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Accepted by Gareth Jones: 5 Mar. 2014; published: 20 Aug. 2014
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PHYTOTAXA
ISSN 1179-3155 (print edition)
ISSN
1179-3163
(online edition)
Copyright © 2014 Magnolia Press
Phytotaxa 176 (1): 102–119
www.mapress.com
/
phytotaxa
/
Article
http://dx.doi.org/10.11646/phytotaxa.176.1.12
Confusion surrounding Didymosphaeria—phylogenetic and morphological
evidence suggest Didymosphaeriaceae is not a distinct family
HIRAN A. ARIYAWANSA
1,2,3
, ERIO CAMPORESI
5
, KASUN M. THAMBUGALA
2,3
, AUSANA MAPOOK
2,3
,
JI-
CHUAN KANG
1
, SITI A. ALIAS
4
, EKACHAI CHUKEATIROTE
2,3
, MARCO THINES
6
, ERIC H.C. McKENZIE
9
& KEVIN D.
.
HYDE
2,3,7,8
1
The Engineering and Research Center for Southwest Bio-Pharmaceutical Resources of National Education Ministry of China,
Guizhou University, Guiyang 550025, Guizhou Province, China
2
School of Science, Mae Fah Luang University, Chiang Rai. 57100, Thailand
3
Institute of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand
4
Institute of Biological Sciences, University of Malaya, 50603, Kuala Lumpur, Malaysia
5
A.M.B. Gruppo Micologico Forlivese “Antonio Cicognani”, Via Roma 18, Forlì, Italy
6
Biodiversity and Climate Research Centre (BiK-F), Senckenberganlage 25, D-60325 Frankfurt (Main), Germany
7
Centre for Mountain Ecosystem Studies (CMES), Kunming Institute of Botany,
8
Chinese Academy of Science, Kunming 650201,
Yunnan, China
8
World Agroforestry Centre, East Asia Office, Kunming 650201, Yunnan, China
9
Manaaki Whenua Landcare Research, Private Bag 92170, Auckland, New Zealand
email: jichuank@yahoo.co.uk; kdhyde3@gmail.com
Abstract
Didymosphaeriaceae is a ubiquitous fungal family that is reported to include saprobic, endophytic and pathogenic
species associated with a wide variety of substrates. The family is characterized by 1-septate ascospores and trabeculate
pseudoparaphyses, mainly anastomosing above the asci. In recent treatments Appendispora, Didymosphaeria,
Roussoella, Phaeodothis and Verruculina were placed in the family. The aim of the present study is to delineate
phylogenetic lineages within Didymosphaeriaceae and allied genera. A new species, Didymosphaeria rubi-ulmifolii, was
isolated and identified based on morphological characters and phylogenetic analyses of partial 18S nrDNA and 28S
nrDNA nucleotide sequence data. Didymosphaeria rubi-ulmifolii clustered with Montagnulaceae as a separate genus,
while two putative strains (HKUCC 5834 and CMW 22186) of D. futilis from GenBank clustered with Cucurbitariaceae
and Didymellaceae, respectively. The new species is characterized by immersed to slightly erumpent ascomata immersed
under a clypeus, a peridium with compressed cells of textura intricata, long trabeculate pseudoparaphyses, anastomosing
mostly above the asci and brown, 1-septate ascospores with granulate ornamentation. Phylogenetic analysis in
combination with morphology and a review of literature show that Appendispora, Phaeodothis, Roussoella and
Verruculina should be excluded from the family. Phaeodothis belongs in Montagnulaceae, Verruculina in Testudinaceae,
while Appendispora and Roussoella belong in Roussoellaceae. The position of Didymosphaeriaceae as a distinct family,
based on 1-septate ascospores and trabeculate pseudoparaphyses, mainly anastomosing above the asci is doubtful. Fresh
collections of more Didymosphaeria strains are needed for epitypification and to obtain sequence data to establish if this
family can be maintained.
Keywords: Didymosphaeriaceae, epitypification, Montagnulaceae, new species
Introduction
Munk (1953) introduced the family Didymosphaeriaceae typified by the genus Didymosphaeria. The family was
characterized by 1-septate ascospores and trabeculate pseudoparaphyses which anastomosed mostly above the asci
(Aptroot 1995). Species can be found mainly in terrestrial habitats as saprobes or parasites on woody branches and
herbaceous stems and leaves, and are also parasitic on other fungi (Hyde et al. 2013). Based on its trabeculate
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CONFUSION SURROUNDING DIDYMOSPHAERIA
pseudoparaphyses, Barr (1990) referred Didymosphaeriaceae to the order Melanommatales, however, the
importance of trabeculate pseudoparaphyses was later disputed (Liew et al. 2000). Due to the uncertainty of the
placement, several authors have referred the family to different higher taxa. von Arx & Müller (1975) treated
Didymosphaeriaceae as a synonym of the Pleosporaceae. Didymosphaeriaceae was maintained as a separate family
within Pleosporales by Aptroot (1995) based on the 1-septate ascospores and trabeculate pseudoparaphyses.
Lumbsch & Huhndorf (2007) assigned Didymosphaeriaceae to Montagnulaceae, while Zhang et al. (2012)
tentatively included Didymosphaeriaceae as a separate family in the Pleosporales under the suborder
Pleosporineae. In the same study it was shown that two strains of Didymosphaeria futilis (HKUCC 5834 and CMW
22186), clustered separately in Cucurbitariaceae and Didymellaceae (Zhang et al. 2012). The understanding of the
type species, Didymosphaeria futilis, and Didymosphaeriaceae is therefore confused.
Based on trabeculate pseudoparaphyses embedded within a gel matrix and brown ascospores, Appendispora
was assigned to Didymosphaeriaceae (Barr 1987, Hyde 1994). Barr (1990) also included Neotestudina in this
family based on the morphology of the ascospores. Kohlmeyer & Volkmann-Kohlmeyer (1990) also assigned
Coronopapilla to Didymosphaeriaceae, while Verruculina enalia (Kohlm.) Kohlm. & Volkm.-Kohlm. (=Lojkania
enalia (Kohlm.) M.E. Barr = Didymosphaeria enalia Kohlm.) was also included in the family, based on its
characteristic ascospores. Lumbsch & Huhndorf (2010) included five genera in Didymosphaeriaceae, i.e.,
Appendispora, Didymosphaeria, Phaeodothis, Roussoella and Verruculina, but Zhang et al. (2012) accepted only
three genera for the family, namely Appendispora, Didymosphaeria and Phaeodothis.
Several asexual morphs have been reported for Didymosphaeriaceae. Sivanesan (1984) stated that
Didymosphaeria has Ascochyta and Periconia asexual states, while Keissleriella also included in
Didymosphaeriaceae by Sivanesan (1984), produces Dendrophoma asexual states. Phylogenetic studies based on
multigene analysis have shown that Keissleriella clusters within Lentitheciaceae, while Dendrophoma cytisporoides
Sacc., the generic type of Dendrophoma clusters with Chaetosphaeria hebetiseta Réblová & W. Gams, and C.
callimorpha (Mont.) Sacc., thus Dendrophoma belongs in Lentitheciaceae (Zhang et al. 2012, Schoch et al. 2009,
Crous et al. 2012). Ellis (1971) reported Periconia asexual states for Didymosphaeria, but this was due to the
misidentification of Lophiostoma as Didymosphaeria (Seifert et al. 2011, Hyde et al. 2013). The generic type of
Ascochyta, A. pisi Lib., clustered within Didymellaceae and, therefore, the placement of Ascochyta in
Didymosphaeriaceae is doubtful (Gruyter et al. 2009, Hyde et al. 2013). Roussoella produces a Cytoplea asexual state
in culture (Hyde et al. 1996). Liu et al. (2014) have introduced a new family Roussoellaceae in Pleosporales to
accommodate Roussoella species. Thus, the placement of the asexual states of Didymosphaeriaceae is also confused.
We have been studying the families of Pleosporales in order to provide a natural classification of this large
order (Ariyawansa et al. 2013a,b,c, Ariyawansa et al. 2014, Zhang et al. 2009, 2012). Some studies have been
based only on morphological characterization, while others have used molecular analysis (Ariyawansa et al.
2013a,b,c, Ariyawansa et al. 2014, Zhang et al. 2011, 2012). Given the considerable taxonomic confusion, the
present study, based on phylogenetic analyses of nrDNA sequence data, was undertaken to (i) validate the familial
placement of Didymosphaeria and assess whether it signifies natural groupings, (ii) verify which morphological
characters are phylogenetically significant by observing the type species of the genera Didymosphaeria and
Phaeodothis, and (iii) establish characters useful for generic delineation.
Materials and methods
Specimen examination
Fresh material of a Didymosphaeria species was collected on dead branches of Rubus ulmifolius in Forlì-
Cesena, Ravaldino, Italy during 2012. Specimens were brought to the laboratory in Zip lock plastic bags®.
Ascomata were removed directly from natural substrates without incubation. Herbarium specimens were obtained
on loan from the Swedish Museum of Natural History (S) and the New York Botanical Garden (NY).The samples
were processed and examined following the method described in Ariyawansa et al. (2013a,b). The fresh and
herbarium materials were examined for fungal fruiting bodies using a Motic SMZ 168 dissecting microscope. Hand
sections of the fruiting structures were mounted in water, examined with a Nikon ECLIPSE 80i compound
microscope and photographed using 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 were processed with Adobe
ARIYAWANSA ET AL.
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Phytotaxa 176 (1) © 2014 Magnolia Press
Photoshop CS3 Extended version 10.0 software (Adobe Systems, The United States). Isolations were made from
single ascospores following a modified method of Chomnunti et al. (2011, 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 overnight. Germinated
ascospores were transferred singly to malt extract agar (MEA) media (Alves et al. 2006, Liu et al. 2011). Voucher
specimens are deposited in the herbarium of Mae Fah Luang University (MFLU), Chiang Rai, Thailand and
cultures are deposited at the Mae Fah Luang University Culture Collection (MFLUCC), Biodiversity and Climate
Research Centre (BiK-F), International Collection of Microorganisms from Plants (ICMP) and BIOTEC Culture
Collection (BCC), the latter under Material Transfer Agreement No. 4/2010 (MTA).
DNA extraction, PCR amplification and sequencing
Fungal isolates were grown on MEA for 28 days at 25°C in the dark. Genomic DNA was recovered from the
growing mycelium using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux®), but using sterile white
quartz sand and a Kontes® battery-powered pestle grinder in 1.5 mL microfuge tubes.
Polymerase chain reaction (PCR) was carried out using known primer pairs. NS1 and NS4 were used to
amplify a region spanning the small subunit rDNA (White et al. 1990). LROR and LR5 primer pairs were used to
amplify a segment of the large subunit rDNA (Vilgalys & Hester 1990) and internal transcribed spacers was
amplified by primer pairs ITS1 and ITS4 (White et al. 1990). 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 MgCl
2
(Cai et al. 2009). Amplification conditions were setup for initial denaturation of 5
min at 95°C, followed by 35 cycles of 45 s at 94°C, 45 s at 48°C and 90 s at 72°C, and a final extension period of
10 min at 72°C (Phillips et al. 2008). 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 & Services Co. (China).
Phylogenetic analysis
The large and small subunits of the nuclear ribosomal RNA genes (LSU, SSU) were included in the analysis.
All sequences obtained from GenBank and those used in Hyde et al. (2013), Schoch et al. (2009) and Zhang et al.
(2012) and are listed in Table 2. Multiple sequence alignments were generated with MAFFT v. 6.864b (http://
mafft.cbrc.jp/alignment/server/index.html). The alignments were checked visually and improved manually where
necessary. Concordance of the SSU and LSU genes datasets was estimated with the partition-homogeneity test
implemented with PAUP v. 4.0b10 (Swofford 2002). Dothidea sambuci was selected as out group taxon. New
sequences are deposited in GenBank (Table 1).
TABLE 1. Synopsis of the characteristics of three species of Didymosphaeria.
Maximum likelihood analyses including 1,000 bootstrap replicates were run using RAxML v. 7.2.6
(Stamatakis & Alachiotis 2006, 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 -21860.18890. The resulting replicates were plotted
on to the best scoring tree obtained previously. Maximum likelihood bootstrap values (ML) equal or greater than
50% are given below or above each node in red (Fig. 1).
MEGA 5.0 (Tamura et al. 2011) was used for minimum evolution (ME) inference, with default settings except
for assuming pairwise deletion, and using the Tamura-Nei nucleotide substitution model. For inferring tree
robustness, 1,000 bootstrap replicates were carried out and bootstrap values (ME) equal or greater than 50% are
given below or above each node in green (Fig. 1).
Species Ascomata (μm) Peridium (μm) Pseudoparaphyses
(μm)
Asci (μm) Ascospores (μm) and
ornamentation
D. decolorans 150–300 × 200–320 30–40 0.8–1.5 55–80 × 5–8 8–13 × 4–6, smooth
D. futilis 100–150 × 120–160 10–20 0.5–1 75–85 × 4–6 7–10 × 3–5, spinulose
D. rubi-ulmifolii 140–250 × 150–280 7–15 0.5–1 60–90 × 4–8 6–10 × 3–6, granulate
Phytotaxa 176 (1) © 2014 Magnolia Press
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CONFUSION SURROUNDING DIDYMOSPHAERIA
FIGURE 1. Best scoring RAxML tree based on a combined dataset of SSU and LSU with bootstrap support values for maximum
likelihood (red) and minimum evolution (green) greater than 50% given below and above the nodes. Dothidea sambuci is the out
group taxon. The original isolate numbers are noted after the species names.
TABLE 2. Isolates used in this study and their GenBank accession numbers. Newly deposited sequences are shown in bold
.
Taxon Voucher/culture LSU SSU
Aigialus grandis JK 5244A GU301793 GU296131
Aigialus parvus A6 GU301795 GU296133
Amniculicola immersa CBS 123083 FJ795498 GU456295
Amniculicola parva CBS 123092 FJ795497 GU296134
Ascocratera manglicola JK 5262C GU301799 GU296136
Bimuria novaezelandiae CBS 107.79 AY016356 AY016338
Boeremia exigua CBS 431.74 EU754183 EU754084
Cochliobolus heterostrophus CBS 134.39 AY544645 AY544727
Cucurbitaria berberidis CBS 363.93 GQ387605 GQ387544
Cucurbitaria berberidis CBS 394.84 GQ387606 GQ387545
Deniquelata barringtoniae MFLUCC 110422 JX254655 JX254656
...... continued on the next page
Pyrenochaetopsis leptospora CBS 101635
Didymosphaeria futilis HKUCC 5834
Pyrenochaeta corni CBS 248.79
Pyrenochaeta cava CBS 115953
Cucurbitaria berberidis CBS 363.93
Cucurbitaria berberidis CBS 394.84
Pleospora herbarum CBS 191.86
Cochliobolus heterostrophus CBS 134.39
Pyrenophora phaeocomes DAOM 222769
Ophiosphaerella herpotricha CBS 620.86
Phaeosphaeria eustoma CBS 573.86
Phaeosphaeria oryzae CBS 110110
Paraphoma radicina CBS 111.79
Didymosphaeria futilis CMW 22186
Boeremia exigua CBS 431.74
Leptosphaerulina australis CBS 317.83
Didymella exigua CBS 183.55
Dothidotthia symphoricarpi CPC 12929
Dothidotthia aspera CPC 12933
Bimuria novae-zelandiae CBS 107.79
Kalmusia scabrispora KT 1023
Kalmusia brevispora KT 1466
Phaeodothis winteri CBS 182.58
Deniquelata barringtoniae MFLUCC 110422
Didymocrea sadasivanii CBS 438.65
Karstenula rhodostoma CBS 690.94
Paraconiothyrium minitans CBS 122788
Paraphaeosphaeria michotii CBS 652.86
Didymosphaeria rubi-ulmifolii MFLUCC 14-0024
Didymosphaeria rubi-ulmifolii MFLUCC 14-0023
Letendraea padouk CBS485.70
Letendraea helminthicola CBS 884.85
Montagnula opulenta CBS 168.34
Massarina eburnea CBS 473.64
Neottiosporina paspali CBS 331.37
Katumotoa bambusicola KT 1517a
Lentithecium aquaticum CBS 123099
Lentithecium fluviatile CBS 122367
Trematosphaeria pertusa CBS 122371
Falciformispora lignatilis BCC 21118
Morosphaeria ramunculicola JK 5304B
Morosphaeria ramunculicola BCC 18405
Morosphaeria velataspora BCC 17059
Pleomassaria siparia
CBS 279.74
Prosthemium canba KT2083-1
Prosthemium betulinum VM20040721
Monotosporella tuberculata CBS 256.84
Melanomma pulvis-pyrius CBS 371.75
Preussia terricola AFTOL-ID 282
Preussia minima CBS 524.50
Eremodothis angulata CBS 610.74
Aigialus grandis JK 5244A
Aigialus parvus A6
Ascocratera manglicola JK 5262C
Amniculicola immersa CBS 123083
Amniculicola parva CBS 123092
Dothidea sambuci DAOM 231303
0.02
100/100
77/54
55/68
65/81
99/100
77/55
99/98
100/100
85/76
57/73
50/57
57/76
50/51
68/55
97/98
60/73
97/84
92/91
50/55
52/58
97/83
70/65
90/100
62/73
--/--
99/91
9 1/100
--/--
99/100
60/79
7 0/73
88/98
6 0/74
90/69
99/91
--/-- 70/90
100/100
100/100
80/91
80/71
96/90
99/98
72/71
100/100
97/80
0/85
86/53
92/70
100/90
100/100
100/100
100/100
100/100
Cucurbitariaceae
Pleosporaceae
Phaeosphaeriaceae
Didymellaceae
Dothidotthiaceae
Montagnulaceae
Massarinaceae
Lentitheciaceae
Trematosphaeriaceae
Morosphaeriaceae
Pleomassariaceae
Melanommataceae
Sporormiaceae
Aigialaceae
Amniculicolaceae
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Phytotaxa 176 (1) © 2014 Magnolia Press
TABLE 2 (continued)
Taxon Voucher/culture LSU SSU
Didymella exigua CBS 183.55 EU754155 EU754056
Didymocrea sadasivanii CBS 438.65 DQ384103 DQ384066
Didymosphaeria futilis HKUCC 5834 GU205219 GU205236
Didymosphaeria futilis CMW22816 EU552123
Didymospheria rubi-ulmifolii MFLUCC 14-0024 KJ436585 KJ436587
Didymospheria rubi-ulmifolii MFLUCC 14-0023 KJ436586 KJ436588
Dothidea sambuci DAOM 231303 AY544681 AY544722
Dothidotthia aspera CPC 12933 EU673276 EU673228
Dothidotthia symphoricarpi CPC 12929 EU673273 EU673224
Eremodothis angulata CBS 610.74 DQ384105 DQ384067
Falciformispora lignatilis BCC 21118 GU371827 GU371835
Kalmusia scabrispora KT 1023 AB524593 AB524452
Karstenula rhodostoma CBS 690.94 GU301821 GU296154
Katumotoa bambusicola KT 1517a AB524595 AB524454
Lentithecium aquaticum CBS 123099 GU301823 GU296156
Lentithecium fluviatile CBS 122367 GU301825 GU296158
Leptosphaerulina australis CBS 317.83 GU301830 GU296160
Letendraea helminthicola CBS 884.85 AY016362 AY016345
Letendraea padouk CBS485.70 AY849951 GU296162
Massarina eburnea CBS 473.64 GU301840 GU296170
Melanomma pulvis-pyrius CBS 371.75 GU301845
Monotosporella tuberculata CBS 256.84 GU301851
Montagnula opulenta CBS 168.34 DQ678086 AF164370
Morosphaeria ramunculicola JK 5304B GQ925854 GQ925839
Morosphaeria ramunculicola BCC 18405 GU479794 GU479760
Morosphaeria velataspora BCC 17059 GQ925852 GQ925841
Neottiosporina paspali CBS 331.37 EU754172 EU754073
Ophiosphaerella herpotricha CBS 620.86 DQ678062 DQ678010
Paraconiothyrium minitans CBS 122788 EU754173 EU754074
Paraphaeosphaeria michotii CBS 652.86 GQ387581 GQ387520
Paraphoma radicina CBS 111.79 EU754191 EU754092
Phaeodothis winteri CBS 182.58 GU301857
Phaeosphaeria eustoma CBS 573.86 DQ678063 DQ678011
Phaeosphaeria oryzae CBS 110110 GQ387591
Pleomassaria siparia CBS 279.74 DQ678078
Pleospora herbarum CBS 191.86 DQ247804 DQ247812
Preussia minima CBS 524.50 DQ678056 DQ678003
Preussia terricola AFTOL-ID 282 AY544686 AY544726
Prosthemium betulinum VM20040721 AB553754
Prosthemium canba KT2083-1 AB553760
...... continued on the next page
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CONFUSION SURROUNDING DIDYMOSPHAERIA
Results
Phylogeny of combined 18S and 28S nrDNA gene datasets
The combined SSU and LSU dataset comprised 57 taxa, including two new strains of Didymosphaeria rubaei.
Results of the partition-homogeneity test (P = 0.107) indicated that the SSU and LSU gene trees reflect the same
underlying phylogeny. Therefore, these datasets were combined and analyzed by using several tree-building
programs and the resulting trees were compared.
Phylogenetic analysis
The combined SSU and LSU gene dataset of 15 families in the Pleosporales is shown (Fig. 1). All trees (ML
and ME) were similar in topology and not significantly different (data not shown). A best scoring RAxML tree is
shown in Fig. 1 with the value of -21860.18890. The strains of Didymosphaeria rubi-ulmifolii (MFLUCC 14-0023
and 14-0024) clustered in the family Montagnulaceae, but were separated from other genera of the family and sister
to Paraphaeosphaeria clade. The two putative strains of Didymosphaeria futilis (HKUCC 5834 and CMW 22186)
from GenBank clustered in Cucurbitariaceae and Didymellaceae, respectively.
Taxo n o m y
Didymosphaeriaceae Munk, Dansk bot. Ark. 15(2): 128 (1953) MycoBank: MB 80702
Notes:—The familial status of the Didymosphaeriaceae is questionable. Lumbsch & Huhndorf (2007) assigned it
to the Montagnulaceae, while von Arx & Müller (1975) treated it as a synonym of the Pleosporaceae. Eriksson &
Hawksworth (1993) listed Didymosphaeria, Montagnula, Verruculina and, doubtfully, Roussoella in this family.
Neotestudina was subsequently referred to the Testudinaceae based on molecular data (Schoch et al. 2009,
Seutrong et al. 2009). Aptroot (1995a) suggested that Didymosphaeriaceae species are probably close to other
families in the Melanommatales with 1-septate ascospores (often recognized at ordinal level and as the
Pyrenulales), viz Requienellaceae, Pyrenulaceae, Trypetheliaceae and Massariaceae and many taxa were excluded
from the Didymosphaeriaceae, i.e. Aaosphaeria, Amphisphaeria, Astrosphaeriella, Dothidotthia,
Flagellosphaeria, Kirschsteiniothelia, Megalotremis, Montagnula, Munkovalsaria, Mycomicrothelia, Parapyrenis
Phaeodothis, and Verruculina. Huhndorf & Lumbsch (2010) treated Didymosphaeriaceae as a separate family and
assigned to Pleosporales and included five genera, viz Appendispora, Didymosphaeria, Roussoella, Phaeodothis
and Verruculina. When considering 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 a distinct family of Pleosporales. However, based on the molecular data the
status of Didymosphaeriaceae as a distinct family is doubtful (Fig. 1). Collections of authentic Didymosphaeria
futilis are needed for epitypification and to obtain sequence data to confirm that Didymosphaeriaceae is a synonym
of Montagnulaceae.
TABLE 2 (continued)
Taxon Voucher/culture LSU SSU
Pyrenochaeta cava CBS 115953 GQ387607 GQ387546
Pyrenochaeta corni CBS 248.79 GQ387608 GQ387547
Pyrenochaetopsis leptospora CBS 101635 GQ387627 GQ387566
Pyrenophora phaeocomes DAOM 222769 DQ499596 DQ499595
Trematosphaeria pertusa CBS 122371 GU301876 GU348999
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Didymosphaeria Fuckel, Jb. nassau. Ver. Naturk. 23-24: 140 (1870) [1869–70]
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.
Notes:—The genus Didymosphaeria was introduced by Fuckel (1870) for three species of ascomycetes with
two-celled ascospores. This genus sensu lato, comprised species having a wide distribution and a broad host range.
Saccardo (1882) restricted the genus to only those species with brown ascospores (Aptroot 1995a). More than 100
species have been excluded from Didymosphaeria by various authors (Barr 1989a,b, 1990, 1992a,b, Hawksworth
1985a,b, Hawksworth & Boise 1985, Hawksworth & Diederich 1988, Scheinpflug 1958). Aptroot (1995a)
included over 400 epithets of Didymosphaeria in his monograph of the genus, after examining over 3,000 species,
and accepted only seven species.
The placement of Didymosphaeria is confused. Initially, the genus comprised three species and most authors
accepted Didymosphaeria epidermidis (Fries) Fuckel as the type but, the type material comprises a coelomycete
(Aptroot 1995a). The type of the only original species, D. rubi Fuckel, could not be found in G or in any other
herbarium (Aptroot 1995a). Considering these facts Hawksworth & David (1989) proposed to conserve the genus
with a lectotype specimen, and selected the widespread and common D. futilis (Fungi Rhenani 1770) as the type for
the Didymosphaeriaceae (Aptroot 1995a). This proposal was accepted by the Yokohama Botanical Congress
(1993), in agreement with the Committee for Fungi of the International Association of Plant Taxonomists. Aptroot
(1995a) listed seven species i.e., D. bisphaerica, D. conoidea, D. dimastospora, D. futilis, D. massarioides, D.
oblitescens, and D. spinosa, which are closely related with the generic type of Didymosphaeria without considering
differences of host or country of origin. Aptroot (1995a) proposed that Didymosphaeria belonged in the family
Didymosphaeriaceae, order Pleosporales based on a peridium containing pseudoparenchymatous cells, a
hamathecium consisting of narrow, trabeculate pseudoparaphyses, mainly anastomosing above the asci and brown,
1-septate ascospores (Kirk et al. 2008).
Asexual stages of Didymosphaeria are unclear. Sivanesan (1984) reported that Didymosphaeria has Ascochyta
and Periconia asexual states, while Kirk et al. (2008) linked Fusicladiella-like and Phoma-like species to the
genus. Linking Didymosphaeria to asexual states should be treated with caution until a lectotype of D. futilis has
been sequenced.
Type species: Didymosphaeria futilis (Berk. & Broome) Rehm, Hedwigia 18: 167 (1879) MycoBank MB 223613
(Fig. 2a–o)
≡ Sphaeria epidermidis Berk. & Broome, Ann. Mag. nat. Hist., Ser. 2 9: 326 (1852)
Saprobic on dead wood. Sexual state: Ascomata 110–140 × 120–160 μm ( = 130 × 140 µm, 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 ( = 15 µm, n = 10) μm wide, 1-layered, composed of hyaline pseudoparenchymatous compressed cells of
textura intricata, fusing at the outside with the host. Hamathecium of dense, 0.5–1 μm ( = 0.8 µm, n = 20) broad,
long, trabeculate pseudoparaphyses, anastomosing frequently above the asci, embedded in mucilage. Asci 75–85 ×
4–6 μm ( = 78 × 6 µm, n = 20), 8-spored, bitunicate, fissitunicate, cylindrical, pedicellate, rounded apex with an
indistinct ocular chamber. Ascospores 7–10 × 3–5 μm ( = 9 × 5 µm, 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: UNITED KINGDOM. England: Norfolk, on dead stem of Rosa sp., March 1850, M.J.
Berkeley (K 147683!, holotype of Sphaeria epidermidis).
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Notes:—The type species of Didymosphaeria, Didymosphaeria futilis (Berk. & Broome) Rehm was initially
described as Sphaeria futilis by Berk. & Broome (1852). Aptroot (1995a) synonymized more than 40 species under
Didymosphaeria futilis. The placement of D. futilis is confused because several molecular studies (e.g., Zhang et
al. 2012) and the present study have shown that the putatively named strains of D. futilis obtained from GenBank
(HKUCC 5834 and CMW 22186) clustered in different families. Therefore, fresh collections of Didymosphaeria
futilis are needed so that molecular data can be used to validate the natural taxonomic affinities of this genus.
FIGURE 2. Didymosphaeria futilis (holotype) a–b. Herbarium packet and specimen. c. Close-up of ascomata. d. Section through
ascoma. e. Close-up of peridium. f. Arrangement of asci and pseudoparaphyses in hamathecium. g. Broad, long trabeculate
pseudoparaphyses, anastomosing mostly above the asci. h–k. Cylindrical asci with an indistinct ocular chamber. l–o. Ascospores with
distinct spinulose ornamentation. Scale bars: c = 100 µm, d–g = 10 µm, h–k = 20 µm, l–0 = 5 µm.
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FIGURE 3. Didymosphaeria decolorans (holotype). a. Herbarium packet and specimen. b–c. Close up of the ascomata. d. Section
through ascoma. e. Close-up of the ostiole g. Long trabeculate pseudoparaphyses. h–l. Immature and mature asci. m–r. Immature and
mature ascospores with smooth wall. Scale bars: b = 500 µm, c = 200 µm, d = 50 µm, h–l = 20 µm, e, f = 10 µm, g, m–r = 5 µm.
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Didymosphaeria decolorans Rehm, Hedwigia 37(Beibl.): (143) (1898) MycoBank: MB 224571 (Fig. 3a–r)
Fungicolous or saprobic on other fungi or dead wood. Sexual state: Ascomata 150–300 × 200–320 μm ( = 280 × 300
µm, n = 10), solitary to aggregated, immersed, subsequently erumpent through host periderm, globose to subglobose,
membraneous, dark brown to black, papillate. Papilla black, with a pore-like ostiole, ostiolar canal filled with
periphyses. Peridium 30–40 ( = 35 µm, n = 10) μm wide, 1-layered, composed of brown pseudoparenchymatous
cells of textura angularis. Hamathecium of dense, 0.8–1.5 μm ( = 1 µm, n = 20) broad, long trabeculate
pseudoparaphyses, anastomosing mostly above the asci, embedded in mucilage. Asci 55–80 × 5–8 μm ( = 75 × 7 µm,
n = 20), 8-spored, bitunicate, fissitunicate, cylindrical, short pedicellate, rounded apex with a minute ocular chamber.
Ascospores 8–13 × 4–6 μm ( = 10 × 5 µm, n = 40), uniseriate, slightly overlapping, ellipsoid with obtuse ends,
brown, 1-septate, slightly to not constricted at the septum, with smooth wall. Asexual state: unknown.
Notes: We observed the holotype of D. decolorans (S, F12021), which was examined by Aptroot (1995a) and
synonymized under D. futilis. D. decolorans grew inside the fruiting bodies of a Leptosphaeria or Pleospora-like
species (Fig. 3b–c). D. decolorans and D. futilis share similar characters in having 1-septate ascospores and
trabeculate pseudoparaphyses, mainly anastomosing above the asci, but differ in the structure of the peridium
(brown pseudoparenchymatous cells of textura angularis versus hyaline pseudoparenchymatous compressed cells
of textura intricata), ascospore wall ornamentation (smooth versus spinulose) and the habitat (fungicolous versus
saprobic), respectively. Fresh collections of D. decolorans and sequence data analysis are needed to confirm if this
is a Didymosphaeria species, as D. decolorans is not typical of the genus because of its fungicolous nature and the
structure of the peridium (composed of brown pseudoparenchymatous cells of textura angularis).
Material examined:—GERMANY. Oberammergau, Bayern, on dry branches of Salix caprea, August 1894
and 1896, leg. Schnabl, determined by H. Rehm (S, F 12021!, holotype).
Didymosphaeria rubi-ulmifolii Ariyawansa, Erio Camporesi & K.D. Hyde, sp. nov. MycoBank MB 808165 (Fig.
4a–l)
Etymology:—The specific epithet rubi-ulmifolii is based on the host genus from which the fungus was isolated.
Saprobic on wood. Sexual state: Ascomata 140–250 × 150–280 μm ( = 210 × 230 µm, n = 10), scattered, or in small
groups, immersed to slightly erumpent under clypeus, globose to sub globose, membraneous, papillate. Papilla black,
with a pore-like ostiole, ostiolar canal filled with periphyses. Peridium 6–12 ( = 10 µm, n = 10) μm wide, 1-layered,
composed of hyaline to light brown pseudoparenchymatous compressed cells of textura intricata, fusing at the outside
with the host. Hamathecium of dense, 0.5–1 μm ( = 0.7 µm, n = 20) broad, long, trabeculate pseudoparaphyses,
anastomosing mostly above the asci, embedded in mucilage. Asci 60–90 × 4–8 μm ( = 75 × 7 µm, n = 20), 8-spored,
bitunicate, fissitunicate, cylindrical, pedicellate, rounded apex with and an indistinct ocular chamber. Ascospores 6–10
× 3–6 μm ( = 10 × 5 µm, n = 40), uniseriate, slightly overlapping, ellipsoid with obtuse ends, brown, 1-septate,
slightly to not constricted at the septum, with granulate ornamentation. Asexual state: not produced in culture.
Material examined:—ITALY. Forlì-Cesena, Ravaldino, on dead branch of Rubus ulmifolius, 14 January 2012,
Erio Camporesi (MFLU 12-2215!, holotype), ex-type living culture (MFLUCC 14-0023 and 14-0024)
Notes:—Based on morphological characteristics, Didymosphaeria rubi-ulmifolii fits in the generic concept of
Didymosphaeria in having 1-septate ascospores and trabeculate pseudoparaphyses, mainly anastomosing above the
asci. Morphologically D. rubi-ulmifolii and D. futilis are similar in having immersed ascomata under clypeus with
single layered peridium comprising hyaline, compressed cells of textura intricata, trabeculate pseudoparaphyses,
anastomosing mostly above the cylindrical asci, and brown, 1-septate ascospores. Differences are in the size of the
ascomata, ornamentation of the ascospores (granulate versus spinulose) and host (Table 1). Based on the original
description of D. rubi, D. rubi-ulmifolii and D. rubi differ in width of the peridium (thin versus broad) and the
ornamentation of the ascospores (granulate versus foveate). We could not observe D. rubi due to the unavailability
of the holotype specimen in G or in any other herbarium and an isotype in W reported only as a coelomycete
(Aptroot 1995a). Didymosphaeria rubicola Berl., differs from D. rubaie in having carbonaceous ascomata and
comparatively large spores (20–23 × 10–12 μm versus 6–10 × 3–6 μm) and the host (Rubus fruticosus versus R.
ulmifolius) (Saccardo 1891). D. futilis, D. decolorans and D. rubaie are compared in Table 1.
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FIGURE 4. Didymosphaeria rubi-ulmifolii (holotype). a. Immersed ascomata on the host surface b. Close-up of the ascomata c.
Section of an ascoma. d. Close-up of peridium. e. Trabeculate, anastomosing and branching pseudoparaphyses. f–h. Asci with 8
spores. i–l. Brown, 1-septate ascospores with granulate ornamentation. Scale bars: c = 100 μm, d = 20 μm, e = 10 μm f–h = 30 μm, i–l
= 10 μm.
Placement of excluded genera
Roussoellaceae J.K. Liu et al., In Press MycoBank: MB 804651
Based on immersed gregarious, clypeate ascomata with trabeculate pseudoparaphyses, embedded in a gel matrix,
long cylindrical bitunicate asci without obvious fissitunicate dehiscence and brown, 1–2-septate ornamented
ascospores, Liu et al. (2014) introduced the new family Roussoellaceae in the order Pleosporales. Most of the taxa
in Roussoellaceae are from bamboo, palms and grasses.
Appendispora K.D. Hyde, Sydowia 46(1): 29 (1994)
Generic description
Saprobic on palms. Sexual state: Ascostromata immersed beneath a stroma, axis horizontal, oblique or vertical
to the host surface, with up to five clustered locules and a central ostiolar canal. Peridium thin, inner layer
composed of hyaline elongated cells and outer layers comprising small brown cells. Hamathecium of dense, long,
trabeculate pseudoparaphyses. Asci 8-spored, bitunicate, fissitunicate, cylindrical, short pedicellate, apically
rounded with an ocular chamber and faint ring. Ascospores uniseriate to partially overlapping, fusoid, brown, 1-
septate, appendaged, slightly constricted at the septum. Asexual state: unknown.
Type species: Appendispora frondicola K.D. Hyde, Sydowia 46(1): 30 (1994)
Notes:—Appendispora was introduced by Hyde (1994) and typified by A. frondicola. Currently two epithets (A.
australiensis and A. frondicola) are listed for Appendispora in Index Fungorum (2014), however, molecular data is
lacking. The genus was described as a saprobe of palms, and is characterized by clustered ascostromata, which are
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immersed beneath a stroma, with their axis horizontal, oblique or vertical to the host surface, with minute ostioles
visible through cracks or blackened dots on the host surface, trabeculate pseudoparaphyses, brown, and 1-septate,
appendaged ascospores with irregular wall striations (Hyde 1994). Hyde (1994) suggested that the genus can be
referred to Didymosphaeriaceae based on trabeculate pseudoparaphyses embedded within gel matrix and brown
ascospores and this was followed by Barr (1987b). Appendispora has similarities with Didymosphaeria, but differs
in having ascospores with reticulate wall ornamentation and appendages (Hyde 1994). Furthermore, ascomata in
Didymosphaeria are immersed under a clypeus with their axis vertical to the host surface, whereas in Appendispora
ascomata are clustered under stromata and their axes are horizontal, oblique or vertical (Hyde 1994). In Hyde
(1994, Fig. 1) the locules appear to be arranged in a ring with a common central ostiole. One Didymosphaeria
species has been reported from the branches of Cytisus multiflorus, with appendaged ascospores (D.
polytrichospora M.T. Lucas & Sousa da Câmara) by Lucas & DaCamara (1953). D. polytrichospora has brown
two-celled ascospores provided with 6-7 appendages at each end, which are illustrated as seta-like (Hyde 1994).
After observing the type species of D. polytrichospora, Aptroot (1994) concluded that this taxon has unitunicate
asci with a J+ subapical ring. The taxonomic position of D. polytrichospora is uncertain, but it is far removed from
Appendispora, which has bitunicate asci lacking a J+ ring. Appendispora shares similarities with Roussoellaceae in
having trabeculate pseudoparaphyses, embedded in a gel matrix, long cylindrical asci and 1-septate, brown
ascospores and furthermore, most of the taxa in Roussoellaceae have been described from bamboo (Hyde et al.
2013, Liu et al. 2014) and palms (Hyde et al. 2013, Liu et al. 2014). Appendispora is also recorded from palms, but
differs from other genera of Roussoellaceae in the arrangement of locules in the ascostromata with a central ostiole
and its appendaged ascospores (Hyde et al. 2013, Liu et al. 2014). Based on the above comparison we place
Appendispora in Roussoellaceae.
Roussoella Sacc., in Saccardo & Paoletti, Atti Ist. Veneto Sci. lett. ed Arti, Sér. 3 6: 410 (1888)
Type species:—Roussoella nitidula Sacc. & Paol., Atti Ist. Veneto Sci. lett. ed Arti, Sér. 3, 6: 410 (1888)
Notes:—Roussoëlla was introduced by Saccardo for the single species R. nitidula (Saccardo & Paoletti 1888). The
genus is characterized by immersed ascomata with long cylindrical asci and brown 1-septate ornamented
ascospores (Hyde et al. 1996). Phylogeny based on multigene analysis has shown that the species of Roussoella,
Roussoellopsis as well as Arthopyrenia salicis form a robust clade, which forms a sister group with pleosporalean
families, but the generic type of Roussoella (R. nitidula) was not included in the phylogenetic study (Tanaka et al.
2009, Zhang et al. 2012). Liu et al. (In Press) will introduce a new family Roussoellaceae to accommodate
Roussoella, a natural classification verified by molecular data and supported by morphology.
Montagnulaceae M.E. Barr, Mycotaxon 77:194 (2001) MycoBank: MB 82111
Barr (2001) introduced the family, Montagnulaceae with three genera including Kalmusia, Montagnula and
Didymosphaerella in the order Pleosporales (Hyde et al. 2013). The family is characterized by ascomata immersed
under a clypeus, a small celled pseudoparenchymatous peridium, cylindric or oblong, fissitunicate, pedicellate asci
and brown ascospores (Hyde et al. 2013).
Phaeodothis Syd. & P. Syd., Annls mycol. 2(2): 166 (1904)
Generic description
Saprobic or parasitic in terrestrial habitats 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, narrow, cellular pseudoparaphyses often in a gelatinous matrix. Asci 8-spored, bitunicate,
cylindrical, short pedicellate. Ascospores fusiform with rounded ends, olivaceous-brown. Asexual state: unknown.
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Type species:—Phaeodothis tricuspidis Syd. & P. Syd., Annls mycol. 2(2): 166 (1904) (Fig. 5)
Saprobic in terrestrial habitats. Sexual state: Ascostromata 235–290 × 120−180 µm ( = 254 × 137 µm, n = 10),
immersed to nearly superficial under clypeus, scattered to gregarious, subglobose to hemispherical, dark brown to
black, papillate, smooth. Papilla black, with a pore-like ostioles, ostiolar canal filled with periphyses. Peridium
15−28 µm ( = 22 µm, n = 20) wide, comprising several layers of hyaline compressed cells. Hamathecium of
dense, 2−4 µm ( = 2 µm, n = 20), aseptate, narrow, cellular pseudoparaphyses often in a gelatinous matrix. Asci
55−80 × 7−12 µm ( = 68 × 10 µm, n = 20), 8-spored, bitunicate, fissitunicate, cylindrical, short pedicellate with a
minute ocular chamber. Ascospores 14−18 × 4−5µm ( = 16 × 5 µm, n = 30), biseriate and partially overlapping,
fusiform with rounded ends, olivaceous-brown, 1-septate, slightly constricted at the septum. Asexual state:
unknown.
FIGURE 5. Phaeodothis tricuspidis (holotype) a. Herbarium packet and specimen. b. Close-up of ascomata. c–d. Sections through
ascoma. 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 = 100 µm, e = 50 µm, f–h = 25 µm, i–l = 5 µm.
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Material examined:—ARGENTINA. San José, Salta, 12 February 1873, P.G. Lo r en t z (S F125876!,
holotype).
Notes:—Phaeodothis is characterized by its immersed to nearly superficial ascostromata, a sparse
hamathecium consisting of thin pseudoparaphyses and 1-septate ascospores (Aptroot 1995). The genus was placed
in Didymosphaeria by Niessl (1875), but Aptroot (1995) transferred the genus to Phaeosphaeriaceae. Phaeodothis
has similarities with Montagnulaceae in having ascomata immersed under a clypeus, a pseudoparenchymatous
peridium with small cells, cylindrical asci and brown ascospores. Phaeodothis however, differs from the other
genera of Montagnulaceae in having a hamathecium consisting of thin, sparse pseudoparaphyses and 1-septate
ascospores. A recent phylogenetic analysis based on multiple genes (LSU, SSU, RBP1, RBP2 and EF-1) concluded
that a strain named Phaeodothis winteri (a synonym of P. tricuspidis Syd. & P. Syd.) clustered within the
Montagnulaceae clade (Schoch et al. 2009). Currently 27 epithets are listed for the genus (Index Fungorum 2014),
while GenBank has seven hits for the genus including the putative strain of Phaeodothis winteri (CBS 182.58).
Based on the above morphological characters and available molecular data, we suggest that Phaeodothis can be
referred to Montagnulaceae.
Testudinaceae Arx, Persoonia 6: 365 (1971) MycoBank: MB 81456
The family Testudinaceae was introduced by von Arx (1971), based on ascomata with a dark peridium, which is
often made up of plates, with bitunicate asci, and dark 2-celled ascospores, about 10 µm long and referred to the
order Pseudosphaeriales (= Pleosporales). Currently the family contains five genera viz Lepidosphaeria,
Neotestudina, Testudina, Verruculina and Ulospora (Schoch et al. 2009, Seutrong et al. 2009, Lumbsch &
Huhndorf 2010, Hyde et al. 2013).
Verruculina Kohlm. & Volkm.-Kohlm., Mycol. Res. 94(5): 689 (1990)
Type species:—Verruculina enalia (Kohlm.) Kohlm. & Volkm.-Kohlm., Mycol. Res. 94(5): 689 (1990)
≡ Didymosphaeria enalia Kohlm., Ber. dt. bot. Ges. 79: 28 (1966)
≡ Lojkania enalia (Kohlm.) M.E. Barr, N. Amer. Fl., Ser. 2, 13: 56 (1990)
Notes:—Verruculina was introduced by Kohlmeyer & Volkmann-Kohlmeyer (1990) to accommodate an obligate
marine species Verruculina enalia (Kohlm.) Kohlm. & Volkm.-Kohlm. The genus is characterized by a single
marine species with immersed, clypeate, carbonaceous, papillate ascomata containing trabeculate
pseudoparaphyses, with cylindrical asci with short pedicels and ellipsoidal, 1-septate, dark brown, verrucose or
verruculose ascospores (Kohlmeyer & Volkmann-Kohlmeyer 1990). Verruculina is similar to Didymosphaeria in
having partly or completely immersed clypeate ascomata, but differs in the dark peridium with thick-walled cells,
forming a textura angularis, and stipitate asci with a clear ocular chamber (Kohlmeyer & Volkmann-Kohlmeyer
1990). Verruculina shows similarities with Testudinaceae in possessing septate, dark brown, verrucose or
verruculose ascospores but can be differentiated based on the immersed, clypeate, carbonaceous papillate ascomata
containing trabeculate pseudoparaphyses and additionally the marine habitat (Hyde et al. 2013, Suetrong et al.
2009, Schoch et al. 2009, Tanaka et al. 2009). Phylogenetic studies based on multigene analysis shows that
Verruculina enalia clustered within Testudinaceae (Hyde et al. 2013, Suetrong et al. 2009).
Discussion
Molecular data play a pivotal role in modern mycological taxonomy, but have some constraints in application. The
most significant and unsettled problem is that the phylogeny inferred from any gene may not reveal the
evolutionary history of the organism (Uilenberg et al. 2004). It is therefore better to incorporate a polyphasic
taxonomy including genotypical and phenotypical characteristics in any studies (Uilenberg et al. 2004). Taxonomic
modifications should not only be based on small portions of the genome, but also use morphological characters
(Uilenberg et al. 2004). The genome also needs to be evaluated (Uilenberg et al. 2004).
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Genera with trabeculate pseudoparaphyses and 1-septate, brown ascospores viz Bicrouania, Caryosporella,
Lineolata, Phaeodothis, Roussoella, Verruculina, were generally classified under Didymosphaeriaceae. Molecular
studies have shown that these particular morphological characters have evolved in different families. Verruculina
enalia clustered within Testudinaceae in order Pleosporales (Suetrong et al. 2009). Lineolata rhizophorae (Kohlm.
& E. Kohlm.) Kohlm. & Volkm.-Kohlm., was originally introduced by Kohlmeyer & Kohlmeyer (1966) as a
species of Didymosphaeria (D. rhizophorae). The genus is characterized by obpyriform ascomata with long
trabeculate pseudoparaphyses and 1-septate, brown ascospores. Three isolates of Lineolata rhizophorae from
varied geographic localities were analyzed by Suetrong et al. (2009) and formed a monophyletic group but in an
unresolved clade with Caryosporella rhizophorae Kohlm., in a sister group (long branch length) in
Dothideomycetidae and thus excluded from Pleosporomycetidae and Pleosporales. Phaeodothis was previously
assigned to Didymosphaeria by Niessl (1875) but recent phylogenetic analysis showed that Phaeodothis winteri (a
synonym of P. tricuspidis Syd. & P. Syd.) nested within the clade of Montagnulaceae (Schoch et al. 2009).
There have been few molecular investigations of Didymosphaeriaceae when compared to morphological
studies. Zhang et al. (2012) accepted only Appendispora, Didymosphaeria and Phaeodothis in the family. The
present study concluded that the phylogenetic trees obtained from maximum likelihood and minimum evolution
inference yielded trees with similar overall topology at subclass and family relationship in agreement with previous
work based on maximum likelihood (Hyde et al. 2013, Schoch et al. 2009, Suetrong et al. 2009, Zhang et al.
2012).The putative strains of Didymosphaeria futilis (HKUCC 5834 and CMW 22186) in GenBank, clustered with
Cucurbitariaceae and Didymellaceae, respectively, in the present study as well as in previous studies (Zhang et al.
2012). In the present study two new strains of the new species 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 the new species, D. rubi-ulmifolii shows that they have similar morphology and
thus represent Didymosphaeria. Therefore, based on the available molecular data and morphology,
Didymosphaeria can be referred to the family Montagnulaceae and Didymosphaeriaceae can be treated as a
probable synonym of Montagnulaceae.
A confusing fact is that the newly generated strains of Didymosphaeria rubi-ulmifolii and the putatively named
strains of the genus type obtained from GenBank (HKUCC 5834 and CMW 22186) clustered in different families.
This is an example of the problems of sequence data in GenBank and other public databases. The morphology and
identification of the putative strains (HKUCC 5834 and CMW 22186) of D. futilis in GenBank as far as we can
ascertain, cannot be checked, as they are not linked to any herbarium material. In this study we provide illustrations
and descriptions of the new species D. rubi-ulmifolii and the holotype of D. futilis which, with the exception of
ascospores ornamentation and host, are identical and should represent the same genus, Didymosphaeria.
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. 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. Hiran Ariyawansa is grateful to A.D Ariyawansa, D.M.K and A. Aptroot for their
valuable suggestions.
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