Fungal Diversity (2014) 68:69–104
DOI 10.1007/s13225-014-0305-6
A molecular phylogenetic reappraisal of the Didymosphaeriaceae
(= Montagnulaceae)
Hiran A. Ariyawansa & Kazuaki Tanaka & Kasun M. Thambugala & Rungtiwa Phookamsak &
Qing Tian & Erio Camporesi & Sinang Hongsanan & Jutamart Monkai & Dhanushka N. Wanasinghe &
Ausanan Mapook & Ekachai Chukeatirote & Ji-Chuan Kang & Jian-Chu Xu & Eric H. C. McKenzie &
E. B. Gareth Jones & Kevin D. Hyde
Received: 18 July 2014 / Accepted: 25 August 2014 / Published online: 26 September 2014
# School of Science 2014
Abstract The ascomycetous families, Didymosphaeriaceae
and Montagnulaceae, have been treated in Pleosporales,
Dothideomycetes, and both include saprobes, endophytes
and pathogens associated with a wide variety of substrates
worldwide. Didymosphaeriaceae was characterized by 1septate ascospores and trabeculate pseudoparaphyses, mainly
anastomosing above the asci, while species in
Montagnulaceae had 1 to multi-septate ascospores and generally cellular pseudoparaphyses. In recent treatments,
Bimuria, Didymocrea, Kalmusia, Karstenula, Montagnula,
Paraphaeosphaeria, Paraconiothyrium and Letendraea were
placed in Montagnulaceae, while only Didymosphaeria has
been placed in Didymosphaeriaceae. New morphological and
molecular data from recent collections have become available
and thus the understanding of the families can be improved.
Based on analyses of concatenated internal transcribed spacer
(ITS) with LSU, SSU and β-tubulin gene sequences, the
taxonomy of the genera classified in these families are reassessed. Our phylogenetic analyses conclude that, the recently introduced Didymosphaeria rubi-ulmifolii with
Pa ra c o ni ot hy r iu m b r as il ie ns e s p e c i e s co m p l e x ,
Alloconiothyrium, Bimuria, Deniquelata, Didymocrea,
Kalmusia, Karstenula, Letendraea, Montagnula,
Neokalmusia, Paraconiothyrium, Paraphaeosphaeria,
Phaeodothis and Tremateia, forms a robust clade named here
D i dy m o s ph a e r i ac e ae . We t h e r e f or e s yn o n ym i z e
Electronic supplementary material The online version of this article
(doi:10.1007/s13225-014-0305-6) contains supplementary material,
which is available to authorized users.
H. A. Ariyawansa : J.<C. Xu : K. D. Hyde (*)
Key Laboratory for Plant Diversity and Biogeography of East Asia,
Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming 650201, People’s Republic of China
e-mail: kdhyde3@gmail.com
H. A. Ariyawansa : K. M. Thambugala : R. Phookamsak : Q. Tian :
S. Hongsanan : J. Monkai : D. N. Wanasinghe : A. Mapook :
E. Chukeatirote : K. D. Hyde
Institute of Excellence in Fungal Research, Mae Fah Luang
University, Chiang Rai 57100, Thailand
H. A. Ariyawansa : J.<C. Xu : K. D. Hyde
World Agroforestry Centre, East Asia and Central, Heilongtan,
Kunming 650201, People’s Republic of China
K. Tanaka
Faculty of Agriculture and Life Science, Hirosaki University, 3
Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan
H. A. Ariyawansa : J.<C. Kang
The Engineering and Research Center for Southwest
Bio-Pharmaceutical Resources of National Education Ministry of
China, Guizhou University, Guiyang 550025, Guizhou Province,
China
E. Camporesi
A.M.B. Gruppo Micologico Forlivese “Antonio Cicognani”, Via
Roma 18, Forlì, Italy
H. A. Ariyawansa : K. M. Thambugala : R. Phookamsak : Q. Tian :
S. Hongsanan : J. Monkai : D. N. Wanasinghe : A. Mapook :
E. Chukeatirote : K. D. Hyde
School of Science, Mae Fah Luang University, Chiang Rai 57100,
Thailand
E. H. C. McKenzie
Manaaki Whenua Landcare Research, Private Bag, 92170 Auckland,
New Zealand
E. B. G. Jones
Department of Botany and Microbiology, King Saudi University,
Riyadh, Saudi Arabia
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Montagnulaceae under Didymosphaeriaceae which is the
oldest name and has priority and provide a update account
of the family. Didymosphaeria is represented by
Didymosphaeria rubi-ulmifolii. A new genus, Neokalmusia,
is introduced in the family to accommodate the
b a m b u s i c o l o u s t a x a K a l m u s i a b re v i s p o r a a n d
K. scabrispora. This introduction is based on molecular data
c o u p l e d w i t h m o r p h o l o g y. O n e n e w s p e c i e s ,
Paraconiothyrium magnoliae and the sexual morph of
Paraconiothyrium fuckelii, is also introduced. Julella and
Barria are assigned to the family based on morphological
similarity with Didymosphaeriaceae. Wilmia (previously referred to the Phaeosphaeriaceae) is synonymized under
Letendraea (Didymosphaeriaceae). Furthermore, a new species, Letendraea cordylinicola, is introduced and placed in
Didymosphaeriaceae based on phylogeny and morphology.
The paraphyletic nature of Paraconiothyrium is partially resolved, but further sampling with fresh collections and molecular data of species in this genus are needed to obtain a natural
classification.
Keywords Didymosphaeriaceae . Montagnulaceae .
New species . Phylogeny . rDNA . β-tubulin
Introduction
Munk A (1953) introduced the family Didymosphaeriaceae,
typified by the genus Didymosphaeria Fuckel. The family was
characterized by 1-septate ascospores and trabeculate
pseudoparaphyses which anastomosed mostly above the asci
(Aptroot 1995; Ariyawansa et al. 2014b). Based on its trabeculate pseudoparaphyses, Barr (1990) referred
Didymosphaeriaceae to the order Melanommatales.
However, the importance of trabeculate pseudoparaphyses
was later disputed (Liew et al. 2000). The confusion surrounding Didymosphaeriaceae and Didymosphaeria was discussed
in detail by Ariyawansa et al. (2014b). They explained that
considering the morphological data only (peridium comprising flattened or irregular cells, 1-septate ascospores and trabeculate pseudoparaphyses mostly anastomosing above the
asci) the family Didymosphaeriaceae appears to be distinct
from Pleosporales. However, on the basis of molecular data
the status of Didymosphaeriaceae as a distinct family was
found to be doubtful as it clustered in Montagnulaceae
(Ariyawansa et al. 2014b). Ariyawansa et al. (2014b) introduced a new species of Didymosphaeria, D. rubi-ulmifolii
Ariyawansa et al., based on morphological comparison with
the generic type, D. futilis (Berk. & Broome) Rehm.
Sequences of Didymosphaeria rubi-ulmifolii clustered within
the Montagnulaceae as a separate genus, thus suggesting that
Montagnulaceae and Didymosphaeriaceae are synonymous.
Fungal Diversity (2014) 68:69–104
Barr (2001) introduced the family Montagnulaceae with
three genera viz. Kalmusia Niessl, Montagnula Berl., and
Didymosphaerella Cooke, in the order Pleosporales, based
on ascomata immersed under a clypeus, a pseudoparenchymatous peridium of small cells, cylindric or oblong,
fissitunicate, pedicellate asci and multi-septate ascospores.
Ascospores in Kalmusia have two to many transverse septa,
in Montagnula they are muriform and in Didymosphaerella
ascospore have a single transverse septum (Barr 2001). The
family was thought to be closely related to Cucurbitariaceae,
but differed in its obtuse ascomatal apex with a minute papillate ostiole and thin-walled, pale ascospores (Barr 2001).
Species in the family Montagnulaceae are parasitic,
saprotrophic or hemibiotropic and also include saprobic species in aquatic environments (Barr 2001; Zhang et al. 2012).
Several recent studies have provided the groundwork for
classification of the family Montagnulaceae (Ariyawansa
et al. 2014b; Verkley et al. 2014). Even though there is a large
body of work comprising taxonomic and phylogenetic studies, most of the genera regarded as members of
Montagnulaceae remain under-studied and therefore poorly
understood for a systematic treatment.
Combined analyses of LSU, SSU, RBP2 and TEF1 gene
data (Schoch et al. 2009; Zhang et al. 2012) showed the family
Montagnulaceae to be well-resolved comprising the genera
Bimuria D. Hawksw et al., Didymocrea Kowalski, Kalmusia
Niessl, Karstenula Speg., Montagnula Berl., and
Paraphaeosphaeria O.E. Erikss. Some species of
Phaeosphaeria, Paraconiothyrium Verkley, and Letendraea
Sacc., also clustered in Montagnulaceae (Schoch et al. 2009;
Zhang et al. 2012). Paraconiothyrium was introduced by
Verkley et al. (2004) to accommodate four species viz.
Parac. estuarinum Verkley & Manuela Silva., Parac.
brasiliense Verkley, Parac. cyclothyrioides Verkley, and
Parac. fungicola Verkley & Wicklow. The sexual morphs of
these species are unknown, but maximum parsimony analysis
of ITS and partial SSU nrDNA sequences showed that these
asexual genera clustered within Pleosporales and grouped in a
clade including Paraphaeosphaeria in Montagnulaceae
(Verkley et al. 2004). In the same study, Coniothyrium
minitans W.A. Campb., C. sporulosum (W. Gams &
Domsch) Aa, and C. minitans W.A. Campb., clustered in the
same clade and were therefore referred to Paraconiothyrium.
Following SSU and ITS analyses, Damm et al. (2008) suggested that Paraconiothyrium can be accommodated within
Paraphaeosphaeria. Zhang et al. (2012) showed that the
asexual morphs of Paraphaeosphaeria michotii (Westend.)
O.E. Erikss. and Paraph. pilleata Kohlm et al. are representatives of Paraconiothyrium in their multi-gene phylogenetic
analysis.
Didymosphaerella has been excluded from the family
Montagnulaceae and placed in Didymellaceae (Zhang et al.
2012). Schoch et al. (2009) and Suetrong et al. (2009) showed
Fungal Diversity (2014) 68:69–104
that the marine ascomycete, Tremateia halophila nested in
Montagnulaceae. Zhang et al. (2012) provided a description
of Montagnula, the type of the family Montagnulaceae, which
also included Bimuria, Didymocrea, Karstenula, Letendraea,
Montagnula, Paraphaeosphaeria and Tremateia, while
Ariyawansa et al. (2013c) added the new genus Deniquelata.
Zhang et al. (2014) neotypified Kalmusia, with K. ebuli
Niessl., to resolve phylogenetic placement of the species
within Kalmusia. Zhang et al. (2014) concluded that
Kalmusia species are polyphyletic within the
Montagnulaceae, with K. ebuli being distant from
K. brevispora (Nagas. & Y. Otani) Y. Zhang et al. and
K. scabrispora (Teng) Kaz. Tanaka et al. The latter two
species appear to represent a different genus.
Verkley et al. (2014) re-assessed Coniothyrium-like taxa
belonging to the Montagnulaceae, based on combine gene
analyses of ITS, LSU, γ-actin and β-tubulin sequences and
proposed Alloconiothyrium Verkley & Stielow, and
Dendrothyrium Verkley & Stielow, as new genera. In the same
study, one new species was described in Paraconiothyrium
while two species so far classified in Paraconiothyrium were
transferred to Paraphaeosphaeria (Verkley et al. 2014).
We have been studying the families of Pleosporales basing
on both morphology and molecular phylogeny in order to
provide a natural classification of this large order
(Ariyawansa et al. 2013a, b, c, 2014a, b, c, d; Hyde et al.
2013; Zhang et al. 2012). This paper aims to clarify the genetic
and taxonomic relationships within the genera of the
Didymosphaeriaceae and Montagnulaceae using a set of isolates that encompasses its genetic, biological and geographic
diversity. Type specimens, or cultures derived from type specimens, have been examined wherever possible.
Material and methods
Specimen examination
Fresh materials of Didymosphaeriaceae and Montagnulaceae
species were collected in Thailand and Italy during 2011-2013.
Specimens were taken to the laboratory in Ziplock plastic bags.
The samples were processed and examined following the method described in Ariyawansa et al. (2013a, b, c). Fresh
and herbarium materials were examined under a Motic SMZ
168 dissecting microscope to locate and isolate ascomata
fruiting bodies. Hand sections of the fruiting structures were
mounted in water for microscopic studies and photomicrography. The taxa were examined using a Nikon ECLIPSE 80i
compound microscope and photographed with a Canon 450D
digital camera fitted to the microscope. Measurements were
made with the Tarosoft (R) Image Frame Work program and
images used for figures processed with Adobe Photoshop CS3
Extended version 10.0 software (Adobe Systems, USA).
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Isolations were made from single ascospores, following a modified method of Ariyawansa et al. (2013b) and Chomnunti et al.
(2014). Contents of the sectioned fruiting body were transferred
to a drop of sterile water on a flame-sterilized slide. Drops of the
spore suspension were pipetted and spread on a Petri-dish
containing 2 % water agar (WA) and incubated at 25 °C.
Germinated ascospores were transferred singly to MEA media
(Alves et al. 2006).
Herbarium specimens were obtained on loan from the
Swedish Museum of Natural History (S) and the New York
Botanical Garden (NY).
Voucher specimens are deposited in the herbarium of Mae
Fah Luang University (MFLU), Chiang Rai, Thailand and
New Zealand Fungarium, Landcare Research (PDD), New
Zealand. The living cultures are deposited at the Mae Fah
Luang University Culture Collection (MFLUCC),
Biodiversity and Climate Research Centre (BiK-F) and
International Collection of Microorganisms from Plants
(ICMP) under Material Transfer Agreement No. 4/2010
(MTA). Each genus is listed along with a description of the
type species, except in cases where there is only a single
species in the genus.
DNA extraction, PCR amplification and sequencing
Single ascospore fungal isolates were grown on MEA or PDA
for 28 days at 25 °C in the dark. Genomic DNA was extracted
from the growing mycelium using the Biospin Fungus
Genomic DNA Extraction Kit (BioFlux®) following the manufacturer’s protocol (Hangzhou, P.R. China). Otherwise DNA
extracted directly from ascomata using a DNA extraction kit
(E.Z.N.A.® Forensic DNA kit, D3591- 01,Omega Bio-Tek)
following Telle and Thines (2008).
The amplification procedure was performed in a 50 μl
reaction volume containing 5–10 ng DNA, 0.8 units Taq
polymerase, 1X PCR buffer, 0.2 mM d’NTP, 0.3 μM of each
primer with 1.5 mM MgCl2 (Cai et al. 2009). The PCR
reactions for amplification of the recently ratified universal
fungal barcode ITS1-5.8S-ITS2 of the nuclear ribosomal
DNA operon (Verkley et al. 2014), were performed under
standard conditions (White et al. 1990; Stielow et al. 2010).
PCR conditions for amplifying the partial SSU and LSU rDNA followed the protocol of Phillips et al. (2008).
Amplification of partial β-tubulin (TUB), covering the variable 5’-end containing four small introns, followed the protocol of Aveskamp et al. (2009) and Carbone and Kohn (1999).
The PCR products were observed on 1 % agarose electrophoresis gels stained with ethidium bromide. Purification and
sequencing of PCR products were carried at Shanghai
Sangon Biological Engineering Technology and Services
Co., (China).
DNA sequence data was obtained from the internal transcribe spacer (ITS), small and large subunits of the nuclear
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ribosomal RNA genes (SSU, LSU) and the protein coding
gene, β-tubulin (TUB). Primer sets used for these genes were
as follows: ITS: ITS5/ITS4 SSU: NS1/NS4; LSU: LR0R/
LR5; TUB4Rd/TUB4Fd (Aveskamp et al. 2009; Carbone
and Kohn 1999). Primer sequences are available at the
WASABI database at the AFTOL website (aftol.org).
Sequences are deposited at NCBI GenBank under the accession numbers provided in Supplementary Table 1. Alignments
are deposited in TreeBASE.
Sequence alignment and phylogenetic analysis
Combined ITS, LSU, SSU and TUB genes were used in the
analysis. All sequences obtained from GenBank and used by
Ariyawansa et al. (2014d), Boonmee et al. (2014), Hyde et al.
(2013), Phookamsak et al. (2014) Schoch et al. (2009),
Suetrong et al. (2009), Verkley et al. (2014), Wijayawardene
et al. (2014) and Zhang et al. (2012) are listed in supplementary Table 1. Multiple sequence alignments were generated
with MAFFT v. 6.864b (http://mafft.cbrc.jp/alignment/server/
index.html). All introns and exons were aligned separately.
Regions containing many leading or trailing gaps were
removed from the ITS, SSU and LSU alignments prior to
tree building. The alignments were checked visually and
improved manually where necessary. Concordance of the
ITS, SSU, LSU and TUB genes data sets was estimated with
the partition-homogeneity test implemented with PAUP v. 4.
0b10 (Swofford 2002).
Maximum parsimonious analysis was performed to obtain
the most parsimonious tree. Trees were inferred using the
heuristic search option with 1,000 random sequence additions
and tree-bisection-reconnection (TBR) branch-swapping algorithm. All gaps were treated as missing data. Maxtrees were
setup to 50,000 and branches of zero length were collapsed
and all multiple parsimonious trees were saved. Descriptive
tree statistics for parsimony (Tree Length [TL], Consistency
Index [CI], Retention Index [RI], Relative Consistency Index
[RC] and Homoplasy Index [HI] were calculated for trees
generated under different optimality criteria. KishinoHasegawa tests (KHT) (Kishino and Hasegawa 1989) were
performed in order to determine whether trees were significantly different. Maximum parsimony bootstrap values (MP)
equal or greater than 50 % are given below or above each
node.
Maximum likelihood analyses including 1,000 bootstrap
replicates were run using RAxML v. 7.2.6 (Stamatakis
2006; Stamatakis et al. 2008) The online tool Findmodel
(http://www.hiv.lanl.gov/content/sequence/findmodel/
findmodel.html) was used to determine the best nucleotide
substitution model for each partition. The best scoring tree
was selected with a final likelihood value of -19492.551787.
The resulting replicates were plotted on to the best scoring tree
obtained previously. Maximum Likelihood bootstrap values
Fungal Diversity (2014) 68:69–104
Fig. 1 RAxML tree based on a combined dataset of SSU, LSU, RPB2
and TEF1, bootstrap support values for maximum likelihood (blue),
maximum parsimony (green) greater than 50 % and bayesian posterior
probabilities greater than 0.90 (black) below and above the nodes.
Halojulella avicenniae is the outgroup taxon. The original isolate numbers are noted after the species names and ex-type strains are in bold. The
type species of each genus is indicated in blue
(ML) equal or greater than 50 % are given below or above
each node in red (Fig. 1).
The model of evolution was performed by using
MrModeltest 2.2 (Nylander 2004). Posterior probabilities
(PP) (Rannala and Yang 1996; Zhaxybayeva and Gogarten
2002) were determined by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v. 3.0b4 (Huelsenbeck and
Ronquist 2001). Six simultaneous Markov chains were run
for 3,000,000 generations and trees were sampled every 100th
generation and 30,000 trees were obtained. The first 6,000
trees, representing the burn-in phase of the analyses, were
discarded while the remaining trees were used for calculating
posterior probabilities in the majority rule consensus tree
(critical value for the topological convergence diagnostic set
to 0.01) (Crous et al. 2006). Bayesian Posterior Probabilities
(BYPP) equal or greater than 0.90 is given below or above
each node (Fig. 1). Phylogenetic trees and data files were
viewed in MEGA v. 5 (Tamura et al. 2011), TreeView v.
1.6.6 (Page 1996) and FigTree v. 1.4 (Rambaut and
Drummond 2008).
Results
Phylogeny of combined ITS, LSU, SSU and TUB gene
datasets
The combined ITS, LSU, SSU and TUB dataset comprises 76
taxa. Halojulella avicenniae is selected as an outgroup taxon.
Results of the partition-homogeneity test (P=0.107) indicate
that the ITS, LSU, SSU and TUB gene trees reflect the same
underlying phylogeny. Therefore, these datasets were combined and analyzed by using several tree-building programs,
the resulting trees compared and the best tree is presented in
Fig. 1. New sequences are deposited in GenBank (Table 1).
Phylogenetic analysis
The combined ITS, LSU, SSU and TUB gene dataset from
five families in the Pleosporales is shown in Fig 1. All trees
(ML, MP and BYPP) were similar in topology and not significantly different (data not shown). The maximum parsimony dataset consists of 2,283 characters and of which 2,437
were constant, 300 variable characters parsimonyuninformative and 776 characters parsimony-informative.
Fungal Diversity (2014) 68:69–104
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Kishino-Hasegawa (KH) test showed length = 3,000 steps, CI
= 0.532, RI = 0.673, RC = 0.358 and HI = 0.468. A best
scoring RAxML tree is shown in Fig. 1, with the value of 19492.551787. Phylogenetic trees obtained from Maximum
Likelihood, Maximum Parsimony and Bayesian analysis
yielded trees with similar overall topology at subclass and
family relationships in agreement with previous work based
on Maximum Likelihood analysis (Ariyawansa et al. 2014d;
Boonmee et al. 2014; Hyde et al. 2013; Phookamsak et al.
2014; Schoch et al. 2009; Suetrong et al. 2009; Verkley et al.
2014; Wijayawardene et al. 2014 and Zhang et al. 2012).
In the multi-locus phylogeny inferred from the combined
dataset shown in Fig. 1, several well-supported sub clades can
be identified in the family Didymosphaeriaceae, which are
interpreted as appropriate for the delimitation of genera, i.e.
Alloconiothyrium, Bimuria, Deniquelata, Didymosphaeria,
Didymocrea, Kalmusia, Karstenula, Letendraea,
Montagnula, Neokalmusia, Paraconiothyrium,
Paraphaeosphaeria, Phaeodothis and Tremateia. Thus the
Didymosphaeriaceae incorporates members of
Montagnulaceae. The phylogenetic data for each genus is
further discussed after the genus notes.
The Didymosphaeria clade comprises the type strain
D. rubi-ulmifolii (MFLUCC 14-0023) plus another strain of
D. rubi-ulmifolii (MFLUCC 14-0024), which clustered with
the type strain of Paraconiothyrium brasiliense Verkley (CBS
100299) and synonymised under D. rubi-ulmifolii and defined
here as D. rubi-ulmifolii sensu stricto. Several strain collections of Paraconiothyrium brasiliense (CBS 122319, CBS
122320, CBS 587.84, CBS 115.92 and CBS 115.92) other
than the type strain, form a distinct clade to D. rubi-ulmifolii
clade, thus treated here as D. rubi-ulmifolii sensu lato. The
closely related Paraconiothyrium variabile (CBS 120014)
forms a separate clade in Didymosphaeria clade which is
synonymised under Didymosphaeria as Didymosphaeria
variabile. Another well-supported (100/1.00/98) clade
forming the major part of the ingroup of the tree comprises
six strains assigned to Paraphaeosphaeria, with three isolates
of Paraph. michotii, the type species of the genus, and the
relatively highly supported clades of the following species:
Paraph. angularis Verkley & van der Aa (CBS 167.70),
Paraph. minitans (W.A. Campb.) Verkley & Stielow (CBS
111750), Paraph. arecacearum Verkley & Stielow (CBS
158.75) and Paraph. viridescens Verkley et al. (CBS
854.73). A putative strain of Karstenula rhodostoma (CBS
690.94) forms a distinct clade sister to Paraphaeosphaeria
clade.
The Paraconiothyrium clade comprises 13 strains with
Parac. estuarinum (CBS 109850T), the type species of
Paraconiothyrium,: Parac. fuckelii (6), Parac. africanum
(CBS 121166), Parac. hawaiiense (CBS 120025), Parac.
fungicola (CBS 113269), Parac. estuarinum (CBS 109850),
Parac. cyclothyrioides (CBS 972.95), Parac. archidendri
Fungal Diversity (2014) 68:69–104
(CBS 168.77) and the new species Parac. magnoliae
(MFLUCC 10-0278) which clusters with Parac. fungicola.
Kalmusia (74/1.00/54) contains five strains, including
Kalmusia ebuli (CBS 123120T), the type species of the genus,
Kalmusia sp. (MFLUCC 14-0560), K. variisporum (CBS
121517T) and K. longisporum (CBS 824.84, 582.83T). The
monotypic asexual genus Alloconiothyrium (CBS 980.95T,
981.95) forms a robust clade sister to Kalmusia.
Letendraea forms a relatively well supported clade within
the family Didymosphaeriaceae. This clade contains five
strains with Letendraea eurotioides (CBS 212.31), the type
species of Letendraea, and the relatively highly supported
clades of the following species: L. padouk (CBS 485.70),
L. helminthicola (CBS 884.85) and the two isolates of the
novel species L. cordylinicola (MFLUCC11-0148 T,
MFLUCC 11-0150) which clusters with L. padouk. The new
genus Neokalmusia forms a robust clade sister to Deniquelata,
Didymocrea and Phaeodothis. Neokalmusia comprises five
strains N. brevispora (CBS 120248, NBRC 106240),
N. scabrispora (CBS 120246, NBRC 106237) and
Neokalmusia sp.. Another well-supported (70/0.91/74) clade
is formed by members of Montagnula, the basal clade in the
family Didymosphaeriaceae, which comprises four stains including Montagnula opulenta (CBS 168.34), M. aloes (CPC
19671), Montagnula sp. (Letendraea helminthicola) (CHTA
R43) and M. anthostomoides (CBS 615.86).
Taxonomy
Didymosphaeriaceae Munk, Dansk bot. Ark. 15(no. 2): 128
(1953), Facesoffungi number: FoF 00200.
= Montagnulaceae M.E. Barr, Mycotaxon 77: 194 (2001)
Saprobic or parasitic in terrestrial or aquatic environments.
Sexual state: Ascomata scattered or gregarious, immersed in
substrate, surrounded with hyphae forming an apical clypeus,
dark brown to black, globose to rarely subglobose, medium to
large sized, and coriaceous. Ostiole immersed to sub-immersed, periphysate when well-developed with a short apex,
narrow or wide, usually circular in section. Peridium two
layered or often three layered in the upper region, composed
of cells of textura angularis or textura intricata, dark brown to
black, coriaceous, with outermost layer consisting of black,
highly pigmented thick-walled conglutinate cells or pseudoparenchymatous cells, with middle layer less pigmented, cells
visible via apex, with innermost layer comprising narrow,
compressed rows of cells. Hamathecium of dense, septate,
narrow, cellular or trabeculate pseudoparaphyses often in a
gelatinous matrix. Asci 2–8-spored, bitunicate, fissitunicate,
cylindric or oblong, pedicellate, with an ocular chamber.
Ascospores uniseriate or biseriate, oblong to narrowly oblong,
hyaline, brown to reddish-brown or dark yellowish-brown,
one to several septate (phragmosporous or muriform),
verruculose, with or without a gelatinous sheath. Asexual
Fungal Diversity (2014) 68:69–104
states: coelomycetous (Barr 2001), Kalmusia and
Paraphaeosphaeria have Coniothyrium-like, Cytoplea,
Microsphaeropsis asexual stages. The generic type of
Paraphaeosphaeria (P. michotii) was linked with
Coniothyrium scirpi Trail (Webster 1955). The
Coniothyrium-like species were subdivided into four groups
by Sutton (1980), viz. Coniothyrium, Microsphaeropsis,
Cyclothyrium and Cytoplea. Paraconiothyrium, which is
closely related to Paraphaeosphaeria based on SSU
sequences phylogeny (Verkley et al. 2004), was introduced
to accommodate Coniothyrium minitans and
C. sporulosum.
Notes: The Didymosphaeriaceae is amended here to include Didymosphaeria and genera from Montagnulaceae including asexual states. We synonymise Montagnulaceae under Didymosphaeriaceae which is the oldest name and has
priority and provide an updated account of the family.
Type: Didymosphaeria Fuckel, Jb. Nassau. Ver. Naturk.
23-24: 140 (1870) [1869-70].
Genera accepted in Didymosphaeriaceae
Alloconiothyrium Verkley et al., Persoonia, 32: 33 (2014).
Type species: Alloconiothyrium aptrootii Verkley et al.,
Persoonia, 32: 33 (2014).
Barria Z.Q. Yuan, Mycotaxon 51: 313 (1994).
Type species: Barria piceae Z.Q. Yuan, Mycotaxon 51:
314 (1994).
Bimuria D. Hawksw. et al., N.Z. Jl. Bot. 17(3): 267 (1979).
Type species: Bimuria novae-zelandiae D. Hawksw. et al.,
N.Z. Jl. Bot. 17(3): 268 (1979).
Deniquelata Ariyawansa & K.D. Hyde, Phytotaxa 105(1):
13 (2013).
Type species: Deniquelata barringtoniae Ariyawansa &
K.D. Hyde, Phytotaxa 105(1): 15 (2013).
Didymocrea Kowalski, Mycologia 57(3): 405 (1965).
Type species: Didymocrea sadasivanii (T.K.R. Reddy)
Kowalski, Mycologia 57(3): 405 (1965).
Basionym: Didymosphaeria sadasivanii K.R.C. Reddy,
Mycologia 53(5): 471 (1962) [1961].
Didymosphaeria Fuckel, Jb. Nassau. Ver. Naturk. 23–24:
140 (1870) [1869-70].
Type species: Didymosphaeria futilis (Berk & Broome)
Rehm, Hedwigia 18: 167 (1879).
Basionym: Sphaeria futilis Berk. & Broome, Ann. Mag.
nat. Hist., Ser. 2 9: 326 (1852).
Julella Fabre, Annls Sci. Nat., Bot., sér. 6, 9: 113. 1879
[1878]. MycoBank MB 2539.
Type species: Julella buxi Fabre, Annls Sci. Nat., Bot., sér.
6, 9: 113. 1878.
Kalmusia Niessl, Verh. nat. Ver. Brünn 10: 204 (1872).
Type species: Kalmusia ebuli Niessl, Verh. nat. Ver. Brünn
10: 204 (1872).
Karstenula Speg., Decades Mycologicae Italicae 7–12: no.
94 (in sched.) (1879).
75
Type species: Karstenula rhodostoma (Alb. & Schwein.)
Speg., Decades Mycologicae Italicae 7–12: no. 94 (1879).
Basionym: Sphaeria rhodostoma Alb. & Schwein., Consp.
fung. (Leipzig): 43 (1805).
Letendraea Sacc., Michelia 2(6): 73 (1880).
Type species: Letendraea eurotioides Sacc., Michelia 2(no.
6): 73 (1880).
Montagnula Berl., Icon. fung. (Abellini) 2: 68 (1896).
Type species: Montagnula infernalis (Niessl) Berl., Icon.
fung. (Abellini) 2(2–3): 68 (1896).
Basionym: Leptosphaeria infernalis Niessl, Inst. Coimbra
31: 13 (1883).
Neokalmusia Kaz. Tanaka et al. (In this study)
Type species: Neokalmusia brevispora (Nagas. & Y.
Otani) Tanaka et al.,
Paraconiothyrium Verkley, Stud. Mycol. 50(2): 327 (2004)
Type species: Paraconiothyrium estuarinum Verkley
et al., Stud. Mycol. 50(2): 327 (2004).
Paraphaeosphaeria O.E. Erikss., Ark. Bot., Ser. 2 6: 405
(1967).
Type species: Paraphaeosphaeria michotii (Westend.)
O.E. Erikss., Cryptogams of the Himalayas 6: 405 (1967).
Basionym: Sphaeria michotii Westend., Bull. Acad. R. Sci.
Belg., Cl. Sci., sér. 2 7(5): 87 (1859).
Phaeodothis Syd. & P. Syd., Annls mycol. 2(2): 166 (1904).
Type species: Phaeodothis tricuspidis Syd. & P. Syd.,
Annls mycol. 2(2): 166 (1904).
Tremateia Kohlm. et al., Bot. Mar. 38(2): 165 (1995).
Type species: Tremateia halophila Kohlm. et al., Bot. Mar.
38(2): 166 (1995).
Key to the genera accepted in Didymosphaeriaceae
1. Parasitic on living leaves.................................. Deniquelata
1. Saprobic on dead wood or leaves.......................................... 2
2. Asci with 2 spores.................................................................... 3
2. Asci with more than 2 spores................................................. 4
3. Asci with short knob-like pedicel and brown, muriform
ascospores................................................................... Bimuria
3. Asci without short knob-like pedicel and hyaline, muriform
ascospores....................................................................... Julella
4. Peridium composed of thin pseudoparenchymatous cells of
textura intricata.............................................. Didymosphaeria
4. Peridium composed of pseudoparenchymatous cells of
textura angularis or textura prismatica................................. 5
5. Ascus with a long, furcate pedicel........................................ 6
5. Ascus with a short furcate pedicel.......................................... 7
6. Clavate ascus with narrowly ovoid to clavate, pale brown, 3septate ascospores..................................................... Kalmusia
6. Cylindro-clavate to clavate asci with oblong to narrowly
oblong, reddish-brown to dark yellowish-brown, muriform
or phragmosporous ascospores............................ Montagnula
7. Ascomatal wall composed only cells of textura intricata.........8
76
7. Ascomatal wall composed both cells of textura angularis and
textura prismatica............................................................ Barria
8. Strictly in marine environments................................ Tremateia
8. In terrestrial environments......................................................... 9
9. Hamathecium consisting of filiform pseudoparaphyses
.................................................................................. Didymocrea
9. Hamathecium consisting of cellular pseudoparaphyses........... 10
10. Ascomata with a pore-like ostiole......................................... 11
10. Ascomata with short papillate ostiole.................................. 12
11. Cylindrical asci with short pedicel and fusiform with rounded
ends, olivaceous-brown, phragmosporous ascospores...................................................................... Phaeodothis
11. Cylindrical to cylindro-clavate asci with short furcate pedicel
and ellipsoid to fusoid, reddish-brown to dark brown
muriform ascospores.............................................. Karstenula
12. Ascomata with hyaline periphyses.................... Neokalmusia
12. Ascomata without hyaline periphyses.........Paraphaeosphaeria
Key to asexual genera of Didymosphaeriaceae
1. Conidiomata pycnidial and with a single cavity, or eustromatic
with several cavities............ Alloconiothyrium
1. Conidiomata eustromatic, simple or complex, rarely pycnidial........................................................... Paraconiothyrium
Alloconiothyrium Verkley et al., Persoonia, 32: 33 (2014),
Facesoffungi number: FoF 00028.
Type species:
Alloconiothyrium aptrootii Verkley et al., Persoonia, 32:
33 (2014), Facesoffungi number: FoF 00029.
Saprobic in terrestrial environments. Sexual state: unknown (Verkley et al. 2014). Asexual state: Conidiomata
300–450μm diam, pycnidial and with a single cavity, or
eustromatic and consisting of complexes reaching 1mm diam.,
with several cavities, with outer surface black, glabrous or
covered by grey mycelium. Conidiomatal wall composed of
an outer layer of brown, thick-walled cells of textura
angularis and an inner layer of hyaline, thick-walled cells of
textura angularis, the outer surface sometimes covered by a
diffuse web of brown hyphae. Conidiogenous cells 4–9×3–
4μm, discrete, often positioned on clumps of cells that protrude into the cavity, broadly ampulliform, holoblastic,
annellidic, often with an elongated neck showing several
distinct percurrent proliferations. Conidia 3–4(–5)×2–3(–
3.5) μm, globose to irregularly ellipsoid, aseptate, initially
hyaline, after secession olivaceous-brown, mature conidial
wall orange-brown, the outer surface verruculose giving the
conidium an irregular outline, with 1 large oil-droplet 1–
1.5μm diam (description from Verkley et al. 2014).
Notes: Alloconiothyrium was introduced to accommodate
Coniothyrium-like species in the Montagnulaceae (Verkley
et al. 2014). Alloconiothyrium aptrootii is known from a soil
sample collected in Papua New Guinea, and all other
Fungal Diversity (2014) 68:69–104
Coniothyrium-like taxa studied in Verkley et al. (2014) were
relatively distantly related. The annellidic conidiogenous cells
and the verruculose conidia resemble Coniothyrium
palmarum, the type species of Coniothyrium, but that species
is characterized by 2-celled conidia and is also genetically
distinct, belonging in the family Leptosphaeriaceae (de
Gruyter et al. 2009). In our phylogeny, Alloconiothyrium
forms a robust clade, sister to the Kalmusia clade. Thus, based
on morphology coupled with DNA data, we confirm that this
genus is well resolved and should be classified under
Didymosphaeriaceae.
Barria Z.Q. Yuan, Mycotaxon 51: 313 (1994).
Type species:
Barria piceae Z.Q. Yuan, Mycotaxon 51: 314 (1994)
(Fig 2), Facesoffungi number: FoF 00031.
Parasitic on Picea schrenkiana. Sexual state: Ascomata
240–300×270–330μm, solitary, sometimes gregarious, immersed, visible as black spots on host surface, uniloculate,
globose, brown to dark brown, with centrally opening ostiole.
Ostiole papillate to depressed, ostiolar canal filled with
periphyses. Peridium 10–23μm wide, composed of two cell
types, outer layers comprising 3–5 layers, thin-walled, dark
brown to black, pseudoparenchymatous cells of textura
angularis or textura prismatica, inner layers composed of 2–
3 layers of hyaline, thin-walled cells of textura angularis.
Hamathecium composed of 1.5–3μm wide, broad, hyaline,
septate pseudoparaphyses, surrounding the numerous asci and
embedded in a gelatinous matrix. Asci (126–) 130–170(–
185)× (15–)17–20(–23) μm (x= 151.2× 19μm, n =25), 8spored, bitunicate, fissitunicate, clavate to cylindric-clavate,
shortly acute pedicel or sub-sessile, apically rounded with a
well-developed ocular chamber, arising from the base of the
ascoma. Ascospores (18.5–)20–24.5(–27) × 9–12.5 μm
(x =22×10.9μm, n=30), overlapping, 1–2-seriate, ellipsoidal
to broadly fusiform, initially hyaline, becoming brown to dark
brown at maturity, 1-septate, constricted at the septum,
smooth to rough-walled, with small guttules, comprising two
layers, endospore thin-walled, epispore thick-walled, mostly
with upper cell larger than lower cell, surrounded by a distinct
mucilaginous sheath. Asexual state: unknown.
Material examined: CHINA, Xinjiang, Urumqi, Tianshan
Mountain, on leaves of Picea schrenkiana Fisch. & C.A. Mey
(Pinaceae), 1 July 1992, Z.Q. Yuan (NY 00164027, isotype).
Notes: The monotypic genus Barria shares similarities with
Didymopleella in ascomatal structure, asci and ascospores
characters, but differs in having a peridium of textura
prismatica and strongly unequal ascospore cells (Munk
1957; Yuan 1994). Recently, Zhang et al. (2012) tentatively
assigned Barria in Phaeosphaeriaceae based on the
ascomata, colour and shape of ascospores. Barria differs from
Phaeosphaeria in having ascomata with thick peridium, clavate, short-pedicellate asci, and didymosporous ascospores,
while Phaeosphaeria has broadly cylindrical to cylindric-
Fungal Diversity (2014) 68:69–104
77
Fig. 2 Barria piceae (NY 00164027, isotype). a Herbarium label and
specimens of Barria piceae. b Appearance of ascomata on host surface. c
Vertical section through ascoma d Section through peridium. e
Pseudoparaphyses in Congo red reagent. f Asci with pseudoparaphyses.
g, h-i Asci. j Ocular chamber in congo red reagent. k-n Ellipsoidal to
broadly fusiform ascospores with a sheath. Scale bars: c=100μm, d-i=
20μm, j-m=10μm
clavate asci with sub-sessile pedicellate and phragmospores
ascospores.
Didymosphaeria shows similarities with Barria in having
immersed to slightly erumpent ascomata under a clypeus,
hyaline pseudoparaphyses, anastomosing frequently above
the asci and 1-septate, ellipsoid ascospores. However, Barria
differs from Didymosphaeria in having brown to dark brown
ascomata comprising several layers of textura angularis to
textura prismatica cells in the peridium, cellular
pseudoparaphyses and asci having well-developed ocular
78
chamber bearing ellipsoidal to broadly fusiform, initially ascospores hyaline, becoming brown to dark brown at maturity,
1-septate with a distinct mucilaginous sheath.
Didymosphaeria has hyaline to pale brown or (rarely) black
peridium, consisting of an internal and external layers, flattened or elongated hyphae, textura intricata with trabeculate
pseudoparaphyses and distoseptate ascospores without a
s h e a t h ( A p t r o o t 1 9 9 5 ) . We p l a c e B a r r i a i n
Didymosphaeriaceae because of its similarities with other
genera in Didymosphaeriaceae. However, fresh collections
of the type species of the genus are needed so that molecular
data can be obtained to verify the natural taxonomic affinities
of this genus.
Bimuria D. Hawksw et al., N. Z. Jl. Bot. 17: 268 (1979).
Type species:
Bimuria novae-zelandiae D. Hawksw. et al., N.Z. Jl. Bot.
17(3): 268 (1979), (Fig 3), Facesoffungi number: FoF 00033.
Saprobic in terrestrial habitats. Sexual state: Ascomata
(185-)200×310(-330) μm diam., semi-immersed or superficial, solitary, scattered, globose, hyaline when young, turning
dark brown to black when mature, ostiolate, the ostiole more
or less sessile or raised into a very short neck. Peridium 5–
8μm diam., comprising 2–3 layers of compressed pseudoparenchymatous cells, outer layer comprising sub-hyaline at first,
but becoming pale brown with age cells of textura angularis,
inner wall composed of hyaline radially compressed cells of
Fungal Diversity (2014) 68:69–104
textura angularis. Hamathecium of dense, 2.5–4μm broad,
septate, cellular pseudoparaphyses, embedded in mucilage,
rarely anastomosing and branching. Asci 82–95×20–33μm
(x = 75 × 29 μm, n = 10), (1-)2(-3)-spored, bitunicate,
fissitunicate, broadly clavate, with a short and small knoblike pedicel, ocular chamber best seen in immature asci.
Ascospores 55–68×25–28μm (x =59×26μm, n=10), accumulating in a subglobose black shiny mass adhering together
outside the ostiole, broadly ellipsoid but becoming narrowed
towards the poles, muriform with (5-) 7 transverse septa, cells
with (0-)l(-2) longitudinal septa in each cell, not constricted at
the septa, dark brown, apical cells paler and lacking longitudinal septa, verruculose. Asexual state: unknown.
Material examined: NEW ZEALAND, North Island,
Wairarapa District, Nutty Farm, isolated from soil, 3 March
1978, C.C. Yen and J.E. Sheridan (CBS 107.79, isotype).
Notes: Bimuria is a monotypic genus characterized by a
very thin peridium, mostly 2-spored, fissitunicate asci and
muriform, dark brown, verrucose ascospores (Hawksworth
et al. 1979). Because of its unique morphological characters,
the familial placement of this genus has been debatable, thus
initially Hawksworth et al. (1979) placed the genus in
Pleosporaceae. Later Barr (1987) transferred Bimuria to
Phaeosphaeriaceae while Lumbsch and Huhndorf (2007)
placed it in Melanommataceae. Morphologically, Bimuria is
most similar to Montagnula (Hawksworth et al. 1979).
Fig. 3 Bimuria novae-zelandiae (CBS 107.79, isotype). a-c Close up of ascoma. d Hyaline, pseudoparaphyses. e-g Two-spored asci with knob like
pedicel. h-j Dark brown, muriform ascospores. Scale bars: a-b=100μm, c=5μm, d=5μm, e-g=30μm, i-j=10μm
Fungal Diversity (2014) 68:69–104
However, the thick carbonaceous peridium distinguishes
Montagnula from Bimuria (Hawksworth et al. 1979). In
addition, the ascospores of Montagnula are discharged
forcibly through the ostiole instead of forming a mass
outside of the ostiole as in Bimuria (Hawksworth et al.
1979). Ascomauritiana lignicola V.M. Ranghoo & K.D.
Hyde has somewhat similar ascospores in 4-spored asci,
but this taxon has unitunicate asci (Ranghoo and Hyde
1999). Hawksworth et al. (1979) suggested that the morphological characters of Bimuria, such as ascospore release
and large, thick-walled ascospores may be an adaptation to
its soil-borne habitat.
Phylogenetic analysis based on combined genes of SSU,
LSU, RPB2 and TEF1 concluded that the type strain of
Bimuria novae-zelandiae (CBS 107.79) nested in the
Montagnulaceae (Schoch et al. 2009). Our phylogenetic study
also showed similar results, with Bimuria forming a monophyletic clade sister to Tremateia. Therefore we include
Bimuria as a well-established genus in Didymosphaeriaceae
based on both morphology and phylogeny.
Deniquelata Ariyawansa & K.D. Hyde, Phytotaxa 105(1):
13 (2013), Facesoffungi number: FoF 00034.
Type species:
Deniquelata barringtoniae Ariyawansa & K.D. Hyde,
Phytotaxa 105(1): 15 (2013) (Fig 4), Facesoffungi number:
FoF 00035.
Pathogenic on living leaves of Barringtonia asiatica.
Sexual state: Ascomata 150–180×164–190μm (x=175×
167μm, n=10), immersed, scattered, globose to subglobose,
black to dark brown, smooth-walled, with a papillate to depressed, elongate ostiole. Peridium 9–17μm diam. (x=12μm,
n=10), composed of 3–5 layers of brown to black, darkly
pigmented, small, thick-walled, 2–5μm wide cells of textura
angularis, with outer wall fused with the host cells, inner wall
consists of 2-layers of polygonal to rectangular, light brownhyaline cells 1–4μm diam. Hamathecium composed of dense,
1–3 μm diam. (x= 2 μm, n = 20), broad, hyaline, septate
pseudoparaphyses, surrounding the numerous asci and embedded in a gelatinous matrix. Asci (-60) 68–80×10–15μm
(x=72×13μm, n=20), 8-spored, bitunicate, fissitunicate, clavate to broadly-clavate, with a 9–17μm, short, broad, furcate,
long pedicel, rounded at apex, with an ocular chamber.
Ascospores 14–16×5–7μm (x=15×6μm, n=40), biseriate or
distichously arranged, partially overlapping, oblong to narrowly oblong, straight or somewhat curved, reddish-brown,
with three transverse septa and 1–2 vertical septa in the central
cells, constricted at the primary and secondary septa at maturity, verruculose, straight or slightly inequilateral, without a
gelatinous sheath. Asexual state: unknown.
Material examined: THAILAND, Chiang Rai Province.,
Muang District, Bandu, Baan Khuakhae, on leaf of
Barringtonia asiatica (L.) Kurz (Lecythidaceae), 18
September 2011, K.D. Hyde (MFLU 12-0303, holotype).
79
Notes: Deniquelata is a monotypic genus characterized by
immersed, dark brown to black ascomata, with bitunicate asci
and brown, muriform ascospores. Deniquelata differs considerably from the type of Montagnula (M. infernalis) both in phylogeny and morphology (Ariyawansa et al. 2013c). Deniquelata
is a pathogenic genus with fruiting bodies scattered in the
necrotic host tissues. Ariyawansa et al. (2013c) concluded that
phylogenetic analysis of the nucleotide sequences of combined
18S and 28S nrDNA provided good evidence that Deniquelata
belongs in Montagnulaceae. In the present study Deniquelata
forms a distinct clade sister to Bimuria and Tremateia. Thus we
accept this genus under Didymosphaeriaceae.
Didymosphaeria Fuckel, Jb. nassau. Ver. Naturk. 23-24:
140 (1870) [1869–70], Facesoffungi number: FoF 00036.
Saprobic on woody branches and herbaceous stems and
leaves. Sexual state: Ascomata solitary, scattered, or in small
groups, immersed to erumpent, globose to ovoid, ostiolate.
Ostiole papillate with a pore-like opening, ostiolar canal filled
with hyaline cells (periphyses). Peridium 1-layered, thin,
composed of brown pseudoparenchymatous cells of textura
intricata. Hamathecium of dense, trabeculate,
pseudoparaphyses, anastomosing mostly above the asci. Asci
8-spored, bitunicate, fissitunicate, cylindrical, with a furcated
pedicel, apically rounded with an indistinct ocular chamber.
Ascospores uniseriate, ellipsoid, brown, 1-septate, wall with
different ornamentations. Asexual state: see notes below.
Type species:
Didymosphaeria futilis (Berk. & Broome) Rehm,
Hedwigia 18: 167 (1879) (Fig 5), Facesoffungi number:
FoF 00037.
Basionym: Sphaeria futilis Berk. & Broome, Ann. Mag.
nat. Hist., Ser. 2 9: 326 (1852).
Saprobic on dead wood. Sexual state: Ascomata 110–140×
120–160μm (x=130×140μm, n=10), scattered, or in small
groups, immersed to slightly erumpent, rarely nearly superficial, under a clypeus, globose to subglobose, membraneous,
papillate. Papilla black, with a pore-like ostiole, ostiolar canal
filled with periphyses. Peridium 10–20 μm (x=15μm, n=10)
wide, 1-layered, composed of hyaline pseudoparenchymatous
compressed cells of textura intricata, fusing at the outside
with the host. Hamathecium of dense, 0.5–1μm (x =0.8μm,
n=20) broad, long, trabeculate pseudoparaphyses, anastomosing frequently above the asci, embedded in mucilage. Asci 75–
85 × 4–6 μm (x= 78 × 6 μm, n = 20), 8-spored, bitunicate,
fissitunicate, cylindrical, pedicellate, rounded at the apex with
an indistinct ocular chamber. Ascospores 7–10×3–5μm (x =
9×5μm, n=40), uniseriate, slightly overlapping, ellipsoid
with obtuse ends, brown, 1-septate, slightly to not constricted
at the septum, with distinctly spinulose ornamentation.
Asexual state: unknown.
Material examined: UK. England: Norfolk, on dead stems
of Rosa sp. (Rosaceae), March 1850, M.J. Berkeley (K
147683, holotype of Sphaeria futilis).
80
Fungal Diversity (2014) 68:69–104
Fig. 4 Deniquelata barringtoniae (MFLU 12-0303, holotype) a-b Fungus on host. c-d Section of ascoma (TS). e Section of peridium comprising a few layers of cells. f Hyaline pseudoparaphyses g-i Eight-spored
asci with short, broad pedicel at the base. j Reddish-brown, muriform,
ascospores. k Ascospores stained in Indian ink. l Germ tubes developing
from ascospore cells. Scale bars: c-d=100μm, e=5μm, f=5μm, g-i=
20μm, j-k=10μm, l=20μm
Notes: Didymosphaeria sensu lato, introduced for three
species of ascomycetes with 2-celled ascospores, comprise
species having a wide distribution and a broad host range.
Saccardo (1882) restricted the genus to only those species with
brown ascospores. Aptroot (1995) included over 400 epithets of
Didymosphaeria in his monograph of the genus, after examining over 3,000 species, but only seven species were accepted.
The placement of Didymosphaeria is confused as described in Ariyawansa et al. (2014b). Sivanesan (1984) reported that Didymosphaeria has Ascochyta and Periconia
Fungal Diversity (2014) 68:69–104
81
Fig. 5 Didymosphaeria futilis (K 147683, holotype of Sphaeria futilis)
a-b Fungus on the host. c Section through ascoma. d Close up of
peridium. e Broad, long trabeculate pseudoparaphyses, anastomosing
mostly above the asci. f-h Cylindrical asci with an indistinct ocular
chamber. i-l Ascospores with distinct spinulose ornamentation. Scale
bars: c=100μm, d=10μm, e, f-h=20μm, i-l=5μm
asexual states, while Kirk et al. (2008) reported Fusicladiellalike and Phoma-like species. Linking Didymosphaeria to
asexual states should be treated with caution until the type of
D. futilis has been sequenced. Ariyawansa et al. (2014b) found
that two strains of the D. rubi-ulmifolii clustered in the family
Montagnulaceae, but were separated from other genera of the
family with high bootstrap support. Comparison of the generic
type, D. futilis, and D. rubi-ulmifolii shows that they have
similar morphology and thus represent Didymosphaeria. Thus
Ariyawansa et al. (2014b) suggested that, based on the available molecular data and morphology, Didymosphaeria can be
referred to Montagnulaceae with Didymosphaeriaceae as the
probable synonym of Didymosphaeriaceae. In this paper, we
formally treat Didymosphaeriaceae as a separate family in the
order Pleosporales and with Montagnulaceae as its synonymy. The generic type of Didymosphaeria, D. futilis needs to
be recollected, epitypified and sequenced so that phylogenetic
analysis can be used to confirm family relationships within
Pleosporales.
In the present study, we observed that the type strain of
Paraconiothyrium brasiliense (CBS 100299) strain forms a
robust clade with D. rubi-ulmifolii. Paraconiothyrium
brasiliense was isolated from coffee fruits in Brazil, but this
species has since been reported from various habitats in other
continents, on woody and herbaceous host plants, such as
Prunus spp. in South Africa (Damm et al. 2008). Nearidentical ITS sequences have been deposited in GenBank for
endophytes isolated from Ginkgo biloba (DQ094168),
Juniperus virginiana (Hoffman and Arnold 2008), and
Ulmus davidiana var. japonica (AB665311), and also from
the herb Alliaria petiolata (EF432267). Strain CBS 395.87
from soil sampled in Italy was identified as Parac. brasiliense
by Verkley et al. (2014). Verkley et al. (2014) showed that the
ACT and TUB sequences of several Parac. brasiliense strains
used in their study were more variable than in other related
species, suggesting that Parac. brasiliense could be a species
complex. In this study we consider that Parac. brasiliense and
Didymosphaeria rubi-ulmifolii are the same species as they
82
cluster with a high bootstrap support. We therefore
synonymise these species. The only available name for this
species is Didymosphaeria rubi-ulmifolii because
Didymosphaeria brasiliensis is already in use.
Didymosphaeria rubi-ulmifolii Ariyawansa et al.,
Phytotaxa.176 (1)111
Basionym: Paraconiothyrium brasiliense Verkley, in
Verkley et al., Stud. Mycol. 50(2): 329 (2004),
Furthermore, in our phylogeny several Parac. brasiliense,
strains form a separate clade sister to the D. rubi-ulmifolii
sensu stricto, thus here we decide to treat it as a separate
species in Didymosphaeria (Didymosphaeria sp.) but without
designating a new species because the morphology is not
available. Paraconiothyrium variabile (CBS 120014) forms
a well-supported clade in Didymosphaeria, thus here we
synonymise Paraconiothyrium variabile under
Didymosphaeria
Didymosphaeria variabile (Riccioni et al.) Ariyawansa &
K.D. Hyde, comb. nov., Index Fungorum number: IF550707.
Basionym: Paraconiothyrium variabile Riccioni et al.
Persoonia 20: 13 (2008).
Julella Fabre, Annls Sci. Nat., Bot., sér. 6 9: 113 (1879)
[1878], Facesoffungi number: FoF 00038.
Saprobic or possibly lichenized. Sexual state: Ascomata
immersed, becoming erumpent to nearly superficial,
sphaeroid, black, coriaceous, ostiolate, formed under a clypeus. Ostiole usually widely porate, with a short neck, ostiolar
canal filled with periphyses. Peridium two layered, outer wall
composed of small, dark brown to black, heavily pigmented,
thick-walled cells of textura angularis and fusing with the
host tissue to the outside, inner wall comprising broad
yellowish-brown cells of textura angularis, thick at the apex
and thinner at the base. Hamathecium of dense broad, septate,
cellular, pseudoparaphyses anastomosing and branching and
embedded in mucilage. Asci 2-spored, bitunicate,
fissitunicate, clavate to cylindrical, with a short, broad,
furcated or knob-like pedicel, rounded at apex and without a
distinct ocular chamber. Ascospores biseriate or partially
uniseriate, asymmetric or nearly symmetric, hyaline, obovoid,
fusoid, or elongate, multi-septate, muriform, verruculose,
thin-walled, surrounded by a mucilaginous sheath, guttulate,
constricted at the septa. Asexual state: unknown.
Type species:
Julella buxi Fabre, Annls Sci. Nat., Bot., sér. 6 9: 113
(1878) (Fig 6), Facesoffungi number: FoF 00039.
Saprobic on woody substrates. Sexual state: Ascomata
200–350×190–250μm (x=300×230μm, n=10), immersed
becoming erumpent to nearly superficial, sphaeroid, black,
coriaceous, ostiolate, formed under a clypeus. Ostiole usually
widely porate, with a short neck, ostiolar canal filled with
periphyses. Peridium two layered, outer wall composed of
small, dark brown to black, heavily pigmented, thick-walled
cells of textura angularis and fusing with the host to the
Fungal Diversity (2014) 68:69–104
outside, inner wall comprising broad yellowish-brown cells
of textura angularis, thick at the apex and thinner at the base.
Hamathecium of dense, 2–4 μm broad, septate,
pseudoparaphyses anastomosing and branching above the asci
and embedded in mucilage. Asci 90–130×25–35μm (x=110×
30μm, n=10), two-spored, bitunicate, fissitunicate, clavate to
cylindrical, with a short, broad, furcated or knob-like pedicel,
rounded at apex and without a distinct ocular chamber.
Ascospores 30–35×10–15μm (x=32×30μm, n=13), biseriate
or partially uniseriate, asymmetric or nearly symmetric, hyaline, obovoid, fusoid, or elongate, multi-septate, muriform,
verruculose, thin-walled, surrounded by a mucilaginous
sheath, guttulate, constricted at the septa. Asexual state:
unknown.
Material examined: FRANCE, Serignan, on Buxus
sempervirens L. (Buxaceae), H. Fabre (S-F5992, holotype).
Notes: Julella has been confused, with some species being
saprotrophic on bark, while others are lichenized, and the
delineation of the genus is poorly defined. Currently, 46
epithets are listed in Index Fungorum (2014). Many of the
species assigned to the genus are facultatively lichenized with
the alga Trentepohlia as the photobiont (Purvis et al. 1992).
Julella shares many common features with Peltosphaeria but
differs in having bisporous asci (Barr 1985). Barr (1985)
proposed Peltosphaeria as a synonym of Julella. Treatments
of Julella are by Barr (1985) who lists three species and one
variety and Aptroot and van den Boom (1995) who considered Julella a predominantly tropical group of bark
saprotrophs, and recognized the species: J. lactea (A.
Massal.) M.E. Barr, J. sericea (A. Massal.) Coppins
(=J. fallcaiosa (Stizenb. ex Arnold) R.C. Harris) and
J. vitrispora (Cooke & Harkn.) M.E. Barr (=J. sublactea),
and regarded other species as synonyms or assigned them to
other genera. After examine the generic type of Julella,
Ariyawansa et al. (2013a) introduced Halojulellaceae and
Halojullela to accommodate J. avicenniae, a marine species
in the suborder Pleosporineae, order Pleosporales,
Dothideomycetes. Justification for the new family was based
on combined gene analysis of the large and small subunits of
the nuclear ribosomal RNA genes (LSU, SSU) and two protein coding genes RPB2 and TEF1, as well as morphological
characters (Ariyawansa et al. 2013a). Combined gene analysis
of nuSSU, nuLSU, mtSSU and TEF1 has shown that the
putative strains of Julella fallaciosa (MPN141 and
MPN547) forms a separate clade in Trypetheliales (Nelsen
et al. 2011), and are therefore excluded from our analysis.
Zhang et al. (2012) suggested that with the exception of
hyaline ascospores, most of the characters of Julella are compatible with Montagnulaceae. Julella is compatible with
Didymosphaeriaceae in having immersed ascomata formed
under a clypeus, short neck, 2-layered peridium composed of
cells of textura angularis and cylindric or oblong, pedicellate,
often with an ocular chamber and oblong to narrowly oblong
Fungal Diversity (2014) 68:69–104
83
Fig. 6 Julella buxi (S-F5992, holotype). a-b Surface view of ascomata
on host substrate (bark). c Longitudinal section of ascoma with clypeus. d
Close up of the peridium. e Cellular pseudoparaphyses. f-g Two spored
asci lacking an ocular chamber. h-j Muriform ascospores. m Ascospore
mounted in Indian ink to show mucilaginous sheath. Scale bars: c=
80μm, d=10μm, e=5μm, f-g=25μm, h-j=15μm
muriform ascospores but differing in having hyaline ascospores. We add Julella to Didymosphaeriaceae but this has
to be confirmed by molecular data.
Kalmusia Niessl, Verh. nat. Ver. Brünn 10: 204 (1872),
Facesoffungi number: FoF 00040
Saprobic on terrestrial habitats. Sexual state: Ascomata
small- to medium-sized, solitary, scattered or in small groups,
immersed to erumpent, globose or subglobose, often laterally
flattened, coriaceous, black-walled, with or without papilla.
Hamathecium of dense, filliform, delicate, septate
pseudoparaphyses, branching and anastomosing between
and above asci, embedded in mucilage. Asci bitunicate, clavate with a long, furcate pedicel. Ascospores narrowly ovoid
to clavate, pale brown, 3-distoseptate. Asexual state:
unknown.
Type species:
Kalmusia ebuli Niessl, Verh. nat. Ver. Brünn 10: 204
(1872) (Fig 7), Facesoffungi number: FoF 00041.
Saprobic on dead stems. Sexual state: Ascomata 290–
360×300–520μm (x=300×430μm, n=10), solitary, scattered,
or in small groups, immersed to erumpent, globose or
subglobose, coriaceous, wall black, with or without papilla,
ostiolate. Papilla small, with small ostioles. Peridium 15–
40μm wide, comprising one cell type of small, pigmented,
dark brown to black, thick-walled cells of textura prismatica
to textura angularis. Hamathecium of dense, 1.5 μm wide,
delicate pseudoparaphyses, 1–septate, branching and anastomosing between and above asci, embedded in mucilage. Asci
75–125 × 10–15 μm (x = 90 × 12 μm, n = 10), 8-spored,
bitunicate, fissitunicate, clavate, with a long, narrow, furcate
pedicel up to 45 μm long, and a shallow ocular chamber.
Ascospores 15–18×5–7μm (x=16×6μm, n=10), biseriate,
narrowly ovoid to clavate, pale brown, 3-distoseptate, without
constriction, smooth-walled. Asexual state: unknown.
Material examined: MORAVIA, Bruno, on dead stems.
(LUX 045054, isotype)
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Fig. 7 Kalmusia ebuli (LUX 045054, isotype) a Hebarium packet. b-c
Surface view of ascomata on host substrate (bark). d Longitudinal section
ascoma with clypeus. e Close up of the peridium. f Broad, septate
pseudoparaphyses. g Long, narrow asci with furcate pedicel. h-i Pale
brown ascospores with 3-distosepta. Scale bars: d=80μm, e=10μm, f=
5μm, g =25μm, h-i=15μm
Notes: Kalmusia was characterized as “immersed,
sphaeroid ascoma with central, stout papilla, surrounded by
hyphae in the substrate, stipitate asci with septate
pseudoparaphyses, and brown, 3-septate, inequilateral ascospores” by Barr (1992). The most morphologically comparable genus to Kalmusia is Thyridaria, which had been treated
as a subgenus under Kalmusia (Lindau 1897), and was subsequently transferred to Platystomaceae in Melanommatales
(Barr 1987, 1990). After comparing their morphological characters, Diapleella and Dendropleella were also considered
synonyms of Kalmusia.
Compared to Thyridaria, Kalmusia has sphaeroid
ascomata, a peridium of small pseudoparenchymatous cells,
basal asci and very thin pseudoparaphyses, thus it was
assigned to Phaeosphaeriaceae by Barr (1990), and the genus
is utilized to accommodate K. ebuli and K. clivensis (Berk. &
Broome) M.E. Barr, as well as closely related species, i.e.
K. utahensis (Ellis and Everh.) Huhndorf & M.E. Barr and
K. coniothyrium (Fuckel) Huhndorf (Barr 1992). But this
proposal is questionable because the clavate asymmetric ascospores as well as the clavate asci with long pedicels of
K. ebuli do not fit the concept of the Phaeosphaeriaceae as
defined by Zhang et al. (2012) and is excluded from the family
by Phookamsak et al. (2014)
Most recent phylogenetic studies indicated that some species of Kalmusia reside outside of the family
Phaeosphaeriaceae (Zhang et al. 2009). Kalmusia ebuli, the
type species of Kalmusia, lacks type material and as such its
phylogenetic position has remained unresolved. As a consequence, the familial position of Kalmusia is based on morphology and molecular phylogeny of species other than the
type (Zhang et al. 2014). Recently, a fresh collection of
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K. ebuli was obtained from decorticated wood of Populus
tremula in the foothills of the French Pyrenees (Zhang et al.
2014). The new collection was designated as neotype to
stabilize the application of the species and genus name.
Zhang et al. (2014) also observed the holotype of K. ebuli f.
sarothamni, and it was found to be a synonym of K. ebuli.
They concluded that the genus Kalmusia is polyphyletic within the family Montagnulaceae, with K. ebuli distantly placed
from K. brevispora and K. scabrispora, which appear to
represent a different genus. During our phylogenetic analysis,
we also obtained similar results, with K. ebuli being distantly
placed from K. brevispora and K. scabrispora and formed a
distinct clade sister to a newly introduced asexual genus
Alloconiothyrium in Didymosphaeriaceae. Furthermore, a recently introduced asexual genus, Dendrothyrium (typified
with D. variisporum (CBS 121517T) and another species of
Dendrothyrium, D. longisporum (CBS 582.83T), clustered
within the Kalmusia ebuli clade. Therefore, we propose to
synonymize Dendrothyrium under Kalmusia by giving priority to the older name and combine two species under
Kalmusia.
Kalmusia longispora (Verkley & Stielow) Ariyawansa &
K.D. Hyde, comb. nov., Index Fungorum number: IF550696.
Basionym: Dendrothyrium longisporum Verkley &
Stielow, Persoonia 32: 35 (2014).
Kalmusia variispora (Verkley & Stielow) Ariyawansa &
K.D. Hyde, comb. nov., Index Fungorum number: IF550697.
Basionym: Dendrothyrium variisporum Verkley &
Stielow, Persoonia, 32: 36 (2014).
Karstenula Speg., Decades Mycologicae Italicae ad no. 94
(in sched.) (1879), Facesoffungi number: FoF 00042.
Saprobic on dead wood and stems in terrestrial habitats.
Sexual state: Ascomata rarely small, usually medium-sized,
immersed usually under thin clypeus, scattered to gregarious,
with flattened top and rounded pore-like ostiole, coriaceous.
Peridium 2-layered, outer layer composed of reddish-brown
to dark brown small cells, with inner layer of pale compressed
cells. Hamathecium of dense, cellular pseudoparaphyses. Asci
bitunicate, fissitunicate, cylindrical to cylindro-clavate with
short furcate pedicel. Ascospores ellipsoid to fusoid, reddishbrown to dark brown muriform. Asexual state: Microdiplodia
(Constantinescu 1993).
Type species:
Karstenula rhodostoma (Alb. & Schwein.) Speg.,
Decades Mycologicae Italicae no. 94. (1879) (Fig 8),
Facesoffungi number: FoF 00043.
Basionym: Sphaeria rhodostoma Alb. & Schwein., Consp.
fung. (Leipzig): 43 (1805).
Saprobic on dead stems in terrestrial habitats. Sexual state:
Ascomata 250–430×450–650μm (x=310×500μm, n=10),
scattered or gregarious, immersed in the subiculum which
sometimes sloths off, globose or subglobose, black, flattened
top often white or reddish and sometimes slightly protruding
85
out of the substrate surface, usually with a wide opening
ostiole after removing the cover, coriaceous. Peridium 30–
40μm wide, comprising two cell types, outer region single
layered, composed of relatively small heavily pigmented
thick-walled compressed cells, inner layer cells larger and
wall thinner, comprising cells of textura angularis, merging
with pseudoparaphyses. Hamathecium of dense, 2–3.5μm
wide, long cellular, septate pseudoparaphyses. Asci 150–
210×12–15μm (x=182×13μm, n=20), 8-spored, bitunicate,
fissitunicate, cylindrical, with a broad, furcate pedicel which is
12–35μm long, and with an ocular chamber. Ascospores 20–
26×7.5–10μm (x=22×8μm, n=20), obliquely uniseriate and
partially overlapping, ellipsoid, reddish-brown, with 3transverse septa and a vertical septum in one or two central
cells, constricted at the septa, verruculose. Asexual state:
unknown.
Material examined: SWEDEN, on dead stems, 1834, E.
Fries (PH 01048835, holotype of Sphaeria rhodostoma Alb.
& Schwein.).
Notes: Karstenula is an ambiguous genus, which has been
synonymised under Pleomassaria by several authors (Lindau
1897; Winter 1885). Karstenula shares similarities with
Didymosphaeria in having ascomata seated in a subiculum
or beneath a clypeal thickening and sometimes apical cells
become reddish or orange-brown (Barr 1990). Barr (1990)
modified the concept of Karstenula (sensu lato), which encompasses some species of Thyridium. However, Barr (1990)
treated Karstenula as having trabeculate pseudoparaphyses
and this is clearly not the case. In most cases, the ascospores
are brown with transverse septa and sparse longitudinal septa.
The ascomata of Karstenula rhodostoma are similar to those
found in Byssosphaeria and Herpotrichia, especially in the
paler area around the ostiole and even in peridium and development under a subiculum. The numerous wide cellular
pseudoparaphyses and cylindrical asci (in Herpotrichia) are
also similar. The main difference between Karstenula and the
other two genera is the 3-septate ascospores with rare longitudinal septa (1-septate in Byssosphaeria and Herpotrichia).
Recent phylogenetic studies have shown that the putative
strain of Karstenula rhodostoma (CBS 690.94) forms a robust
clade with Phaeodothis winteri (Niessl) Aptroot, Didymocrea
sadasivanii, Bimuria novae-zelandiae, Montagnula opulenta,
Curreya pityophila (J.C. Schmidt & Kunze) Arx & E. Müll.,
and some species of Letendraea and Paraphaeosphaeria
(Kodsueb et al. 2006a; Zhang et al. 2009a). Thus Zhang
et al. (2012) tentatively included Karstenula in
Montagnulaceae.
Our phylogenetic analysis reveals that the putative strain of
Karstenula rhodostoma (CBS 690.94) resides in
Didymosphaeriaceae, sister to the Paraphaeosphaeria clade.
Therefore we tentatively refer Karstenula to
Didymosphaeriaceae. However, the placement of Karstenula
in Didymosphaeriaceae can only be confirmed by phylogenetic
86
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Fig. 8 Karstenula rhodostoma (PH 01048835, holotype of Sphaeria
rhodostoma). a-b Herbarium packet and specimen. c. Ascomata on
substrate. d Section thought ascoma. e Asci arrangement. f Close up of
the peridium. g Hamathecium comprising cellular pseudoparaphyses. h-i
Asci when immature. j Ascus at maturity. k-m Ellipsoid, reddish-brown
ascospores. Scale bars: d=100μm, e, h–j=50μm. f, k–m=20μm, g=
10μm
work including sequencing the generic type of Karstenula
(K. rhodostoma).
Letendraea Sacc., Michelia 2(6): 73 (1880), Facesoffungi
number: FoF 00044.
Saprobic in terrestrial habitats. Sexual state: Ascomata
rarely small, usually medium-sized, immersed usually under
a thin clypeus, scattered to gregarious, with flattened top and
rounded pore-like ostiole, coriaceous. Peridium 2-layered,
outer layer composed of reddish-brown to dark brown small
cells, inner layer of pale compressed cells. Hamathecium of
dense, cellular pseudoparaphyses. Asci cylindrical to cylindro-
clavate with short furcate pedicel. Ascospores one septate,
ellipsoid to fusoid, reddish-brown to dark brown. Asexual
state: unknown.
Type species:
Letendraea eurotioides Sacc., Michelia 2(6): 73 (1880)
(Fig 9), Facesof fungi number: FoF 00045.
Saprobic in terrestrial habitats. Sexual state: Ascomata
100–130μm diam., semi-immersed to superficial, globose to
subglobose, solitary, bright coloured, with papillate, central
ostiole (from illustration). Hamathecium of dense, filliform,
hyaline pseudoparaphyses. Asci (63-)73–75×13–18μm (x =
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87
Fig. 9 Letendraea eurotioides
(NY0091436, lectotype) a Semi
immersed to superficial, globosesubglobose ascomata. b
Hamathecium. c Young and
immature aci. d-f Asci with eight
spores. Scale bars: a-b=20μm,
c=50μm, d-f=10μm, g=20μm
71×15μm, n=8), 8-spored, bitunicate, fissitunicate, cylindricalclavate, slightly curved, sessile. Ascospores 13–16×5–6μm
(x = 14×5μm, n=20), 1–2-seriate, ellipsoidal to fusoid, uppercell wider toward base narrow and rounded ends, 1-septate,
olivaceous brown, guttulate, smooth. Asexual state: unknown.
Material examined: USA, on dead branches of Rubi
fruticosi (NY0091436, lectotype).
Notes: Letendraea is characterized by superficial, globose
to conical ascomata, filliform pseudoparaphyses, obclavate to
cylindrical, 8-spored asci, and fusoid to oblong, 1-septate
ascospores (Saccardo 1880). Because of its similar morphology with Karstenula rhodostoma, Kodsueb et al. (2006b)
assigned Letendraea to Melanommataceae.
Subsequent multigene phylogenetic analysis indicated that
both L. helminthicola (Berk. & Broome) Weese. and
L . p a d o u k N i c o t & P a rg . - L e d u c . n e s t e d w i t h i n
Montagnulaceae (Schoch et al. 2009; Zhang et al. 2009), thus
Zhang et al. (2012) referred Letendraea to Montagnulaceae.
In our phylogeny we obtained the same results and apart from
the putative strains of L. helminthicola (CBS 884.85) and
L. padouk (CBS 485.70), we included the putative strain of
L. eurotioides (CBS 212.31), the generic type of Letendraea
in our analysis. Therefore, we treat Letendraea as a separate
genus in Didymosphaeriaceae.
During our study we collected a Dothideomycetes species
(Letendraea cordylinicola) from living and dead leaves of
Cordyline sp. from Thailand, associated with leaf spot
disease, and showing similar morphology to Wilmia
brasiliensis. Dianese et al. (2001) referred Wilmia in
Phaeosphaeriaceae based on morphological characters.
Wilmia is similar to other genera in Phaeosphaeriaceae viz.
Barria, Lautitia and Metameris, with its didymosporous ascospores and coelomycete asexual morph (Dianese et al.
2001; Zhang 2012). Zhang et al. (2012) tentatively placed
Wilmia in Phaeosphaeriaceae as the genus is poorly known
and has no supporting molecular phylogenetic evidence,
although Zhang et al. (2012) mentioned that the genus might
be accommodated in Leptosphaeriaceae based on its dicotyledonous host habit.
Our phylogenetic analysis showed that a novel Letendraea
species (Letendraea cordylinicola) nested within Letendraea
in the family Didymosphaeriaceae. Letendraea and Wilmia
share similar characters in having immersed to superficial,
globose-subglobose ascomata with filliform
pseudoparaphyses and ellipsoidal to fusoid 1-septate, olivaceous brown ascospores with smooth wall. Therefore, based
on morphology of the type species of Wilmia, W. brasiliensis
and novel Wilmia-like (Letendraea cordylinicola) species,
88
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coupled with molecular data we exclude Wilmia from
Phaeosphaeriaceae and transfer it to Didymosphaeriaceae.
Furthermore, we propose to synonymize Wilmia under
Letendraea by giving priority to the older name. The type
species, Letendraea brasiliensis and the novel species
L. cordylinicola are described below.
Letendraea brasiliensis (Dianese et al.), Phookamsak,
Ariyawansa & K.D. Hyde comb. nov., Index Fungorum
number: IF550698 (Fig 10), Facesoffungi number: FoF
00046.
Basionym: Wilmia brasiliensis Dianeseet al., Mycologia
93(5): 1014 (2001).
Fig. 10 Letendraea brasiliensis (UB 8438, holotype of Wilmia
brasiliensis) a Herbarium label and specimens. b Ascomata on host
surface. c Section through ascomata stained by cotton blue reagent. d
Section through peridium. e Pseudoparaphyses stained by Congo red
reagent. f-h Ascospores. i-k Asci. l Section through conidiomata stained
by cotton blue reagent. m Conidiogenous cells. n Conidia. o-q Conidia.
Scale bars: c=100μm, l=50μm, d-k=20μm, m=10μm, f-n=5μm, o-q=
2μm
Fungal Diversity (2014) 68:69–104
Parasitic on Memora pedunculata. Sexual state:
Ascomata 140–200×90–125μm, scattered, rarely clustered,
immersed to erumpent through host surface, globose to
subglobose, brown to dark brown, central ostiole. Ostiole
papillate with elongated periphyses. Peridium 6–21μm wide,
composed of 2–5 layers, thin-walled with equal thickness,
small, flattened pseudoparenchymatous, brown to dark brown
cells of textura angularis to textura prismatica. Hamathecium
dense of 1.5–3 μm wide, cellular pseudoparaphyses,
unbranched, not -constricted at the septa, hyaline, embedded
in a mucilaginous matrix. Asci 70–90(-98)×(11-)13–15(-17)
μm (x = 82.9 × 13.8 μm, n = 30), 8-spored, bitunicate,
fissitunicate, cylindric-clavate, short-pedicellate, apically
rounded with indistinctly ocular chamber. Ascospores
(18-)20–24×6–7(-7.5) μm (x=21.9×6.9μm, n=30), overlapping, 1–2-seriate, didymosporous, fusiform with obtuse ends, brown, 1-septate, constricted at the septum,
smooth-walled, with indistinct appendage at the ends,
surrounded by mucilaginous sheath. Asexual state:
cohabit with sexual state on leaf lesions. Conidiomata
80–220μm high, 100–165μm diam., pycnidial, scattered
to clustered, solitary, immersed to erumpent through
host surface as black dots, visible as slightly raised or
bumped, small, pale brown to brown spots on host
surface, uniloculate, globose to sub globose, brown to
dark brown, central ostiole, papillate with large
periphyses. Conidiomata walls 8–14μm wide, composed
of 2–5 layers, thin-walled cells with equal thickness,
small, flattened pseudoparenchymatous cells of textura
angularis to textura prismatica, brown to dark brown.
Conidiophores reduced to conidiogenous cells.
Conidiogenous cells 5–10×(4.5-) 5–7(-10) μm (x =7×
6.4μm, n=20), enteroblastic, phialidic, ampulliform to
lageniform, hyaline, smooth-walled, aseptate. Conidia
(12-)14–20(-26.5) × 5–7 μm (x = 15.9 × 6.4 μm, n = 30),
oblong to obovoid or ellipsoidal with rounded or obtuse
ends, hyaline, indistinctly 1-distoseptate, rough-walled.
Material examined: BRAZIL, Distrito Federal Vargem
Bonita, Fazenda Áqua Limpa, on leaves of Memora
pedunculata Vell. (Bignoniaceae), 18 May 1995, C.A.
Inácio (UB 8438, holotype of Wilmia brasiliensis).
Letendraea cordylinicola Phookamsak, Ariyawansa &
K.D. Hyde, sp. nov., Index Fungorum number: IF550695
(Fig 11), Facesoffungi number: FoF 00047.
Etymology: The specific epithet cordylinicola is based on
the host genus from which the fungus was isolated.
Saprobic or parasitic on Cordyline sp. Sexual state:
Ascomata 85–115 × 100–160 μm, scattered, immersed to
semi-immersed, pale yellowish to reddish-brown on leaf lesions, globose to subglobose, pale yellowish to brown, central
ostiole. Ostiole papillate and ostiolar canal filled with long
periphyses. Peridium 9.5–20μm wide, composed of 2–5
l a y e r s , t h i n - w a l l e d w i t h e q u a l t h i c k n e s s , l a rg e
89
pseudoparenchymatous cells of textura angularis to textura
prismatica, hyaline to pale yellowish. Hamathecium dense of
2–3(-4) μm wide, numerous, broadly cellular
pseudoparaphyses, unbranched, anastomosing, indistinctly
constricted at the septum, embedded in a mucilaginous matrix.
Asci (51-) 55–60(-68)×(8-) 10–12μm (x=59.1×10.4μm, n=
20), 8-spored, bitunicate, fissitunicate, cylindric-clavate, short
pedicellate, apically rounded with an indistinct ocular chamber, arising from the base of the ascoma. Ascospores 13–15×
3–4μm (x =13.9×3.4μm, n=30), overlapping, 1–3-seriate,
didymosporous, fusiform, pale brown to brown, 1-septate,
constricted at the septum, often enlarged near septum in the
upper cell, smooth-walled, with distinct appendage at both
ends, surrounded by a mucilaginous sheath. Asexual state:
unknown.
Culture characters: Colonies on potato dextrose agar
(PDA) 73–82mm diam. after 30 days at 25-30 °C, white to
pale orange at the margin, white to orange or grey at the
centre; reverse white orangish to pale orange at the margin,
with zonate brown to black in the centre, medium dense,
irregular shape, flatten, separate from agar, dull with undulate
edges, fluffy to velvety, margin with well-defined to slightly
radiating, forming ascomata on colony after 8 weeks, producing light brown pigment tinted agar.
Material examined: THAILAND, Mae Jai District, Pa
Faek village, on dead leaves of Cordyline sp., 5 September
2010, R. Phookamsak, RP0064 (MFLU 11-0184, holotype),
ex-type living culture = MFLUCC 11-0148; Chiang Rai,
Muang District, Pakha village, on living Cordyline sp., 15
August 2010, S. Wikee, RP0066 (MFLU 11-0186), living
culture = MFLUCC 11-0150; Phan District, Muang Phan,
on living leaves of Cordyline sp. 7 November 2010, K.
Niraphai, RP0097 (MFLU 11-0217), living culture =
MFLUCC 11-0181.
Notes: Letendraea brasiliensis and the generic type
L. eurotioides share similar characters such as having
globose-subglobose ascomata, cylindric-clavate asci with 1septate brown ascospores, but differ in the nature of the
pseudoparaphyses (cellular versus filliform), size of the asci
(small versus large) and nature of the ascospores (with distinct appendage versus without distinct appendage).
Letendraea cordylinicola shares similarities with
L. brasiliensis in having immersed to semi-immersed
ascomata with central ostiole filled with long periphyses,
cylindric-clavate, short pedicellate asci bearing fusiform, 1septate, brown ascospores and both cause leaf spots on their
host. However, L. brasiliensis differs from L. cordylinicola in
having large ascomata and indictinct appendages and sheath
while L. cordylinicola has small ascomata, nice appendages
and a sheath. Based on morphology coupled with DNA
sequence data, we accept L. brasiliensis and
L. cordylinicola in the genus Letendraea because of their
similarities with other species in this genus.
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Fig. 11 Letendraea cordylinicola (MFLU11-0184, holotype) a
Ascomata on host surface. b Section through ascomata. c
Section through peridium. d Pseudoparaphyses stained by cotton blue
reagent. e Asci embedded in pseudoparaphyses. f-h Asci. i-k Ascospores
with polar appendages that eventually expand into a sheath. l Ascospore
stained in Indian ink with a prominent sheath. m Germination of ascospore. Scale bars: b=50μm, c=20μm, d-h, m=10μm, i-l=5μm
Montagnula Berl., Icon. fung. (Abellini) 2: 68 (1896),
Facesoffungi number: FoF 00048.
Saprobic on dead wood and leaves in terrestrial habitats.
Sexual state: Ascomata small to medium size, immersed to
erumpent, gregarious or grouped, globose to subglobose,
black. Hamathecium of dense, narrowly cellular, septate
pseudoparaphyses. Asci bitunicate, fissitunicate, usually
cylindric-clavate to clavate with a long pedicel. Ascospores
oblong to narrowly oblong, straight or somewhat curved,
reddish-brown to dark yellowish-brown, muriform or
phragmosporous. Asexual state: unknown.
Type species:
Montagnula infernalis (Niessl) Berl., Icon. fung.
(Abellini).2: 68 (1896) (Fig 12), Facesoffungi number: FoF
00049.
Basionym: Leptosphaeria infernalis Niessl, Inst. Coimbra
31: 13 (1883).
Saprobic on dead leaves in terrestrial habitats. Sexual
state: Ascomata 220–280×250–310μm (x = 250×280μm,
n=5), immersed to erumpent, gregarious or clustered, globose
to subglobose, sometimes triangular in dried material, short
ostiole always filled with hyaline closely adhering cells.
Peridium 40–55μm thick at sides, up to 80μm thick near the
apex, 3-layered, outer layer composed of heavily pigmented
thick-walled small cells of textura angularis, apex thicker
with smaller cells and thicker cell wall, thinner near the base;
mid layer less pigmented, innermost layer of narrow compressed rows of cells, merging with pseudoparaphyses.
Hamathecium of dense, 2–4.5μm broad, narrow, septate cellular pseudoparaphyses. Asci 153–170×18–22μm (x = 157×
Fungal Diversity (2014) 68:69–104
91
Fig. 12 Montagnula infernalis (M 1183, holotype of Leptosphaeria
infernalis). a Fungus on the herbarium material. b-c Dry black ascomata
on host surface. d Ascoma in horizontal section. e Layers of peridium. f
Cellular pseudoparaphyses g immature ascus. h mature ascus with long
pedicel. g-h Eight spored asci not visible. i-j Asci bearing muriform
ascospores. Scale bars: a-c=0.5mm, d=100μm, e-f=10μm, g-j=20μm
20μm, n=10), 8-spored, bitunicate, fissitunicate, cylindroclavate to clavate, pedicel 28–60μm long, with an ocular
chamber best seen in immature ascus. Ascospores 24–29×9–
11μm (x = 26×10μm, n=20), biseriate, oblong to narrowly
oblong, straight or somewhat curved, reddish-brown to dark
yellowish-brown, verruculose, with five transverse septa and
one vertical septum in each middle cells, constricted at the
primary and secondary primary septa. Asexual state:
unknown.
Material examined: PORTUGAL, Coimbra lusitania, on
leaves of Fourcroya longava (Agavoideae), February 1881,
Moller (M 1183, holotype of Leptosphaeria infernalis).
Notes: Montagnula was introduced by Berlese (1896) in
order to separate two dictyosporous species, M. infernalis
(Niessl) Berl., and M. gigantean (Mont.) Berl. from
Pleospora, based on the presence of hyphal stromatic tissue
over the ascomata and asci with a long pedicel (Barr 2001).
Wehmeyer (1957) had placed Montagnula as a subgenus of
Pleospora. Crivelli (1983) again treated Montagnula as a
separate genus and divided the genus into two sub genera,
i.e. Montagnula and Rubiginospora, which are distinguished
based on dark brown ascospores located on Agavaceae and
reddish-brown ascospores on Poaceae, respectively (Barr
2001), but this proposal was not accepted by many mycologists. Subsequently, Leuchtmann (1984) and Aptroot (1995)
included some phragmosporous and didymosporous species
in the genus and eventually it became heterogenic (Zhang
et al. 2012). Apart from the type species, some Montagnula
species produce Aschersonia Mont. asexual morphs (Hyde
et al. 2011). The genus presently has 28 epithets (Index
Fungorum 2014). GenBank has 19 hits for the genus including putative strains of M. opulenta (De Not.) Aptroot (CBS
168.34), M. aloes Crous et al. (CPC 19671), M. rhodophaea
(Bizz.) Leuchtm. (CBS 616.86), M. dura (Niessl) Crivelli
(CBS 380.54), M. spartii (Fabre) Aptroot (CBS 183.58) and
M. anthostomoides (Rehm) Leuchtm. (CBS 615.86).
One striking character of Montagnula infernalis is the very
long ascal pedicel which develops once it is released from the
ascomata. However, this character appears to have evolved
more than once and can be found in Kirschsteiniothelia
elaterascus Shearer which clusters with Helicascus (Shearer
et al. 2009). The same character is also found in Xenolophium
and Ostropella in the Platystomaceae (Mugambi and
Huhndorf 2009). Montagnula opulenta is a didymosporous
species, but phylogenetically closely related to those
dictyosporous (Karstenula rhodostoma) and phragmosporous
(Paraphaeosphaeria michotii) members of Montagnulaceae
(Zhang et al. 2009). This might indicate that compared to
other morphological characters, ascospore type is not a good
character at the family level classification. Recent phylogeny
based on multi-gene analysis has shown that the putative
strain of M. opulenta forms a robust phylogenetic clade with
species of Bimuria, Curreya, Didymocrea, Letendraea,
Paraphaeosphaeria, Phaeodothis and Karstenula, which
92
might represent a familial group (Schoch et al. 2006; Zhang
et al. 2009, 2012).
Our phylogenetic data also shows that the putative strains
of Montagnula rhodophaea (CBS 616.86) and M. spartii
( C B S 1 8 3. 58 ) cl us t e r e d i n L e n t i t h e c i a c e a e an d
Massarinaceae, respectively. The morphology and identification of these putative strains in GenBank as far as we can
ascertain, cannot be checked, as they are not linked to any
herbarium material, therefore we placed them in
Lentitheciaceae incertae sedis and Massarinaceae incertae
sedis, respectively. Montagnula dura (CBS 380.54) was excluded from our final analysis because in the preliminary
analysis we observed that this strain clustered within
Pleosporaceae (details not shown). Our phylogenetic results
indicated that the putative strains of M. opulenta (CBS
168.34), M. aloes (CPC 19671) and M. anthostomoides
(CBS 615.86) nested within Montagnulaceae and form a
separate clade sister to the Kalmusia and Alloconiothyrium
clades. The strain named Letendraea helminthicola (CHTA
R43) resides in Montagnula. This is probably a misidentification of Montagnula sp. as Letendraea helminthicola.
The morphology and identification of the putative strain
(CHTAR43) of Letendraea helminthicola in GenBank as
far as we can determine, cannot be checked, as they are not
linked to any herbarium material thus preliminarily reidentified as Montagnula sp. Therefore based on morphology, coupled with available molecular data, we keep
Montagnula as a distinct genus in Didymosphaeriaceae.
Fresh collections of the M. infernalis and further molecular
and morphological studies are desirable to confirm our
results.
Neokalmusia Kaz. Tanaka et al., gen. nov., Index
Fungorum number: IF550700, Facesoffungi number: FoF
00050.
Etymology: The generic epithet, neo (Lat., new), refers to
the similarity to Kalmusia.
Saprobic on culms of bamboo in terrestrial habitats. Sexual
state: Ascomata immersed under black clypeus-like structure
composed of host epidermis and fungal mycelia, subglobose
to oblong on host surface, hemispherical, with several perithecia arranged in single or double rows along the long axis of
the culms. Ostiole absent or short papillate, with numerous
periphyses. Peridium at side composed of several layers of
polygonal, thin-walled, brown cells; at the rim composed of
vertically orientated, hyaline, cells of textura angularis.
Hamathecium composed of numerous, broadly cellular
pseudoparaphyses, branching, anastomosing, with slime coating. Asci (4–) 8-spored, bitunicate, fissitunicate, cylindricclavate, with a long stipe, apically rounded with welldeveloped ocular chamber, arising from basal ascomata.
Ascospores overlapping, uniseriate or biseriate,
phragmospores, fusiform, slightly curved, initially hyaline,
becoming yellowish-brown to reddish-brown at maturity, 3–
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5-septate, thick-walled, verrucose, with a sheath. Asexual
state: unknown.
Type species:
Neokalmusia brevispora (Nagas. & Y. Otani) Kaz. Tanaka,
Ariyawansa & K. D. Hyde, comb. nov., Index Fungorum
number: IF550701. (Fig. 13), Facesoffungi number: FoF
00051.
Basionym: Phaeosphaeria arundinacea var. brevispora
Nagas. & Y. Otani, Rep. Tottori Mycol. Inst. 15: 38 (1977).
Saprobic on culms of Sasa spp. Sexual state: Ascomata
200–370(–400)×190–330(–440) μm immersed under black
clypeus-like structure composed of host epidermis and fungal
mycelium, scattered to grouped, subglobose to oblong on host
surface, 1–3.5mm long, 0.4–0.7mm wide. Ostiole absent or
short papillate, 50–75(-110) μm long, with hyaline periphyses.
Peridium 15–20μm diam., composed of 2- layers, outer layer
composed of 2–4 layers, light brown to dark brown pseudoparenchymatous cells of textura angularis, inner wall consist
of 1–2 layers, hyaline cells of textura angularis.
Hamathecium of 1.5–2.5μm dense thick, cellular, hyaline,
septate pseudoparaphyses. Asci 80–118×10.5–15μm (x=
100.2×13.4μm, n=32), 4–8-spored, cylindrical to clavate,
bitunicate, fissitunicate, with a 10–25(–30) μm (x =19.8μm,
n=30) a short pedicel. Ascospores 18–24(-26.5)×4–7μm (x=
20.1×5.2μm, n=76), uniseriate or partially overlapping, fusiform, 3-septate, with a mostly submedian (0.48–0.56; x=0.52,
n=46) primary septum, reddish-brown, thick-walled, verrucose, with 2–5μm thick sheath. Asexual state: unknown.
Material examined: JAPAN, Fukushima, Minamiaizu, Ose
pond, on dead twigs of Sasa sp. (Poaceae), 30 Aug. 2003, N.
Asama, (KT 1466 = HHUF 28229), living culture CBS
120248 = JCM 13543 = MAFF 239276; Hokkaido, Isl.
Rishiri, Afutoromanai river, on dead twigs of Sasa kurilensis,
29 July 2007, K. Tanaka & G. Sato, (KT 2313 = HHUF
30016), living culture = NBRC 106240.
Notes: A new genus Neokalmusia is established to accommodate two bambusicolous taxa, N. brevispora and
N. scabrispora. These species have been previously placed in
Kalmusia (Tanaka et al. 2005; Zhang et al. 2009), but subsequent
molecular studies (Hyde et al. 2013) as well as in the present
study (Fig. 1), do not support this placement. Morphological
studies of Kalmusia based on the holotype of K. ebuli (type of
this genus) indicated that the genus is characterized by solitary
sphaeroid ascomata, a peridium of small pseudoparenchymatous
cells, clavate basal asci with very long pedicels, very thin
pseudoparaphyses and distoseptate, smooth-walled ascospores
(Zhang et al. 2012, this study). The two Neokalmusia species
on bamboo have subglobose to oblong ascomata including several perithecia in a row, clypeus-like structure composed of thinwalled cells and verrucose ascospores.
Neokalmusia scabrispora (Teng) Kaz. Tanaka et al., comb.
nov., Index Fungorum number: IF550702. (Fig. 14),
Facesoffungi number: FoF 00052
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93
Fig. 13 Neokalmusia brevispora (KT 1466). a-b Ascomata on host surface. c-d Vertical section through ascomata. e Section through peridium. f
Pseudoparaphyses. g-h Asci. i Ocular chamber. j Germinating ascospore. k-o Ascospores. Scale bars: a-b=1mm, c-d=100μm, e-o=10μm
Basionym: Leptosphaeria scabrispora Teng, Sinensia,
Shanghai 4: 378 (1934).
Saprobic on culms of bamboo. Sexual state: Ascomata
200–300×130–500μm, immersed under black clypeus-like
structure composed of host epidermis and fungal mycelium,
subglobose to oblong on host surface scattered to grouped.
Ostiole absent or slightly papillate, about 85–100 μm long,
with numerous periphyses. Peridium 7.5–20μm thick at sides,
composed of 3–6 layers of brown polygonal thin-walled cells
of 5–10×2.5–6.5μm, surrounded by wedge-shaped stromatic
region (250–400μm wide at sides) composed of vertically
orientated hyaline cells of textura angularis. Hamathecium
1–2μm wide narrowly cellular, numerous, septate, branched
and anastomosing, embedded in a mucilaginous matrix. Asci
123.5–160×(15.5-)17–22μm (x =142.1×1 8.8μm, n=50), 8spored, bitunicate, clavate, rounded at the apex and with an
apical chamber, with a short stipe. Ascospores 29–40.5×7–
10μm (x =34.8×8.5μm, n=68), biseriate, fusiform to ellipsoid, slightly curved, 5 (rarely 7) -septate, with a median
primary septum 0.48–0.53μm (x =0.50μm, n=57) wide,
slightly constricted at the septa, with third cell from the apex
enlarged, penultimate cells shortest, brown to yellowishbrown, verrucose, with 10–20μm wide sheath. Asexual state:
unknown.
Material examined: JAPAN, Tochigi, Kanuma, near Ooashi
river, on dead twigs of Phyllostachys bambusoides (Poaceae),
6 March 2003, N. Asama (KT 1023 = HHUF 28608), living
culture CBS 120246 = JCM 12851 = MAFF 239517;
Kagoshima, Kumagegun, Isl. Yakushima, Miyanoura river
(riverbank), on dead twigs of Phyllostachys bambusoides
(Poaceae), 17 Mar. 2007, K. Tanaka & H. Yonezawa (KT
2202 = HHUF 30013), living cultures = NBRC 106237.
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Fig. 14 Neokalmusia scabrispora (KT 1023) a-b Ascomata on host
surface. c-d Vertical section through ascomata. e Section through peridium. f Pseudoparaphyses. g-h Asci. i Ocular chamber. j Germinating
ascospore. k-o Ascospores. a-b, f-o from KT 2202, c-e from KT 1023.
Scale bars: a-b=1mm, c-d=100μm, e-o=10μm
Notes: This fungus was originally described as a species of
Leptosphaeria (Teng 1934) and later transferred to
Massariosphaeria (Shoemaker and Babcock 1989) or
Kalmusia (Tanaka et al. 2005). Neokalmusia scabrispora,
however, does not belong to the Leptosphaeriaceae typified
by Leptosphaeria doliolum or to the Thyridariaceae
encompassing the type species of Massariosphaeria
(M. phaeospora) (Hyde et al. 2013). Neokalmusia
scabrispora shares similar characters to Neokalmusia
brevispora in having immersed ascomata under black
clypeus-like, cellular pseudoparaphyses, 8-spored, bitunicate,
clavate, asci rounded at the apex and fusiform to ellipsoid,
brown to yellowish-brown, verrucose ascospores with a thick
sheath. Neokalmusia scabrispora differs in the thickness of
the peridium (7.5–20 μm versus 15–20 μm) and the number
of septa in ascospores (5–7 versus 3).
Paraconiothyrium Verkley, Stud. Mycol. 50(2): 327
(2004), Facesoffungi number: FoF 00053.
Saprobic or parasitic in terrestrial habitats. Sexual state:
unknown (Verkley et al. 2004). Asexual state: Conidiomata
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eustromatic, simple or complex, rarely pycnidial,
Conidiogenous cells discrete or integrated, phialidic, sometimes percurrent. Conidia aseptate, sometimes 1-septate, thinwalled, smooth-walled or minutely warted, hyaline when
liberated, later brown (Verkley et al. 2004).
Type species:
Paraconiothyrium estuarinum Verkley & Manuela Silva,
in Verkley et al., Stud. Mycol. 50(2): 327 (2004),
Facesoffungi number: FoF 00054.
Saprobic in terrestrial environments. Asexual state:
Conidiomata 0.2–0.5(-1) mm diam., mostly submerged in
the agar, but also superficial and on the aerial mycelium,
eustromatic, globose or flattened, dark brown to black, with
several merging cavities, ostioles absent, opening by dissolution of upper cells. Conidiomatal wall 30–45μm thick, composed of outer layer of isodiametric or more flattened cells
with hyaline to reddish-brown walls thickened up to 1.5μm,
lined by a 35–60(-75) μm thick inner layer of textura
angularis, surface of the conidiomatal wall often covered
under brown entangling hyphae. Conidiogenous cells 4–
6.5×2.5–3.5(-4) μm, discrete, assembled into protruding
masses of cells, or integrated in very compact conidiophores,
ampulliform to subcylindrical, hyaline, indeterminate,
phialidic with an inconspicuous periclinal thickening and
collarette, later often with a single percurrent proliferation,
mostly. Conidia narrowly ellipsoidal or short-cylindrical,
straight or slightly curved, rounded at both ends, 1-celled,
with one or two small, polar guttules, and with thin and
smooth walls that are hyaline at secession, but soon becoming
olivaceous- or yellowish-brown. Sexual state: unknown
(Verkley et al. 2004).
Notes: Paraconiothyrium was introduced by Verkley et al.
(2004) to accommodate four new species, Parac. estuarinum,
Parac. brasiliense, Parac. cyclothyrioides, and Parac.
fungicola. Based on the combination of ITS and partial SSU
nrDNA sequences Verkley et al. (2004), included
Coniothyrium minitans and the ubiquitous soil fungus
Coniothyrium sporulosum in Paraconiothyrium. In the same
study, the asexual morphs of Paraphaeosphaeria michotii and
P. p i l l e a t a w e r e r e g a r d e d a s r e p r e s e n t a t i v e o f
Paraconiothyrium, but remained formally unnamed.
Paraconiothyrium species are phylogenetically distant from
typical members of the other coelomycete genera such as
Coniothyrium, Microsphaeropsis, Cyclothyrium, and
Cytoplea. In Paraconiothyrium, the conidiomata generally
are complex, eustromatic and relatively thick-walled. They
may appear as simple pycnidia, but then they usually lack a
well differentiated ostiole. In Coniothyrium, the conidiomata
are true pycnidia, which may merge in vitro but then always
produce well-developed, sometimes even papillate ostioles.
The most distinctive Coniothyrium feature is the annellidic
conidiogenous cells, which percurrently proliferate after secession of each conidium. Coniothyrium conidia are thick-
95
walled and verruculose, with a truncate base and sometimes a
basal frill (Sutton 1980). Microsphaeropsis species are also
pycnidial, but their conidiogenous cells are discrete, Phomalike phialides, which only rarely proliferate percurrently.
Microsphaeropsis olivacea, the type species, has pale brown,
1-celled, thin- and smooth-walled conidia (Verkley et al.
2004).
Câmara et al. (2001) assessed morphological data in relation to ITS sequences for nine species of Paraphaeosphaeria,
and identified three lineages, which were later confirmed by
SSU data (Câmara et al. 2003). They found that only one
species, Paraph. pilleata, was congeneric with the type species Paraph. michotii (Verkley et al. 2004). Thus, only two
species were retained in Paraphaeosphaeria sensu stricto. For
the other species the genera Phaeosphaeriopsis and
Neophaeosphaeria were erected. Following multi-locus
DNA analysis and detailed morphological study, Verkley
et al. (2014) were able to delimit and formally propose nine
new species and two new genera among the fungi in the
Montagnulaceae formerly recognizable as Coniothyrium-like
asexual morphs. Verkley et al. (2014) suggested that the genus
Paraconiothyrium appears to be paraphyletic within the family Montagnulaceae but proposed that it should not be split up
to further genera.
In the present study, we also observed similar phylogeny,
where Paraconiothyrium appears to be paraphyletic within
the family Didymosphaeriaceae. Several sexual stages of
some Paraconiothyrium species were reported during the
study, i.e. the sexual state of Paraconiothyrium fuckelii
(MFLUCC 13-0043) and novel species of
Paraconiothyrium, Parac. magnoliaee introduced from
Thailand and are described below.
Paraconiothyrium fuckelii (Sacc.) Verkley & de Gruyter, in
Gruyter et al., Stud. Mycol. 75: 25 (2012) (Fig. 15),
Facesoffungi number: FoF 00055.
Basionym: Coniothyrium fuckelii Sacc., Fungi venet. nov.
vel. Crit., Sér. 5: 200 (1878).
Saprobic on dead wood in terrestrial habitats. Sexual state:
Ascomata 290–360×300–520μm (x=300×430μm, n=10),
solitary, scattered, or in small groups, immersed to erumpent,
globose or subglobose, coriaceous, wall black, with or without
papilla, ostiolate. Papilla small, with small ostioles. Peridium
15– 40 μm wide, comprising one cell type of small,
pigmented, thick-walled cells of textura prismatica to textura
angularis. Hamathecium of dense, 1.5μm broad, delicate
pseudoparaphyses, 1-septate, branching and anastomosing
between and above asci, embedded in mucilage. Asci 75–
125×10– 15μm (x =90×12μm, n=10), 8-spored, bitunicate,
fissitunicate, clavate, with a long, narrowed, furcate pedicel
which is up to 45 μm long, and a low ocular chamber.
Ascospores 15–18×5–7μm (x =16×6μm, n=10), biseriate,
narrowly ovoid to clavate, pale brown, 3-septate, constricted
at the middle septum, smooth-walled. Asexual state:
96
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Fig. 15 Paraconiothyrium fuckelii. (MFLU 14-0305) a-b Appearance
of ascomata on the host surface. c Section of an ascoma. d Section of
peridium. e Clavate ascus with a short, narrow pedicel. f-h Subglobose to
ellipsoid or obovoid ascospores. i-j Conidiogenous cells. k Orangebrown conidia. Scale bars: c=100μm, d=25μm, e=10μm, f-k=5μm
Conidiomata pycnidial 300–400μm diam. and with a single
cavity, more often eustromatic. Conidiomatal wall 3-layered,
outer layer composed cells of textura angularis with somewhat thickened, brown walls, and an inner layer composed
cells of textura angularis-globulosa with somewhat thickened, hyaline walls. Conidiogenous cells 4–10(–13)×3–
5μm, discrete or integrated in short, simple, 1–2-septate conidiophores, broadly ampulliform to globose, holoblastic, often annellidic with 1 or 2 percurrent proliferations noticeable
by the distinct scars on a somewhat elongated neck, hyaline.
Conidia 3–4×2–3(–3.5) μm variable in shape, subglobose to
ellipsoid or obovoid, rarely more cylindrical, initially hyaline,
olivaceous-brown soon after secession, smooth, orangebrown, aseptate.
Material examined: THAILAND, Chiang Rai, Bandu, on
dead wood, 28 September 2012, K.M. Thambugala (MFLU
14-0305), living culture = MFLUCC 13-0043.
Paraconiothyrium magnoliae Monkai, Ariyawansa &
K.D. Hyde, sp. nov., Index Fungorum number: IF550703
(Fig. 16), Facesoffungi number: FoF 00056.
Etymology: The specific epithet magnoliae is based on the
host genus from which the fungus was isolated.
Saprobic on dead leaves in terrestrial environments. Sexual
state: Ascomata 130–200×150–250 μm (x = 150×320μm,
n=10) small to medium-sized, immersed to semi-immersed,
depressedglobose, ostiolate. Ostiole papillate, black, smooth,
with beak, ostiolar canal lined without hyaline periphyses.
Peridium 10–17μm (x =14μm, n=20) wide, usually with 3-
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97
Fig. 16 Paraconiothyrium
magnolia (MFLU 12-1414,
holotype) a-b Fungus on host
substrate. c Section of an ascoma.
d Cellular, hyaline, septate, broad,
pseudoparaphyses. e-g
Cylindrical asci with a short,
broad pedicel. h-j Yellowishbrown ascospores with bipolar
appendage. k Germinating
ascospores. Scale bars: c=50μm,
d=5μm, e-g=30μm, h-j=5μm,
k=10μm
5 layers, composed cells of textura prismatica. Hamathecium
of dense 2–4μm (x =2μm, n=20) wide, cellular, septate,
hyaline, pseudoparaphyses. Asci 16–27×4–6μm (x =23×
5μm, n=30), 8-spored, bitunicate, fissitunicate, cylindrical
with a short, broad pedicel with a minute ocular chamber.
Ascospores 15–30×4–7μm (x = 24×5, n=40), uniseritate or
partially overlapping, broadly elliptical, yellowish-brown, 3septate (rarely 4-septate), with small guttules, with bipolar
appendage at the tip and one polar appendage on the base,
surrounded by a gelatinous sheath. Asexual state: unknown.
Material examined: THAILAND, Chiang Mai, Doi
Suthep-pui, on dead leaves of Magnoliae liliifera
(Magnoliaeceae) 18 September 2011, J. Monkai (MFLU 140306, holotype), ex-type living culture = MFLUCC 10-0278
Notes: Paraconiothyrium magnoliae resembles many species of Phaeosphaeria in having 3-septate ascospores with a
gelatinous sheath. Phaeosphaeria eustoma and
Phaeosphaeria parvula have ascospores which are very similar in size to the morphology of P. magnoliaee. In addition,
Parac. magnoliae is similar to Phaeosphaeria typharum and
Phaeosphaeria barriae in having gelatinous sheathed ascospores (with appendaged ascospores only in Phaeosphaeria
barriae), but differ in the shape of the ascospores. The phylogenetic analysis of combined ITS, LSU, SSU nrDNA and
TUB sequences provides strong evidence that Parac.
magnoliae belongs in Didymosphaeriaceae, where it forms a
separate clade along with Parac. fungicola within the clade of
Paraconiothyrium with relatively high bootstrap support
(Fig 1), thus a new species is proposed.
Paraphaeosphaeria O.E. Erikss., Ark. Bot., Ser. 2 6: 405
(1967), Facesoffungi number: FoF 00057.
Saprobic in terrestrial habitats. Sexual state: Ascomata
small to medium-sized, immersed to semi-immersed, depressed, globose, ostiolate. Ostiole with a short beak, without
periphyses. Peridium usually with 3-5 layers, composed cells
of textura prismatica. Hamathecium composed of filamentous, hyaline, septate, broad, dense pseudoparaphyses. Asci 8spored, bitunicate, fissitunicate, cylindrical with a short pedicel. Ascospores uniseritate or partially overlapping, multi
septate, broadly elliptical, yellowish-brown, with small
guttules, smooth, with a thin uniform sheath. Asexual state:
Coniothyrium-like, Conidiomata eustromatic or pycnidial.
Conidiogenous cells discrete or integrated, phialidic, or
annellidic with one or two percurrent proliferations. Conidia
aseptate or 1-septate, smooth to verrucose (Verkley et al.
2014).
Type species:
Paraphaeosphaeria michotii (Westend.) O.E. Erikss.,
Cryptogams of the Himalayas 6: 405 (1967) (Fig. 17),
Facesoffungi number: FoF 00058.
Basionym: Sphaeria michotii Westend., Bull. Acad. R. Sci.
Belg., Cl. Sci., sér. 2 7(5): 87 (1859).
Saprobic on dead leaves of Poaceae. Sexual state:
Ascomata 130–200×150–250μm (x = 150×320μm, n=10)
98
Fungal Diversity (2014) 68:69–104
Fig. 17 Paraphaeosphaeria
michotii. (MFLU 12-2210,
epitype) a Ascomata on host
substrate. b Section of ascoma. c
Close up of the peridium. d Ascus
surrounded by cellular
pseudoparaphyses. e-g Asci with
short, broad pedicel bearing 8
spores. h-j Mature ascospores
with thin uniform sheath. k
Ascospores mounted in Indian
ink. Scale bars: b=100μm,
c=50μm, d=20μm, f-h=60 μm,
i-k=10μm
small to medium-sized, immersed to semi immersed, depressed globose, ostiolate. Ostiole papillate, black, smooth,
with beak and without periphyses. Peridium 10–17μm (x=
14μm, n=20) wide, usually with 3–5 layers, composed of
cells textura prismatica. Hamathecium of dense 2–3μm (x=
2 μm, n = 20) filamentous, hyaline, septate, broad,
pseudoparaphyses. Asci 60–85×12–28μm (x=77×20μm,
n=20), 8-spored, bitunicate, fissitunicate, cylindrical with a
short, broad pedicel with minute ocular chamber. Ascospores
15–30×4–7μm (x = 24×5, n=40), uniseritate or partially
overlapping, 2-septate, broadly elliptical, yellowish-brown,
with small guttules, smooth, with a thick uniform sheath.
Asexual state: not observed in the culture.
Material examined: ITALY, Forlì-Cesena, Montevescovo,
on dead leaves of Poaceae, 3 February 2012, E. Camporesi
(MFLU 12-2210, epitype), ex-type living culture = MFLUCC
13-0349.
Notes: Paraphaeosphaeria has been separated from
Leptosphaeria (Eriksson 1967), and it is quite comparable
with Phaeosphaeria. Paraphaeosphaeria can be distinguished from Phaeosphaeria by its ascospores. Ascospores
of Paraphaeosphaeria michotii have two septa, and they are
biseriate, straight, subcylindrical with broadly rounded ends,
rather dark brown and punctate. The primary septum is laid
down closer to the distal end than to the proximal, and the
larger, proximal hemispore is divided by one transverse
septum. There are more septa in the proximal hemispore of
other species such as Paraph. castagnei (Durieu & Mont.)
O.E. Erikss., Paraph. obtusispora (Speg.) O.E. Erikss. and
Paraph. vectis (Berk. & Broome) Hedjar. Asexual characters
can also distinguish Paraphaeosphaeria and Phaeosphaeria.
Paraphaeosphaeria produces Coniothyrium-like asexual
states, but Phaeosphaeria has Hendersonia-Phaeoseptoria
asexual states (Eriksson 1967). Câmara et al. (2001) provided
descriptions of sexual and asexual morphs of Paraph. michotii
and Paraph. pilleata, while other species treated under
Paraphaeosphaeria were transferred later to
Neophaeosphaeria and Phaeosphaeriopsis (Câmara et al.
2003). None of the amerosporic coniothyrium-like species
associated with these sexual morphs has been assigned with
a formal name. Shoemaker and Babcock (1985) redescribed
some Canadian and extralimital species, and excluded
Paraph. longispora (Wegelin) Crivelli and Paraph. oblongata
(Niessl) Crivelli from Paraphaeosphaeria based on their longitudinal septa as well as beak-like papilla and wall structures.
Molecular phylogenetic results based on a multi-gene study
indicate that Paraphaeosphaeria should belong to
Montagnulaceae (Zhang et al. 2009). Recently, Ariyawansa
et al. (2014d) epitypified Paraph. michotii from a fresh collection and thus the placement of the Paraphaeosphaeria in
Montagnulaceae is confirmed. In our phylogenetic analysis,
Paraphaeosphaeria forms a well-supported clade sister to
Fungal Diversity (2014) 68:69–104
Didymosphaeria, thus Paraphaeosphaeria is treated as a separate genus in the Didymosphaeriaceae.
Phaeodothis Syd. & P. Syd., Annls mycol. 2(2): 166
(1904), Facesoffungi number: FoF 00059.
Saprobic or parasitic on dead wood or grasses in terrestrial
environments. Sexual state: Ascostromata subglobose to
hemispherical, immersed, scattered to gregarious, dark brown
to black. Peridium comprising several layers of hyaline compressed cells. Hamathecium of dense, cellular
pseudoparaphyses often in a gelatinous matrix. Asci 8-spored,
bitunicate, fissitunicate, cylindrical, short pedicellate.
Ascospores fusiform with rounded ends, olivaceousbrown.
Asexual state: unknown.
Type species:
Phaeodothis tricuspidis Syd. & P. Syd., Annls mycol. 2(2):
166 (1904) (Fig. 18), Facesoffungi number: FoF 00060.
Saprobic on dead wood or grasses. Sexual state: Ascomata
235–290×120−180μm (x=254×137μm, n=10), immersed to
nearly superficial under clypeus, scattered to gregarious,
subglobose to hemispherical, dark brown to black, papillate,
Fig. 18 Phaeodothis tricuspidis
(S F125876, holotype) a
Herbarium packet and specimen.
b Close up of ascomata. c-d
Sections through ascomata. e
Peridium comprising hyaline
compressed cells. f-h Mature and
immature asci surrounded by
pseudoparaphyses. i-l Fusiform,
olivaceous brown ascospores.
Scale bars: c-d=100μm, e=
50μm, f-h=25μm, i-l=5μm
99
smooth. Papilla black, with pore-like ostioles, ostiolar canal
filled with periphyses. Peridium 15−28μm (x=22μm, n=20)
wide, comprising several layers of hyaline compressed cells.
Hamathecium of dense, 2−4μm (x=2.5μm, n=20), aseptate,
cellular pseudoparaphyses often in a gelatinous matrix. Asci
55 − 80 × 7 − 12 μm (x =68 × 10 μm, n = 20), 8-spored,
bitunicate, fissitunicate, cylindrical, short pedicellate, apically rounded with a minute ocular chamber. Ascospores
14.5−17.5×4−5μm (x=15.8×4.4μm, n=30), biseriate and
partially overlapping, fusiform with rounded ends, olivaceous brown, 1-septate, slightly constricted at the septa.
Asexual state: unknown.
Material examined: ARGENTINA, San José, Salta, 12
February 1873, P.G. Lorentz (SF125876, holotype).
Notes: Phaeodothis is characterized by its immersed to
nearly superficial ascomata, a sparse hamathecium consisting
of cellular pseudoparaphyses and 1-septate ascospores
(Aptroot 1995). Phaeodothis was placed in Didymosphaeria
by von Niessl (1875), but Aptroot (1995) transferred it to
Phaeosphaeriaceae. Phaeodothis is similar to genera in the
100
Didymosphaeriaceae in having ascomata immersed under a
clypeus, a pseudoparenchymatous peridium with small cells,
cylindrical asci and brown ascospores. However, Phaeodothis
differs from other genera of Didymosphaeriaceae in having a
hamathecium consisting of sparse pseudoparaphyses and 1septate ascospores. Phylogenetic analysis based on LSU,
SSU, RBP1, RBP2 and EF-1 sequences concluded that a
strain named Phaeodothis winteri (a synonym of
P. tricuspidis Syd. & P. Syd.) clustered within the clade of
Montagnulaceae (Schoch et al. 2009). Currently 27 epithets
are listed for the genus in Index Fungorum (2014), while
GenBank has seven hits for the genus including the putative
strain of P. winteri (CBS 182.58). Based on the above morphological characters and available molecular data, we suggest that Phaeodothis can be referred to Didymosphaeriaceae.
Tremateia Kohlm., Volkm.-Kohlm. & O.E. Erikss., Bot.
Mar. 38(2): 165 (1995). Facesoffungi number: FoF 00223
Type species:
Tremateia halophila Kohlm et al., Bot. Mar. 38(2): 166
(1995) (Fig. 19), Facesoffungi number: FoF 00062.
Saprobic on dead wood in marine habitats. Sexual state:
Ascomata 225–320×330−410μm (x=260×355μm, n=10),
solitary, scattered, brown to black, immersed in the host tissue,
globose to subglobose. Peridium 15–25μm diam., composed
of two layers, outer layer composed of irregular, thick-walled,
brown cells of textura angularis. The inner layer composed of
hyaline, larger cells of textura angularis. Hamathecium dense
of 2–2.5 μm cellular, septate long, hyaline pseudoparaphyses.
Asci 120–170 × 24 − 30 μm (x= 150 × 26 μm, n = 20), 4–8spored, bitunicate, clavate to broadly clavate, short pedicellate, thin-walled, apically rounded with a broad ocular chamber. Ascospores 25–42×12−20μm (x=30×16μm, n=20),
uniseriate or biseirate, ellipsoid to fusiform, light brown to
brown, muriform, constricted at central septum, with 2layered cells, sometimes rounded at both sides with a 15–
25μm wide, large uniform sheath. Asexual state: Phoma-like
(Kohlmeyer et al. 1995).
Material examined: USA, North Carolina: Broad Creek, on
senescent culms of Juncus roemerianus Scheele (Juncaceae),
28 November, 1993, J. J. Kohlmeyer (NY 01353617,
isotype).
Notes: Tremateia, introduced as a facultative marine genus
(Kohlmeyer et al. 1995), is characterized by depressed globose, immersed ascomata, numerous cellular
pseudoparaphyses, fissitunicate and clavate asci, ellipsoid
muriform ascospores, and a Phoma-like asexual stage
(Kohlmeyer et al. 1995). Based on these morphological characters, Kohlmeyer et al. (1995) referred Tremateia to
Pleosporaceae. Furthermore, Kohlmeyer et al. (1995) suggested that Lewia M.E. Barr & E.G. Simmons and
Diademosa Shoemaker & C.E. Babc., are superficially similar
Fungal Diversity (2014) 68:69–104
to Tremateia. DNA sequences based phylogenies of recent
literatures, and also in the present study, have shown that
T. halophila nested in Montagnulaceae sister to Bimuria
novae-zelandiae. (Schoch et al. 2009; Suetrong et al. 2009),
thus familial status of Tremateia is verified.
Discussion
Molecular data play a pivotal role in modern mycological
taxonomy, but have some constraints in application
(Ariyawansa et al. 2014a; Boonmee et al. 2014; Hyde et al.
2014; Nilsson et al. 2014; Phookamsak et al. 2014; Schoch
et al. 2014; Thambugala et al. 2014; Wijayawardene et al.
2014). The most significant and unsettled problem is that the
phylogeny inferred from any gene may not reveal the evolution history of the organism (Uilenberg et al. 2004). It is
therefore better to incorporate a polyphasic taxonomy including genotypical and phenotypical characteristics in all future
studies (Ariyawansa et al. 2014a; Udayanga et al. 2014;
Uilenberg et al. 2004). The genome also needs to be evaluated
(Ariyawansa et al. 2014a; Uilenberg et al. 2004).
There have been only a few molecular investigations of
Didymosphaeriaceae compared to morphological studies
(Ariyawansa et al. 2014b). Genera with trabeculate
pseudoparaphyses and 1-septate, brown ascospores viz
Bicrouania, Caryosporella, Lineolata, Phaeodothis,
Roussoella and Verruculina, were generally classified under
Didymosphaeriaceae. Molecular studies have shown that these particular morphological characters have evolved in different families (Ariyawansa et al. 2014b). The position to the
taxa treated in the present study, it is required to first ascertain
the phylogenetic placement of the fungus in the
Montagnulaceae, as similar fungi occur in other
Pleosporales as well; LSU can be used to verify the order
and mostly also the family and genus to which the fungus
belongs. ITS alone might be adequate for a precise
identification of most species, as it is adequately variable
among most closely associated taxa in Montagnulaceae, but
it fails to distinguish all species. Furthermore we used TUB
gene for our combine gene phylogeny, which was proposed
by Verkley et al. (2014) to resolve the molecular taxonomy of
taxa in Montagnulaceae.
By combining multi-locus DNA sequencing with detailed
morphological analyses, we were able to delimit and formally
propose one new species, one sexual morph and one new
genus among the taxa in the family Didymosphaeriaceae,
previously classified in different families and genera in the
order Pleosporales. Importantly, we synonymise
Montagnulaceae under Didymosphaeriaceae by giving the
Fungal Diversity (2014) 68:69–104
Fig. 19 Tremateia halophila (NY 01353617, isotype) a-b, d Vertical
hand sections of ascomata. c, e Vertical hand section of ostiole and
peridium. f-j Asci with muriform ascospores. k Cellular
101
pseudoparaphyses. l-q Fusiform, light brown to brown, muriform ascospores. Scale bars: a-d=50μm, c, e=20μm, f-i, j-k=10μm, l-k=5μm
102
priority to the oldest name. The paraphyletic nature of
Paraconiothyrium is partially resolved by classifying some
species with their sexual states and combining both morpholo g y a n d p h y l o g e n y. B e c a u s e t h e b r a n c h e s o f
Paraconiothyrium clade that conflict with its monophyly are
insufficiently supported, it was decided not to split it up into
further genera. Morphological characters traditionally used to
define genera in coelomycetes include conidiomatal structure,
structure of the conidiophores, conidiogenesis and conidial
characters such as pigmentation, septal structure and number,
and conidial appendages (Sutton 1980; Nag Raj 1993). Recent
molecular studies have shown that these features are not
always suitable in delimiting genera as natural entities, and
they may vary even between sibling species (Crous et al.
2012). Generic boundaries drawn in the present study are
based primarily on statistically well-supported branches in a
multi-locus phylogeny. Some of the characters mentioned
above are thus overlapping between the accepted genera.
For example, phialidic and annellidic conidiogenesis occur
both in Paraconiothyrium and Paraphaeosphaeria (Verkley
et al. 2004, 2014; Damm et al. 2008). Recent studies based on
multi gene analysis have proven that the morphology of the
Coniothyrium-like scatter throughout the order Pleosporales
i.e. species with setose pycnidia and conidiogenesis with
elongated conidiophores occur in other genera, such as,
Cucurbitariaceae, Didymellaceae, Phaeosphaeriaceae,
Leptosphaeriaceae, Montagnulaceae and Sporormiaceae, indicating convergent evolution (Gruyter et al. 2013).
Neokalmusia was introduced in this study to accommodate
Kalmusia brevispora and K. scabrispora based on molecular
analysis in combination with morphology and a review of the
literature. By introducing the new genus Neokalmusia, we
were able to sort out the paraphyletic nature of Kalmusia in
the family Didymosphaeriaceae. Furthermore, Barria included in the family Didymosphaeriaceae, have been previously
referred to Phaeosphaeriaceae. Wilmia was synonymized
under Letendraea based on the fresh collection of new species
Letendraea cordylinicola (MFLU11-0184).
A confusing fact is that the putatively named strains of
Montagnula, M. spartii (CBS 183.58) and M. rhodophaea
(CBS 616.86) obtained from GenBank, clustered in
Massarinaceae and Lentitheciaceae, respectively. This is an
example of the problems of sequence data in GenBank and
other public databases. The morphology and identification of
the putative strains of M. spartii (CBS 183.58) and
M. rhodophaea (CBS 616.86) in GenBank cannot be checked,
as they are not linked to any herbarium material.
Acknowledgments MFLU grant number 56101020032 is thanked for
supporting studies on Dothideomycetes. We are also grateful to the
Mushroom Research Foundation, Chiang Rai, Thailand for funding.
Kevin D. Hyde thanks the Chinese Academy of Sciences, project number
2013T2S0030, for the award of Visiting Professorship for Senior International Scientists at Kunming Institute of Botany. Jian-Chu Xu would
Fungal Diversity (2014) 68:69–104
like to thank Humidtropics, a CGIAR Research Program that aims to
develop new opportunities for improved livelihoods in a sustainable
environment, for partially funding this work. H.A Ariyawansa and J.C.
Kang are grateful to the Agricultural Science and Technology Foundation
Of Guizhou Province (Nos. NY[2013]3042), the International Collaboration Plan of Guizhou Province (No. G [2012]7006) and the innovation
team construction For Science and Technology Of Guizhou Province
(No. [2012]4007) from the Science and Technology Department of
Guizhou Province, China. K. Tanaka would like to thank the Japan
Society for the Promotion of Science (JSPS, 25440199) for financial
support. Gareth Jones is supported by the Distinguished Scientist Fellowship Program (DSFP), King Saud University, Saudi Arabia. Hiran
Ariyawansa is grateful to A.D Ariyawansa, D.M.K Ariyawansa
Dhanuska Udayanaga and Dimuth Manamgoda for their valuable
suggestions.
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