
Xenostigmina aceris (Dearn. & Barthol.) A. Hashim. & Kaz.
Tanaka, comb. nov. MycoBank MB821403.
Basionym:Cercosporella aceris Dearn. & Barthol., Mycologia 9:
362. 1917.
Synonyms:Mycopappus aceris (Dearn. & Barthol.) Redhead &
G.P. White, Canad. J. Bot. 63: 1430. 1985.
Petrakia aceris (Dearn. & Barthol.) Jaklitsch & Voglmayr,
Sydowia 69: 90. 2017.
Stigmina zilleri A. Funk, Canad. J. Bot. 65: 482. 1987.
Xenostigmina zilleri (A. Funk) Crous, Mycol. Mem. 21: 155. 1998.
Mycosphaerella mycopappi A. Funk & Dorworth, Canad. J. Bot.
66: 295. 1988.
HASHIMOTO ET AL.
198
Fig. 5. Pseudodidymella fagi. A, B. Ascomata on substrate. C. Ascoma in longitudinal section. D. Peridium of ascoma. E. Ascus. F. Apex of ascus. G. Stipe of ascus. H, I.
Ascospores. J–L. Spermatogonia in culture. M. Spermatogonium in longitudinal section. N. Perdium of spermatogonium. O, P. Spermatogenous cells. Q, R. Spermatia. S, T.
Leaves of Fagus crenata with necrotic brown spots. U, V. Propagules on the leaf surface. W, X. Propagules. Y–AB. Appendages of propagule. A–Ifrom HHUF 22903. J–R
from culture CBS 142917 = MAFF 245738. S, T from HHUF 30553. U, X, AA, AB from HHUF 30516. V, W, Z from HHUF 23672. Yfrom HHUF 30517. Scale bars: A, J,
T = 500 μm; B, K, L, U, V = 250 μm; C, M, W, X = 50 μm; D, E, N, Y–AB = 10 μm; F–I, O –R=5μm.
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Didymella mycopappi (A. Funk & Dorworth) Crous, Mycol. Mem.
21: 152. 1998.
Notes:Xenostigmina zilleri is the name that has been commonly
used for this pathogen, although the epithet of Cercosporella
aceris is older than that of Stigmina zilleri (Crous 1998, Crous
et al. 2009, Phookamsak et al. 2014, Tian et al. 2015, Gross
et al. 2017). Therefore, we proposed a new combination, Xen-
ostigmina aceris.
Incertae sedis
Alpinaria Jaklitsch & Voglmayr, Sydowia 69: 84. 2017.
Saprobic on dead twigs of woody plants. Sexual morph: Asco-
mata globose to ovoid, immersed to superficial, gregarious,
sometimes confluent, ostiolate. Peridium composed of elongate,
thin-walled, brown cells, at base composed of elongate, hyaline
cells. Pseudoparaphyses septate, branched and anastomosed.
Asci bitunicate, cylindrical, 8-spored. Ascospores fusiform, multi-
septate, smooth. Asexual morph: Conidiomata pseudopycnidial,
globose to cylindrical, sometimes deformed, septate, confluent,
multiloculate, scattered, semi-immersed, black, with one to two
non-papillate ostiole. Peridium rectangular, brown cells. Co-
nidiophores absent. Conidiogenous cells holoblastic, cylindrical,
hyaline, smooth. Conidia cylindrical with rounded ends, hyaline,
smooth, aseptate.
Type species:Alpinaria rhododendri (Niessl) Jaklitsch &
Voglmayr.
Alpinaria rhododendri (Niessl) Jaklitsch & Voglmayr, Sydowia
69: 84. 2017. Fig. 7.
Basionym:Cucurbitaria rhododendri Niessl, Verh. Nat. Ver.
Brünn 10: 200. 1872.
Synonyms:Gibberidea rhododendri (Niessl) Petr., Ann. Mycol.
32: 330. 1934; nom. illegit.
Melanomma rhododendri Rehm, Ber. Naturhist. Ver. Augsburg
26: 48. 1881.
Fig. 6. Pseudodidymella minima. A–C. Leaves of Fagus japonica with necrotic brown spots. D–F. Propagules on the leaf surface. G, H. Propagules. I. Appendages of
propagule. A–C, H from HHUF 30552. D, E, G, I from HHUF 30551. Ffrom culture CBS 142921 = MAFF 246249. Scale bars: D –F = 250 μm; G, H = 50 μm; I = 5 μm.
HASHIMOTO ET AL.
200
Gibberidea rhododendri (Rehm) Petr., Krypt. Forsch. (München)
2: 160. 1931.
Gibberidea rhododendri (Rehm) Kirschst., Hedwigia 81: 206,
1944; nom. illegit.
Saprobic on dead twigs of ericaceous plants. Sexual morph:
Ascomata globose to ovoid, immersed, becoming largely
erumpent to superficial, gregarious, sometimes confluent,
140–190 μm high, 110–250 μm diam. Ostiolar neck short
papillate, composed of carbonaceous, thick-walled, black cells.
Peridium 55–75 μm thick at side composed of elongate, thin-
walled, 12–13 × 5–6.5 μm, brown cells, 87–102 μm thick at
base composed of elongate, thin-walled, 4 –6μm diam, hyaline
cells. Pseudoparaphyses trabeculate, 1–1.5 μm wide, septate,
branched and anastomosed. Asci bitunicate, cylindrical,
100–118 × 7 –9μm(
x= 109.5 × 7.8 μm, n = 11), with a short
stipe (3.5–10 μm long, x=7μm, n = 11). Ascospores fusiform,
13–21 × 5–6μm(
x= 16.5 × 5.6 μm, n = 50), l/w 2.2–4.2
(x= 3.0, n = 50), 3-septate, with a primary septum nearly median
(0.42–0.57, x= 0.50, n = 50) and constricted, smooth, without
sheath. Asexual morph: Conidiomata pseudopycnidial, globose
to cylindrical, sometimes deformed, septate, confluent, multi-
loculate, scattered, semi-immersed, black, up to 190 μm high,
110 –250 μm diam. Ostiolar neck mainly single, occasionally two,
non-papillate. Peridium 20–25 μm wide, composed of
7.5–11.5 × 5–7μm, rectangular, brown cells. Conidiophores
reduced to conidiogenous cells. Conidiogenous cells holoblastic,
6–10.5 × 3–4.5 μm, cylindrical, hyaline, smooth. Conidia cylin-
drical with rounded ends, 2–4×1–2μm(
x=3×1.6μm, n = 50),
l/w 1.1–2.6 (x= 1.9, n = 50), hyaline, smooth, aseptate, guttulate
when young.
Culture characteristics: Colonies on PDA attaining 26–31 mm
diam within 21 d, velvety, wet, olivaceous black, smoke grey at
margin; reverse olivaceous black at centre (Fig. 8F); asexual
morph formed.
Specimen examined:Japan, Iwate, Hachimantai, Yakeyama, near Goshogake
spa, on leaf bud of Rhododendron brachycarpum, 9 Jul. 2008, Y. Harada, KT
2520 (HHUF 30554; culture CBS 142901).
Notes: The ascospore size in the material mentioned above is
identical to that of A. rhododendri reported by Jaklitsch &
Fig. 7. Alpinaria rhododendri. A, B. Ascomata on substrate. C. Ascoma in longitudinal section. D. Lateral peridium of ascoma. E. Ascus. F. Apex of ascus. G. Stipe of ascus. H.
Pseudoparaphyses. I–K. Ascospores. L, M. Conidiomata in culture. N. Conidiomata in longitudinal section. O. Peridium of conidioma. P, Q . Conidiogenous cells. R. Conidia. S.
Germinating conidia. A–Kfrom HHUF 30554. L–Sfrom culture CBS 142901. Scale bars: A, L = 500 μm; B, C, M, N = 100 μm; D, E, O = 10 μm; F –K, P–S=5μm.
PSEUDODIDYMELLACEAE AND ALLIED GENERA
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Voglmayr (2017), who designated the epitype of this species.
The ITS, tef1 and rpb2 sequences from our material are
completely identical to those from the ex-epitype strain of this
species (CBS 141994). This species has been reported from
twigs or buds of Rhododendron spp. in the Asia (R. chrysanthum;
Müller 1959), Europe (R. ferrugineum and R. hirsutum;Jaklitsch
& Voglmayr 2017), and North America (Rhododendron sp.;
Mugambi & Huhndorf 2009). In addition, we collected this spe-
cies on R. brachycarpum from the subalpine zone in Japan.
Alpinaria rhododendri appears to be a relatively common species
in the subalpine to alpine zone worldwide.
Alpinaria was recently established to accommodate a single
species A. rhododendri, which was transferred from Mela-
nomma because this species is phylogenetically distinct from
the type species of Melanomma, and possesses ascomata
with a roughened surface view of textura prismatica and tex-
tura angularis (Jaklitsch & Voglmayr 2017). Furthermore, they
treated the genus as a member of Melanommataceae
(Jaklitsch & Voglmayr 2017). Although no asexual morph was
reported for this species (Müller 1959, Mugambi & Huhndorf
2009, Jaklitsch & Voglmayr 2017), we newly observed its
asexual morph in culture (Fig. 7L–S). As a result of our
observation of the asexual morph, as well as the sexual
morph, we clarified that this species has atypical features for
Melanommataceae; its ascomata are composed of hyaline
cells at the base, and are pseudopycnidial. Confluent con-
idiomata are not found in sexual/asexual morphs of Mela-
nommataceae. In our phylogenetic tree, the genus placement
is confirmed outside Melanommataceae sensu stricto (Fig. 1).
Therefore, we treat Alpinaria as incertae sedis in Pleosporales
in this study; additional taxa related to this genus will be
needed to resolve its familial placement.
DISCUSSION
Re-circumscription of Melanommataceae sensu
stricto
Melanommataceae has been extensively studied in recent years
based on phylogenetic evidence (Mugambi & Huhndorf 2009,
Schoch et al. 2009, Wijayawardene et al. 2012, 2014, Butin
et al. 2013, de Gruyter et al. 2013, Su et al. 2015, Tian et al.
2015, Li et al. 2016, Gross et al. 2017, Jaklitsch & Voglmayr
2017). The characters emphasised for members of this family
include a carbonaceous peridium of ascomata and trabecular
pseudoparaphyses. These species are known saprobes on
decaying plant material, or, rarely, as plant pathogens. The fa-
milial concept of Melanommataceae was revised and expanded
after in a study by Mugambi & Huhndorf (2009), who applied a
molecular approach. A recent monograph of Melanommataceae
was based on morphological and multi-gene phylogenetic data
(Tian et al. 2015). Although monophyly of Melanommataceae
was confirmed in previous studies, statistical support for Mela-
nommataceae sensu lato was lacking (Mugambi & Huhndorf
Fig. 8. Colony characters of Melanomma spp. and Pseudodidymellaceae spp. used in this study on PDA within 3 wk at 20 °C in the dark (left: upper, right: reverse). A.
Melanomma japonicum (CBS 142905 = JCM 13124 = MAFF 239634, ex-holotype culture). B. Me. pulvis-pyrius (CBS 142908). C. Mycodidymella aesculi (CBS 142914, ex-
holotype culture). D. Pseudodidymella fagi (MAFF 245740, ex-holotype culture of Pycnopleiospora fagi). E. Pseudod. minima (CBS 142921 = MAFF 246249, ex-holotype
culture). F. Alpinaria rhododendri (CBS 142901). Scale bar: A–F=1cm
HASHIMOTO ET AL.
202
2009, Schoch et al. 2009, Tian et al. 2015). Additionally, previous
authors did not examine the asexual morphs, although various
asexual morphs, such as those with mononematous, synnem-
atous, and pycnidial conidiomata, are known to occur in this
family. Two of the most striking genera are Petrakia and Xeno-
stigmina, which have mycopappus-like propagules as asexual
morphs, and were reported to be foliicolous necrotrophs (Funk
1986, Funk & Dorworth 1988, Crous 1998, Crous et al. 2009,
Butin et al. 2013), whereas species of Melanomma, the type
genus of this family, have aposphaeria-like pycnidial asexual
morphs and are known to be saprobes on twigs of various plant
hosts (Chesters 1938, Romero 1998, Zhang et al. 2008). Our
multi-gene phylogenetic analyses of this family clearly showed
the poly- and paraphyletic nature of Melanommataceae sensu
lato (Fig. 1), and morphological observations of sexual and
asexual morphs led to the conclusion that Melanommataceae
should be restricted to the type genus Melanomma. In addition,
four genera with mycopappus-like propagules in their asexual
morphs (Mycodidymella,Petrakia,Pseudodidymella, and Xen-
ostigmina) are separated from Melanommataceae sensu stricto,
and we thus establish a new family, Pseudodidymellaceae,to
accommodate these genera.
Relationships among genera in
Pseudodidymellaceae
Mycodidymella and Xenostigmina are retained as natural
genera in the present study. Butin et al. (2013) found that the
sexual morph of Mycodidymella is similar to that of Petrakia, and
thus recognised Petrakia in a broad sense and included
Mycodidymella as a synonym. This treatment was supported by
a later study (Li et al. 2016). Gross et al. (2017) showed these
three genera are closely related based on an ITS phylogeny, but
no taxonomic conclusions about their generic validities were
made. Recently, Jaklitsch & Voglmayr (2017) proposed that
Mycodidymella and Xenostigmina are synonyms of Petrakia.
They considered that phylogenetic relatedness of Xenostigmina
and Petrakia, and morphological similarity of the sexual morph
and mycopappus-like propagules among these genera are
strong arguments for synonymising them (Jaklitsch & Voglmayr
2017). Our phylogenetic analysis including Mycodidymella as
well as Xenostigmina and Petrakia clarified that their mono-
phyletic status was not well supported in any analyses (below
60 % ML BP/ 0.95 Bayesian PP, Fig. 1). Their sexual morphs
are superficially similar as indicated by Jaklitsch & Voglmayr
(2017),butMycodidymella has deeper and more well-
developed ascomata (up to 210 μm high) than those of Petra-
kia (up to 150 μm high) and Xenostigmina (upto100μm high).
Additionally, their morphological characters of their synasexual
morphs are also different; hyaline, up to 20 μm thick spor-
odochia, holoblastic conidiogenous cells, and sigmoid, multi-
septate, thin-walled, hyaline conidia (Mycodidymella;this
study); brown, up to 30 μm thick sporodochia, annellidic con-
idiogenous cells, and globose to ovoid, dictyosporus, thick-
walled, brown conidia with cellular appendages (Petrakia;
Butin et al. 2013, Li et al. 2016); and brown to black, up to 45 μm
high sporodochia, holoblastic conidiogenous cells, and clavate
with a short rostrum, dictyosporus, thick-walled, brown conidia
(Xenostigmina;Funk 1986, Crous 1998).Therefore, we treat
these genera as distinct based on morphological differences of
sexual and synasexual morphs.
Synasexual morphs of these three genera are produced after
leaves fall in late autumn (Funk & Dorworth 1988, Wei et al.
1997, Butin et al. 2013, Gross et al. 2017). Conidia of syna-
sexual morphs were not observed on overwintered leaves for
Petrakia and Mycodidymella, and their function in the disease
cycle during the winter season has not been clarified (Wei et al.
1997, Butin et al. 2013). No synasexual morph is known from
Pseudodidymella, despite their close relationship to the other
three genera. Further studies on the Pseudodidymella syna-
sexual morph are needed to elucidate the whole life cycle of this
genus and produce robust taxonomic classifications for
Pseudodidymellaceae.
Form and function of mycopappus-like
propagules
The genus Mycopappus was established based on its type
species Mycop. alni (on Alnus,Betula,Crataegus, and Pyrus;
Redhead & White 1985, Braun et al. 2000, Takahashi et al.
2006), which produces epiphyllous, multicellular propagules in
its asexual morph (Redhead & White 1985). Later, three species
were assigned to in this genus: Mycop. aceris (on Acer macro-
phyllum;Redhead & White 1985), Mycop. aesculi (on Aesculus
turbinata;Wei et al. 1998), and Mycop. quercus (on Quercus
acutissima;Suto & Kawai 2000). Two species, Mycop.alni and
Mycop.quercus, produce microconidia and sclerotia in culture
(Redhead & White 1985, Suto & Kawai 2000), and the sexual
morph of the latter species is characterised by stipitate apothecia
and inoperculate asci (Suto & Suyama 2005). Mycopappus alni
was suggested to be a member of Sclerotiniaceae (Helotiales,
Leotiomycetes) based on its sclerotial morph and phylogenetic
analyses using ITS sequences (Takahashi et al. 2006). The two
other species, Mycop. aceris and Mycop. aesculi, were excluded
from Mycopappus sensu stricto, because their sexual morphs
belong to the dothideomycetous taxa, namely Xenostigmina
aceris (Funk & Dorworth 1988, Crous 1998, Crous et al. 2009)
and Mycodidymella aesculi (Wei et al. 1998), respectively.
Morphological differences in mycopappus-like propagules
among these lineages were indicated in a previous study (Suto &
Kawai 2000). The main bodies of sclerotiniaceous species
(Mycop. alni and Mycop. quercus) are composed of multi-septate
claviform cells (Suto & Kawai 2000, Suto & Suyama 2005,
Takahashi et al. 2006), whereas those of dothideomycetous
species (Mycod. aesculi and X. aceris) are composed of asep-
tate globose cells (Redhead & White 1985, Wei et al. 1998). The
morphological resemblance of mycopappus-like propagules be-
tween leotiomycetous and dothideomycetous lineages appears
to be the result of convergent evolution due to similar ecological
function, such as rain-splash dispersal across the leaf surface. A
similar situation was reported in two phylogenetically distinct
genera, Spiroplana (Dothideomycetes) and Spirosphaera (Leo-
tiomycetes), which have spirally coiled, buoyant conidia that
resulted in adaptation to water dispersal in terrestrial or aero-
aquatic environments (Voglmayr et al. 2011).
The mycopappus-like propagules of Pseudodidymellaceae
may contribute to secondary infection of host leaves with high
inoculum potential. Wei et al. (1998) suggested that this morph
plays an important role in disease development. Morphological
variation of the propagules at the generic level was observed, but
the taxonomic significance was not been examined in several
studies (Redhead & White 1985, Wei et al. 1998, Butin et al.
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2013, Gross et al. 2017, Jaklitsch & Voglmayr 2017). Our ob-
servations revealed that morphological features of propagules
differed between Mycodidymella,Petrakia, and Xenostigmina
(with few appendages), and Pseudodidymella (with numerous
appendages). The hyphal appendages of Pseudodidymella
could enhance fungal encounters with Fagus leaves that have
conspicuous wax ornamentation (Denk 2003), as is the case of
asexual fungi with conidial appendages (Nag Raj 1993,
Hashimoto et al. 2015a). The morphological variation of propa-
gules is also observed at the species level: Pseudod. fagi on
F. crenata has a larger main body with longer appendages
(Fig. 5W and X), and Pseudod. minima on F. japonica has a
smaller main body with shorter appendages (Fig. 6G and H).
These morphological variations of their propagules may be
correlated with presence (in F. japonica) or absence (in
F. crenata) of leaf papillae (Denk 2003) as a result of adaptation
to the host surface.
A phoma-like morph is known in the life cycle in Petrakia
(Butin et al. 2013). This morph is also observed in Mycodidy-
mella and Pseudodidymella after fructification of mycopappus-
like propagules (Fig. 4J–R and 5J–R). The conidial-like struc-
tures of this morph appear to be spermatia, because they do not
germinate in water agar or glucose agar.
Speciation through host switching and host
jumping
Plant pathogens frequently infect phylogenetically related hosts
(Jackson 2004, Giraud et al. 2008, Walker et al. 2010, 2012,
Mejía et al. 2011). The genus Pseudodidymella was originally
established as a monotypic genus composed of the type spe-
cies Pseudod. fagi, which was reported to be a pathogen of
F. crenata (Fagaceae,Fagales) in Japan (Wei et al. 1997). Most
recently, this species was re-discovered and reported to be a
disease agent of F. sylvatica in Germany and Switzerland
(Gross et al. 2017). A new species of this genus, Pseudod.
minima, occurs on F. japonica. Members of Pseudodidymella
appear to be host-specificonFagus. Close host/fungus asso-
ciations and coevolution were reported in members of Gno-
moniaceae,Phaeosphaeriaceae, and Sclerotiniaceae (Jackson
2004, Walker et al. 2012, Ertz et al. 2015). Although ITS se-
quences of Pseudod. fagi were 100 % identical among isolates
from F. crenata and F. sylvatica (Gross et al. 2017), those of
Pseudod. minima differed from Pseudod. fagi based on six
nucleotide positions and one gap in ITS sequences (this study).
This result was compatible with host phylogeny: F. crenata and
F. sylvatica are closely related to each other, but F. japonica is
phylogenetically distantly related to the other species (Denk
et al. 2005).
Alternatively, three genera, Mycodidymella,Petrakia, and
Xenostigmina, are host-specific for Acer spp. or Aesculus
(Sapindaceae,Sapindales), which are distantly related to
Fagales (APG IV 2016). It has been recognised that several plant
pathogens switch to unrelated host plants (Reddy et al. 1998,
Takamatsu et al. 2000, Jackson 2004). Gross et al. (2017)
also found that host switching occurred in members of Pseu-
dodidymellaceae, and members of this family evolutionally
diversified by host switching. Similar evolutionary processes that
led to speciation through host jumping are known from Clav-
icipitaceae, which includes plant pathogens, insect pathogens,
and mycoparasites (Kepler et al. 2012).
Future studies
The asexual genus Seifertia on Rhododendron spp. is charac-
terised by synnematous conidiomata with cladosporium-like con-
idia (Li et al. 2016). Phylogenetic relatedness of this genus to
members of Pseudodidymellaceae was suggested (Li et al. 2016,
Gross et al. 2017). However, we prefer to not include this species in
Pseudodidymellaceae and place it incertae sedis, because of the
lack of mycopappus-like propagules in the life cycle. This genus
might represent a new family; however, analysis of its sexual
morph and further taxa related to this genus are needed to
determine its familial placement. Another genus, Alpinaria,was
originally established to accommodate the type species,
A. rhododendri, which was segregated from Melanomma (Jaklitsch
& Voglmayr 2017). They regarded the genus as a member of
Melanommataceae, based on phylogenetic analyses (Jaklitsch &
Voglmayr 2017). In the present study, we newly observed the
asexual morph of Alpinaria, which had not been reported in pre-
vious studies (Müller 1959, Holm 1968, Mugambi & Huhndorf
2009, Jaklitsch & Voglmayr 2017). According to our phylogenetic
analyses and morphological observations, this species is distantly
related to Melanommataceae sensu stricto (Fig. 1) and has atyp-
ical features for Melanommataceae, such as hyaline cells at the
base of ascomata and pseudopycnidial conidiomata. Several
melanomma-like fungi that possess well-developed carbonaceous
ascomata may have evolved several times within Pleosporales,
such as in Cyclothyriellaceae,Ohleriaceae,Nigrogranaceae,Tei-
chosporaceae,Thyridariaceae (Jaklitsch & Voglmayr 2016,
Jaklitsch et al. 2016b). It seems that familial circumscriptions
based merely on sexual morph characters is insufficient to
distinguish the members of Melanommataceae sensu lato.
The present study revealed unexpected diversity of Mela-
nommataceae sensu Tian et al. (2015). Our approaches, which
combined morphological features of both sexual and asexual
morphs with molecular phylogenetic analyses, enabled a re-
circumscription of Melanommataceae sensu stricto and the
establishment of Pseudodidymellaceae. To build a comprehen-
sive taxonomic framework, further discovery of more specimens
along with additional morphological and molecular data would
help elucidate other unresolved lineages of Melanommataceae
sensu lato.
ACKNOWLEDGEMENTS
This work was supported by funding from the Japan Society for the Promotion of
Science (JSPS 26291084, 15H04491, 16J07243, and 16K07474). We thank Y.
Harada for his help with collection of fungal specimens, and anonymous re-
viewers for their valuable comments and suggestions.
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