A Molecular and Morphological Reassessment of Diademaceae

Abstract

We revisit the family Diademaceae based on available sequence data and morphology. Diademaceae is characterized by ascomata opening with a flat circular lid and fissitunicate, short orbicular frequently cylindrical, pedicellate asci. Ascospores are frequently circular in section but narrowing to one end with three or more transverse septa, without longitudinal septa, and mostly with a thick sheath. In recent treatments Clathrospora, Comoclathris, Diadema, Diademosa, and Graphyllium were placed in the family. Following molecular and morphological study, Clathrospora, Comoclathris, and Diademosa, are excluded from the family and referred to Pleosporaceae. Graphyllium is excluded from Diademaceae, based on hysterothecium-like ascomata with a longitudinal opening, and tentatively placed in Hysteriaceae with uncertainty; species with hysterothecia have now been accommodated in at least five families. The study accepts only Diadema in the family. The status of Diademaceae as a distinct family, based on the ascomata opening by a flat circular lid, is thought to be doubtful. Fresh collections of Diadema are needed for epitypification and to obtain sequence data to establish if this is a well-resolved family.

Full text

Research Article
A Molecular and Morphological Reassessment of Diademaceae
Hiran A. Ariyawansa,1,2,3 Rungtiwa Phookamsak,2,3 Saowaluck Tibpromma,2,3
Ji-Chuan Kang,1and Kevin D. Hyde2,3
1e Engineering and Research Center for Southwest Bio-Pharmaceutical Resources of National Education Ministry of China,
Guizhou University, Guiyang, Guizhou 550025, China
2School of Science, Mae Fah Luang University, Chiang Rai 57100, ailand
3Institute of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, ailand
Correspondence should be addressed to
Ji-Chuan Kang; jichuank@yahoo.co.uk and Kevin D. Hyde; kdhyde@gmail.com
Received  August ; Accepted  October ; Published  January 
Academic Editors: R. Jeewon and S. J. Suh
Copyright ©  Hiran A. Ariyawansa et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
We revisit t h e f a m i l y Diademaceae based on available sequence data and morphology. Diademaceae is characterized by ascomata
opening with a at circular lid and ssitunicate, short orbicular frequently cylindrical, pedicellate asci. Ascospores are frequently
circular in section but narrowing to one end with three or more transverse septa, without longitudinal septa, and mostly with a
thick sheath. In recent treatments Clathrospora,Comoclathris,Diadema,Diademosa,andGraphyllium were placed in the family.
Following molecular and morphological study, Clathrospora,Comoclathris,andDiademosa, are excluded from the family and
referred to Pleosporaceae.Graphyllium is excluded from Diademaceae, based on hysterothecium-like ascomata with a longitudinal
opening, and tentatively placed in Hysteriaceae with uncertainty; species with hysterothecia have now been accommodated in at
least ve families. e study accepts only Diadema in the family. e status of Diademaceae as a distinct family, based on the
ascomata opening by a at circular lid, is thought to be doubtful. Fresh collections of Diadema are needed for epitypication and
to obtain sequence data to establish if this is a well-resolved family.
1. Introduction
Based on the ascomata opening by a at circular lid, Shoe-
maker and Babcock []introducedDiademaceae,whichthey
considered to be a unique family in the order Pleosporales.
Initially ve genera, that is, Clathrospora,Comoclathris,
Diadema,Diademosa,andMacrospora, were included in
the family []. Other than the ascomata opening by a at
circular lid, the family was characterized by bitunicate and
ssitunicate, clavate or ellipsoidal, short pedicellate asci,
and applanate or rarely cylindrical ascospores with three or
more transverse septa with or without longitudinal septa and
usually with a thick sheath and frequently circular in section
but narrowing to one end [,].
Species of the order Pleosporales with applanate asco-
spores can be found in three families (Diademaceae,Hys-
teriaceae,andPleosporaceae), which dier in the way the
ascomata open []. Ascomata openings by a at circular lid
are characteristic of Diademaceae.InspeciesofHysteriaceae
ascomataopenviaalongnarrowslitandspeciesofPleospo-
raceae open by a central pore []. Various authors have
included and excluded dierent genera in Diademaceae by
giving priority to dierent morphological characters [,,].
Platyspora had been referred to this family by various authors
[]orwasconsideredasynonymofGraphyllium [,]
or Comoclathris []. Lumbsch and Huhndorf []assigned
Macrospora to Pleosporaceae,asthegenuswasconsideredto
be a synonym of Pyrenophora andthistreatmentwasfollowed
by Zhang et al. [,].Inthesamestudy,Lumbschand
Huhndorf []hadalsoreferredGraphyllium to Diademaceae.
Sequencedataisnowavailableforsomeofthesegenera
thus the importance of their morphological characters and
overall relationships can now be tested based on molecular
phylogeny.
We have been studying the families of Pleosporales
in order to provide a natural classication via morpho-
logical characterization together with molecular analysis
Hindawi Publishing Corporation
e Scientific World Journal
Volume 2014, Article ID 675348, 11 pages
http://dx.doi.org/10.1155/2014/675348

e Scientic World Journal
[–,]. e family Diademaceae has been poorly studied and
presently comprises ve genera [], but this has not changed
since the family was introduced by Shoemaker and Babcock
[]. Given the considerable taxonomic confusion we revisited
thisfamilybasedonphylogeneticanalysesofrDNAsequence
data coupled with morphological characters. e aims of the
study are to (i) discuss the familial placement of the genera
in Diademaceae and assess whether they represent natural
groups, (ii) determine which morphological characters are
useful for generic delineation by observing the type species of
each genera, and (iii) illustrate the genera to stimulate fresh
collections being made so that molecular data can be used to
resolve the systematic relationships of the family.
2. Materials and Methods
2.1. Specimen Examination. e basic methodology used in
this study was the same as Ariyawansa etal. []. e type spec-
imens were loaned from the US National Fungus Collections
(BPI), Agriculture and Agri-Food Canada (DAOM), and
New York Botanical Garden (NY). Ascomata were rehydrated
in % KOH prior to examination and sectioning. Hand
sections of the fruiting structures were mounted in water for
microscopic studies and photomicrography. e fungus was
examined in a Nikon ECLIPSE i compound microscope
and photographed by a Cannon D digital camera tted to
the microscope. Measurements were made with the Taroso
(R) Image Frame Work program and images used for gures
were processed with Adobe Photoshop CS Extended version
. soware (Adobe Systems Inc., USA).
2.2. Phylogenetic Analysis. e large and small subunits of
the nuclear ribosomal RNA genes (LSU, SSU) were included
in the analysis. All sequences obtained from GenBank were
used in Schoch et al. []andZhangetal.[]andarelistedin
Table . Sequences were aligned using Bioedit v.. version
[] and ClustalX v. . []. e alignments were checked
visually and improved manually where necessary.
Maximum Likelihood analysis was performed in RAxML
[] implemented in raxmlGUIv..b []. e search strat-
egy was set to rapid bootstrapping and the analysis was
carried out using the GTRGAMMAI model of nucleotide
substitution. e number of replicates was automatically
inferred using the stopping criterion []. Maximum Likeli-
hood bootstrap values equal or greater than % are given
below or above each node (Figure ). Phylogenetic trees were
drawn using Treeview v. .. [Page ].
3. Results
3.1. Molecular Phylogeny Based on Combined nrSSU and
nrLSU. e combined  S and  S nrDNA data set com-
prised  taxa including strains of Clathrospora elynae (CBS
. and CBS .), Comoclathris magna (CBS .),
Clathrospora heterospora (CBS .), and Comoclathris
compressa (CBS . and CBS .) with Dothidea sam-
buci as the out-group taxon. e  taxa analyzed in the
cladogram formed  familial clades. Maximum Likelihood
analysis used  bootstrap replicates and yielded a tree
with the likelihood value of ln:-.and the following
model parameters: alpha: . and invar: .;
Π(A): ., Π(C): ., Π(G): ., and Π(T):
.. Phylogenetic trees obtained from maximum likeli-
hood analyses yielded trees with similar overall topology at
family and generic relationship in agreement with previous
work [,,].
3.2. Molecular Phylogeny of Diademaceae. Tw o puta tive
strains of Clathrospora elynae (CBS . and CBS .)
which had been previously referred to Diademaceae by
Lumbsch and Huhndorf [] and Shoemaker and Babcock
[] were clustered in the family Pleosporaceae but separated
from other genera of the family with a relatively high
bootstrap value (%). e type species of Comoclathris,C.
lanata,wasnotavailableforstudy,butthetwoComoclathris
compressa strains cluster in a well supported clade within the
Pleosporaceae,outsidetheAlternaria complex. erefore we
confer with Zhang et al. [] and Woudenberg et al. []in
transferring these two genera to Pleosporaceae.Twoputative
strains of Comoclathris magna (CBS .) and Clathrospora
heterospora (CBS .) were clustered within the Alternaria
complex as in Woudenberg et al. []. Woudenberg et
al. [] have tentatively considered Comoclathris magna
(CBS .) and Clathrospora heterospora (CBS .) as
Alternaria species. ere is, however, confusion concerning
the CBS . strain, because Dong et al. []usedthe
name Comoclathris baccata in their paper for strain CBS
. but submitted sequences to GenBank under the name
Clathrospora diplospora []. In their study, Woudenberg
et al. [] have synonymised Comoclathris baccata with
C. heterospora.Wecouldnotlocatethetypespeciesof
Diadema,Diadema tetramerum,andDiademosa,Diademosa
californiana, for phylogenetic analysis due to the unavailabil-
ity of sequence data. erefore recollection, epitypication,
and sequence data of Diadema,Diadema tetramerum,and
Diademosa,Diademosa californiana,arenecessarytovalidate
Diademaceae genera and species relationships.
3.3. Taxonomy
Diademaceae. Shoemaker & C.E. Babc., Can. J. Bot. ():
 (), MycoBank: MB .
Parasitic or saprobic in stems and leaves. Sexual state:
Ascomata subepidermal or subcuticular and later become
supercial, globose, opening via at circular lid, dark brown
to black. Peridium thin, consisting of small pigmented thick-
walled cells of textura angularis.Hamathecium of dense cellu-
lar pseudoparaphyses. Asci -spored, bitunicate, ssitunicate,
clavate or ellipsoidal, short orbicular pedicel, without an
ocular chamber. Ascospores partially overlapping to biseriate,
fusiform, brown, with three or more transverse septa with-
out longitudinal septa, mostly terete (cylindrical; frequently
circular in section but narrowing to one end), mostly with a
thick sheath. Asexual state: Unknown.
Type : D i adema . Shoemaker & C.E. Babc.

e Scientic World Journal 
T : Taxa used in the phylogenetic analysis and their corresponding GenBank numbers. Culture and voucher abbreviations are indicated
where available.
Taxo n Cu l t u re SSU L S U
Aigialus grandis JK A GU GU
Aigialus parvus BCC  GU GU
Alternaria alternata CBS . KC DQ
Amniculicola immersa CBS  GU FJ
Amniculicola parva CBS  GU FJ
Ascocratera manglicola JK C GU GU
Bimuria novae-zelandiae CBS . AY AY
Boeremia exigua CBS . EU EU
Byssothecium circinans CBS . AY AY
Clathrospora elynae CBS . KC KC
Clathrospora elynae CBS . KC KC
Clathrospora heterospora (Alternaria sp.) CBS . KC KC
Cochliobolus heterostrophus CBS . AY AY
Comoclathris compressa CBS . KC KC
Comoclathris compressa CBS . KC KC
Comoclathris magna (Alternaria sp.) CBS . KC DQ
Didymella exigua CBS . EU EU
Dothidea sambuci DAOM    AY   AY   
Dothidotthia aspera CPC  EU EU
Dothidotthia symphoricarpi CPC  EU EU
Halojulella avicenniae BCC  GU GU
Halojulella avicenniae BCC  GU GU
Helicascus nypae BCC  GU GU
Katumotoa bambusicola MAFF  AB AB
Lentithecium aquaticum CBS  GU GU
Lentithecium uviatile CBS  GU GU
Leptosphaeria doliolum CBS . GU GU
Leptosphaeria dryadis CBS . GU
Leptosphaeria maculans DAOM  DQ DQ
Leptosphaerulina australis CBS . GU GU
Massarina eburnea CBS . GU GU
Montagnula opulenta CBS . AF DQ
Morosphaeria ramunculicola BCC  GQ GQ
Morosphaeria ramunculicola JK B GU GU
Neophaeosphaeria lamentosa CBS  GQ GQ
Neottiosporina paspali CBS . EU EU
Ophiosphaerella herpotricha CBS . DQ DQ
Phaeosphaeria eustoma CBS . DQ DQ
Phoma radicina CBS . EU EU
Pleomassaria siparia CBS . DQ DQ
Pleospora betae CBS  EU EU
Pleospora calvescens CBS . EU EU
Pleospora chenopodii CBS . JF JF
Pleospora herbarum CBS . DQ DQ
Pleospora incompta CBS . GU GU
Pleospora typhicola CBS . JF JF

e Scientic World Journal
T  : C on t i nued.
Taxo n Cu l t u re SSU L S U
Preussia terricola DAOM     AY    AY     
Prosthemium betulinum CBS  AB AB
Prosthemium canba JCM  AB AB
Pyrenophora phaeocomes DAOM  DQ DQ
Sporormiella minima CBS . DQ DQ
Sporormiella minima CBS . DQ DQ
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
--
--
--
86
0.02
60
92
78
98
55
57 90
59
60
85
96
75
99
75
50
55
92
52
74
55
93
99
79
84
Paraphoma radicina CBS 111.79
Leptosphaeria dryadis CBS 643.86
Leptosphaeria doliolum CBS 505.75
Leptosphaeria maculans DAOM 229267
Boeremia exigua CBS 431.74
Leptosphaerulina australis CBS 317.83
Didymella exigua CBS 183.55
Dothidotthia aspera CPC 12933
Hallojulella avicenniae BCC 20173
Bimuria novae-zelandiae CBS 107.7 9
Kalmusia scabrispora MAFF 239517
Neottiosporina paspali CBS 331.37
Katumotoa bambusicola HHUF28661
Lentithecium aquaticum CBS 123099
Lentithecium uviatile CBS 122367
Morosphaeria ramunculicola JK 5304B
Helicascus kanaloanus A237 H
Helicascus nypae BCC 36752
Prosthemium canba JCM 16966
Pleomassaria siparia CBS 279 .74
Amniculicola immersa CBS 123083
Aigialus grandis BCC 18419
Aigialus parvus BCC 18403
Pleospora herbarum CBS 191.86
Pleosporaceae
Phaeosphaeriaceae
Leptosphaeriaceae
Didymellaceae
Halojulellaceae
Montagnulaceae
Massarinaceae
Lentitheciaceae
Morosphaeriaceae
Sporormiaceae
Amniculicolaceae
Aigialaceae
Alternaria alternate CBS 916.96
Pyrenophora phaeocomes DAOM 2227 69
Cochliobolus heterostrophus CBS 134.39
Neophaeosphaeria lamentosa CBS 102202
Dothidotthia symphoricarpi CPC 12929
Hallojulella avicenniae BCC 18422
Montagnula opulenta CBS 168.34
Massarina eburnea CBS 473.64
Massarina ramunculicola BCC 18405
Prosthemium betulinum CBS 1274 68
Amniculicola parva CBS 123092
Ascocratera manglicola JK 5262C
Dothidea sambuci DAOM231303
Clathrospora heterospora (Alternaria sp.) CBS 175.52
Comoclathris magna (Alternaria sp.) CBS 174.52
Comoclathris compressa CBS 157.53
Comoclathris compressa CBS 156.53
Pleospora typhicola CBS 132.69
Pleospora incompta CBS 467.76
Pleospora calvescens CBS 246.79
Pleospora chenopodii CBS 206.80
Clathrospora elynae CBS 196.54
Clathrospora elynae CBS 161.51
Ophiosphaerella herpotricha CBS 620.86
Phaeosphaeria eustoma CBS 573.86
Phaeosphaeria oryzae CBS 110110
55
Phoma betae CBS 109410
Dothidotthiaceae
F : RAxML tree based on a combined dataset of SSU and LSU. Bootstrap support values >% are shown above or below the branch.
e putative strains of Clathrospora elynae (CBS . and CBS .) and Comoclathris compressa (CBS . and CBS .) are indicated
in red. Dothidea sambuci is the out-group taxon. e original isolate numbers are noted aer the species names.

e Scientic World Journal 
Shoemaker and Babcock []introducedDiademaceae
which they considered to be a distinctive family compris-
ing Clathrospora,Comoclathris,Diadema,Diademosa,and
Macrospora whose species have ascomata opening by a at
circular lid []. e feature of ascomata opening via a at
circular lid was considered to be an adaptation to the alpine
habitat []. Ascospores are fusiform, brown, with three or
more transverse septa, with or without longitudinal septa, and
frequently terete, usually with a thick sheath [,,].
Lumbsch and Huhndorf []excludedMacrospora from
Diademaceae andassignedittoPleosporaceae,asitwascon-
sidered to be a synonym for Pyrenophora.Wehaveseentype
material of Macrospora scirpicola and it is neither diadema-
ceous nor pleosporaceous and therefore will be considered as
subject of a future paper. Lumbsch and Huhndorf []also
included Graphyllium in the family Diademaceae,butthis
classicationhasnotbeenfollowedbymanyauthors.Shoe-
maker and Babcock []andZhangetal.[] referred Graphyl-
lium to the family Hysteriaceae based on its hysterothecium-
like ascomata forming a longitudinal, slit-like opening. Shoe-
maker and Babcock []assignedClathrospora to Diademaceae
based on ascomata opening with an intraepidermal discoid
lid and muriform applanate ascospores with more than one
row of longitudinal septa. Ascomata, however, have slightly
papillate ostioles and Alternaria-like asexual morphs, and
recent molecular data shows that Clathrospora has an anity
with the family Pleosporaceae [,]. Platyspora has been
referred to Diademaceae [] and was considered a synonym
of Graphyllium [,]orasasynonymofComoclathris [].
Species of the order Pleosporales with applanate asco-
spores were previously separated into three families (Diadem-
aceae,Hysteriaceae,andPleosporaceae)whichdierinthe
way the ascomata open []. Ascomata openings by a at
circularlidwerecharacteristicofthefamilyDiademaceae,
while species of Hysteriaceae openviaalongnarrowslitand
species of Pleosporaceae open by a central pore []. Based on
the above discussion we exclude Clathrospora,Comoclathris,
Diademosa,andGraphyllium from the Diademaceae.Based
on morphology and/or molecular data and at this time, we
accept only Diadema, which has mostly terete ascospores
(except D. obtusa which has attened ascospores), in the
family. Diademaceae is, however, not supported by molecular
data, but no sequence data is available for the generic
type Diadema. Further studies are required to resolve the
phylogenetic relationship in the Pleosporales.Inthelightof
all of the above, we retain the Diademaceae to include a
single genus Diadema which has immersed, intraepidermal
ascomata, opening via a at circular lid, and asci with a short
orbicular pedicel without an ocular chamber and ascospores
are reddish-brown, usually cylindrical, and frequently cir-
cularinsectionbutnarrowingtooneendwithadistinct,
mucilaginous sheath.
3.4. Accepted Genus in Diademaceae
Diadema. Shoemaker & C.E. Babc., Can. J. Bot. (): 
(), MycoBank: MB .
Saprobic on culms of grasses (Poaceae). Sexual state:
Ascomata scattered, immersed, intra-epidermal, globose to
subglobose, black to brown, smooth-walled and opening via
aatcircularlid.Peridium -layered, composed of small
pigmented thick walled compressed cells, base composed
of small pigmented thick-walled cells of textura angularis.
Hamathecium of dense, numerous, septate, hyaline, cellu-
larpseudoparaphyses. Asci -spored, numerous, bitunicate,
ssitunicate, broadly-clavate, with a short orbicular pedicel,
without an ocular chamber. Ascospores obliquely biseriate,
broadly fusiform, usually cylindrical; frequently circular in
section but narrowing to one end, brown to reddish-brown,
without longitudinal septa, guttulate, smooth-walled or nely
punctate, with wide, distinct mucilaginous sheath. Asexual
state:Unknown.
Type S p ecie s : Diade m a tetrame r um. Shoemaker & C.E. Babc.
[as “tetramera”], Can. J. Bot. ():  (), MycoBank:
MB  (see Figure ).
Saprobic on culms of grasses (Poaceae). Sexual state:
Ascomata – ×– 𝜇m(
𝑥 = 190×250 𝜇m, 𝑛=10),
scattered, immersed, intra-epidermal, globose to subglobose,
black to brown, smooth-walled and opening via a at circular
lid. Peridium – 𝜇m(
𝑥=16,𝑛=20), -layered, composed
of small, pigmented, thick-walled, compressed cells, base
composed of small, pigmented, thick-walled cells of textura
angularis.Hamathecium of dense, - 𝜇mdiam(
𝑥=2,𝑛=
20), numerous, septate, hyaline, cellularpseudoparaphyses.
Asci – ×– 𝜇m(
𝑥 = 110 × 22𝜇m, 𝑛=20), -
spored, numerous, bitunicate, ssitunicate, broadly-clavate,
with a short orbicular pedicel, rounded at apex without an
ocular chamber. Ascospores – ×– 𝜇m( ̄
𝑥=44×
13𝜇m, 𝑛=40), obliquely biseriate, broadly fusiform, brown
to reddish-brown, -transseptate, without longitudinal septa,
guttulate, smooth-walled or nely punctate, with a distinct,
- 𝜇mwide,mucilaginoussheath.Asexual state:Unknown.
Material Examined. USA,California,Mt.Shasta,ridgesouth
of Horse Camp, elevation  , on culms of Tr i setu m
spicatum (L.) Richter,  July  W.B. Cooke  (DAOM,
holotype).
Shoemaker and Babcock []introducedDiadema and
characterized the genus by large ascospores without longitu-
dinal septa with a distinct mucilaginous sheath and ascomata
with a circular lid-like opening. Currently eight species of
Diadema arelistedinIndexFungorum[]. Six species were
included when the genus was introduced and another two
species (Diadema ahmadii,Kaz.Tanaka&S.H.Iqbal,and
Diadema sieversiae (Peck) Huhndorf) were later added [,
]. e nature of the ascomata appears to be an important
character of this genus and family. Except D. obtusa all
other species of Diadema have terete; that is, ascospores are
cylindrical, frequently circular in section but narrowing to
one end. We observed D. tetramerum,thegenerictypeof
Diadema and besides ascomata opening via a circular lid, asci
with the short orbicular pedicel without an ocular chamber
and trans-septate, ascospores, lacking longitudinal septa, and
surrounded by a very broad sheath narrowed to a waist near

e Scientic World Journal
(a) (b) (c)
(d)
(e) (f) (g)
(h)
(i)
(j)
F : Diadema tetramerum (holotype). (a) Ascomata on substrate opening via a at circular lid. (b) Vertical section of ascoma. (c)
Closeup of the peridium. (d) Hyaline, septate pseudoparaphyses. (e) Apical part of the asci, ((f)-(g)) Asci with short orbicular pedicel. ((h)–
(j)) Reddish-brown ascospores with broad sheath. Scale bars: (b) =  𝜇m, (c) =  𝜇m, ((d)–(g)) =  𝜇m, and ((h)–(j)) =  𝜇m.
the middle septum are considered to be signicant for the
genus.
No molecular data is available for the type or other
species of Diadema. erefore recollection, epitypication,
andsequencedataisessentialtoestablishfamilyandspecies
relationships.
3.5. Excluded Genera
Clathrospora. Rabenh., Hedwigia ():  ().
Saprobic on wood and stems. Sexual state:Ascomata semi-
immersed, scattered on putrid host stems and foliage, brown
to blackish brown, subglobose or nearly globose, with a
central sunken ostiole open via a circular lid, asci and pseu-
doparaphyses forming at the base of the peridium. Peridium
composed of – layers of brown, relatively thick-walled cells
of textura angularis, inner cells attened, thin-walled and
lighter. Hamathecium composed of dense, hyaline, liform,
pseudoparaphyses which are longer than the asci. Asci -
spored, bitunicate, ssitunicate, thick-walled, cylindrical to
clavate, with a short pedicle and shallow ocular chamber.
Ascospores biseriate, fusiform -transseptate, two or many
rows of longitudinal septa, muriform, constricted only at the
centralseptum,darkbrowntobrown,surroundedbyathin,
hyaline mucilaginous sheath. Asexual State:Alternaria-like.
Type Species: Clathrospora elynae. Rabenh., Hedwigia  : 
() (see Figure ).
Saprobic on wood and stems. Sexual State:Ascomata 
×– × 𝜇m(
𝑥 = 170 × 150𝜇m, 𝑛=10), semi-
immersed, scattered on the putrid host stems and foliage,
subglobose or nearly globose, brown to blackish brown, with
acentralsunkenostioleopenviaacircularlid,asciand
pseudoparaphyses forming on the base of the peridium.
Peridium – 𝜇m(
𝑥=38,𝑛=20), composed of – layers
of brown, relatively thick-walled cells of textura angularis,
inner cells attened, thin-walled and lighter. Hamathecium
composed of dense, - 𝜇mdiam(
𝑥=2,𝑛=20), hyaline,
liform, pseudoparaphyses, longer than the asci. Asci –
 ×– 𝜇m(
𝑥 = 190 × 35𝜇m, 𝑛=20), -spored,
bitunicate, ssitunicate, thick-walled, cylindrical to clavate,
with a short pedicle and ocular chamber. Ascospores –
 ×– 𝜇m(
𝑥=53×23𝜇m, 𝑛=40), biseriate,
fusiform, -transseptate, two or many rows of longitudinal
septa, muriform, constricted only at the central septum, dark

e Scientic World Journal 
(a) (b)
(c) (d) (e)
(f) (g) (h)
(i)
(j)
(k)
F : Clathrospora elynae (isotype). (a) Herbarium material. (b) Closeup of ascomata. (c) Section of the ascomata. (d) Closeup of the
peridium (e) Hyaline, liform, and pseudoparaphyses. ((f)–(h)) Cylindrical to clavate asci with a short pedicle and ocular chamber. ((i)–(k))
Dark brown to brown muriform ascospores surrounded by a thin, hyaline mucilaginous sheath. Scale bars: (b) =  𝜇m, (c) =  𝜇m, ((d)–(g))
=𝜇m, and ((h)–(j)) =  𝜇m.
brown to brown, surrounded by a thin, hyaline mucilaginous
sheath. Asexual State:Alternaria-like.
Material Examined. Switzerland, on the stem of Carex
curvula, September , Winter (BPI , isotype).
Shoemaker and Babcock []assignedClathrospora to
Diademaceae and included an additional nine species and
providedakeytothegenusbasedonthenumberof
septa and length of ascospores. Clathrospora was character-
ized by circular lid-like opening and applanate, muriform
ascospores. Currently,  Clathrospora species are listed in
the genus in Index Fungorum []. Molecular studies based
on combine gene analysis showed that two putative strains of
Clathrospora, C. elynae (CBS .) and C. diplospora (IMI
), were clustered in Pleosporaceae [,]. We obtained
similar results in the phylogenetic tree produced from
combined nrLSU and nrSSU sequence analysis (Figure ).
Clathrospora elynae the type of Clathrospora formed a sepa-
rate clade with relatively high bootstrap support (%) within
Pleosporaceae. Based on the phylogenetic result together
with the morphological characters (slightly papillate ostiole
and Alternaria-like asexual morph) we refer Clathrospora to
Pleosporaceae.
Comoclathris. Clem., Gen. fung. (Minneapolis): ,  ()
≡Platyspora Weh m., World Mon ograph of the Ge nus
Pleospora and its Segregates:  ().

e Scientic World Journal
Habitat saprobic on dead wood or stems. Sexual state:
Ascomata semi-immersed to supercial, scattered or aggre-
gated, subglobose or nearly globose, brown to blackish brown
coriaceous, ascomata opening via a large circular aperture or
lid. Peridium comprising - layers of brown, relatively thick-
walled cells of textura angularis.Hamathecium composed
of dense, hyaline, liform, septate pseudoparaphyses. Asci
-spored, bitunicate, ssitunicate, cylindrical to cylindro-
clavate, with an ocular chamber. Ascospores uniseriate or
partially overlapping, fusiform, muriform, brown to reddish-
brown, surrounded by a thick, hyaline, mucilaginous sheath.
Asexual State:Alternaria-like.
Type Species: Comoclathris lanata. Clem. [as “Comochla-
tris”], Gen. fung. (Minneapolis): – (). MycoBank:
MB .
Comoclathris,typiedbyComoclathris lanata,wasintro-
duced by Clements (). e genus is characterized by
ascomata with circular lid-like openings and applanate
reddish-brown to dark reddish-brown, muriform ascospores,
with single longitudinal septa []. Zhang et al. []tenta-
tively placed Comoclathris in the Pleosporaceae based on
Alternaria-like asexual morphs and this was followed by
Woudenberg et al. []. Comoclathris shares common char-
acters with Pleospora herbarum, the type of Pleospora,in
having cylindrical to cylindroclavate asci with an ocular
chamber and muriform, brown or pale brown, with or
without sheath ascospores. Comoclathris and Pleospora dier
in the opening of ascomata (opening via a large circular
aperture or lid versus open by a central pore). Comoclathris
and Pleoseptum share similar characters in having globose,
black, ascomata, and cylindrical to cylindroclavate asci with
muriform, yellowish to dark brown ascospores. Comoclathris
diers from Pleoseptum in having supercial ascomata with
circular lid-like openings composed of comparatively thin
peridium and applanate and fusiform ascospores surrounded
by a distinct hyaline, mucilaginous thick sheath [,]. In
Pleoseptum ascomata are immersed, usually with a papillate
apex, with a relatively broad peridium and ovoid to fusoid
ascospores [,]. Comoclathris was considered to dier from
Clathrospora as in the latter genus species have two or more
rows of longitudinal septa as compared with a single row
in Comoclathris []. Shoemaker and Babcock []provideda
key to  species of Comoclathris.Presentlyepithetsare
listed for Comoclathris in Index Fungorum []. Molecular
data for Comoclathris lanata,thetypespeciesofComoclathris,
is not available. Two strains of Comoclathris compressa (CBS
. and CBS .), however, cluster together in a well-
supported clade within the family Pleosporaceae []. Based
on the phylogenetic result coupled with the morphological
characters (Alternaria-like asexual morph) we agreed with
Zhang et al. [] and Woudenberg et al. []toplaceComo-
clathris in Pleosporaceae. is is, however, based on a species
and recollectionof the type species is essential to establish the
correct placement of the genus.
Diademosa. Shoemaker & C.E. Babc., Can. J. Bot. (): 
().
Saprobic on stems and wood. Sexual state:Ascomata
immersed, initially erumpent becoming supercial, scattered,
depressed-globose,someattenedatthebase,openingadisc-
like lid of brown prismatic cells with setae. Peridium com-
posed of brown pseudoparenchyma cells of textura angularis.
Hamathecium of numerous, dense, septate, hyaline, cellu-
larpseudoparaphyses. Asci -spored, bitunicate, ssitunicate,
clavatewithshortnarrowpedicelandminuteocularchamber.
Ascospores biseriate, partially overlapping, fusiform, straight,
frequently circular in section but narrowing to one end, with
transverse and vertical septa, pale brown to dark brown,
smooth walled. Asexual state:Unknown.
Type S p ecie s : Diade m osa califo r nian a. (M.E. Barr) Shoemaker
& C.E. Babc. [as “californianum”], Can. J. Bot. (): 
() ≡Graphyllium californianum M.E.Barr,Mem.N.Y.
bot. Gdn :  () (see Figure ).
Saprobic on stem and wood. Sexual state:Ascomata
– ×– 𝜇m(
𝑥 = 315 × 275𝜇m, 𝑛=10),
immersed, initially erumpent becoming supercial, scattered,
depressed-globose, some attened at the base, opening a
disc-like lid of brown prismatic cells with setae. Peridium
– 𝜇m(
𝑥=32,𝑛=20), composed brown pseudo-
parenchyma cells of textura angularis.Hamathecium of dense,
- 𝜇mdiam(
𝑥=2,𝑛=20), numerous, septate, hyaline,
cellularpseudoparaphyses. Asci – ×– 𝜇m(
𝑥=
150 × 26𝜇m, 𝑛=20), -spored, bitunicate, ssitunicate,
clavatewithshortnarrowpedicelandminuteocularchamber.
Ascospores – ×– 𝜇m(
𝑥=57×29𝜇m, 𝑛=40),
biseriate or discontinuously arranged, partially overlapping,
fusiform, straight, cylindrical; frequently circular in section
but narrowing to one end, with transverse and vertical septa,
muriform, constricted at rst septum, pale brown to dark
brown, smooth walled. Asexual state:Unknown.
Material Examined. USA, Bump-Cold Boiling Lake Trail,
Lassen Volcanic National Park, Shasta, California, on branch
of Wyet hia,July,W.B.Cooke&D.L.Hawksworth.
(NY, holotype).
Diademosa was established by Shoemaker and Babcock
[]andtypiedbyD. californiana, based on the ascoma open-
ing via a circular lid and ascospores being frequently circular
in section, but narrowing to one end. Diademosa californiana
was initially introduced as Graphyllium californianum by Barr
[]andreferredtoHysteriaceae based on the pore or slit like
opening. Reexamination of the type specimens by Shoemaker
and Babcock []concludedthatDiademosa opened by a at
lid similar to Diadema andassigneditintoDiademaceae.e
lidishardtoobserveinsectionsunlesstheyaremounted
directly in lactic acid because excessive swelling occurs in
water []. Diademosa diers from Comoclathris in having
cylindrical, frequently circular in section, but narrowing to
one end ascospores compared with attened ascospores of
Comoclathris.Diademosa and the generic type of Pleospo-
raceae,Pleospora share common characters. Both Diademosa
and Pleospora comprise narrowly oblong ascomata with
cellular pseudoparaphyses and cylindrical to clavate asci
with muriform, brown or pale brown ascospores. However,
Diademosa diers from Pleospora in having an ascomata

e Scientic World Journal 
(a) (b) (c)
(d) (e) (f) (g)
(h) (i) (j) (k)
(l)
(m)
(n)
F : Diademosa californiana (holotype). ((a)-(b)) Ascomata on host substrate. (c) Side view of the ascomata. ((d)-(e)) Section of
ascomata. (f) Section of peridium. (g) Septate, hyaline, and cellularpseudoparaphyses. (h) Light to dark brown seta. ((i)–(k)) Ascus with
minute pedicel bearing irregularly arranged  ascospores. (l)–(n) Ascospores. Scale bars: ((d)-(e)) =  𝜇m, ((f )–(h)) =  𝜇m, ((i)–(k)) =
 𝜇m, and (l)–(n) =10𝜇m.
opening via a circular lid, covered with setae and asci with
short narrow pedicel, while Pleospora species have ascomata
opening by a central pore without setae and asci with a
short, thick, furcated pedicel. Except the ascomata opening
via disc-like lid, Diademosa resemblessomecharactersof
Pyrenophora.atis,bothDiademosa and Pyrenophora
have supercial ascomata with setae and muriform, smooth-
walled, light brown to dark brown ascospores. Currently
four species of Diademosa are listed in Index Fungorum
[], but no molecular data is available for the genus. We
place Diademosa in Pleosporaceae because of its similarities
with other genera in this family, but conrmation of the
phylogenetic status of this genus depends on recollecting the
fungus and epitypication with molecular sequences.
Graphyllium. Clem., Botanical Survey of Nebraska :  ().
Habitat saprobic on woody stems. Sexual state:Ascomata
semi-immersed, hysteriform, black to brown, subglobose
to ovoid. Peridium comprising - layers of brown, rela-
tively thick cells of textura angularis,innercellsattened,
thin-walled and lighter. Asci spored,bitunicate,ssitu-
nicate, clavate. Ascospores biseritate overlapping, muriform,
applanate, obpyriform, straight, with - transverse septa, –
 longitudinal septa or no longitudinal septa, brown to olive
green. Asexual state:Unknown.
Type S pecie s :Graphyllium chlo¨
es. Clem., Bot. Surv. Nebraska
:  () ≡Pleospora chlo¨
es (Clem.) Petr., Sydowia (-):
 ().
Initially Graphyllium was placed in the Hypodermiaceae
by Cl´
emencet () and described as “Hysterothecium
innate, then erumpent, linear, simple, membranaceous-
plectenchymatous, black; asci ovoid or cylindrical-clavate, -
spored; spores brown, elliptical to oblong, with transverse-
and longitudinal septa, but not muriform; pseudoparaphyses
simple or branched, septate, forming an epithecium.” Later

 e Scientic World Journal
Barr [] transferred the genus to order Pleosporales and
referred to Phaeosphaeriaceae.Platyspora was considered as
a synonym of Graphyllium []. Shoemaker and Babcock
[]assignedGraphyllium to Hysteriaceae considering the
ascomatal characters along with applanate ascospores that are
at least -septate in side view and have some longitudinal
septa in front view. Lumbsch and Huhndorf []included
Graphyllium in the family Diademaceae,butZhangetal.[]
referred to Hysteriaceae. We examined the generic type of
Graphyllium,G. chlo¨
es we also agreed to refer Graphyllium
tentatively in Hysteriaceae because of its hysterothecium-
like ascomata forming a longitudinal opening which is
clearly deviated from the lid-like opening in Diademaceae.
However the correct placement of this taxon still depends on
epitypication with molecular data.
4. Concluding Remarks
e importance of molecular data in determining the impor-
tance of morphological characters and relationship of micro-
fungi cannot be overstressed and has proved signicant at
establishing genus and species relationships [,]and
resolving cryptic species in important plant pathogenic gen-
era, for example, Diaporthe []andPestalotiopsis []. Shoe-
maker and Babcock []introducedDiademaceae which they
considered to be a distinctive family based on the ascomata
opening via a at, circular lid and comprising Clathrospora,
Comoclathris,Diadema,Diademosa, and Macrospora [].
Recent studies based on molecular phylogeny [,], includ-
ing this study, conclude that Clathrospora and Comoclathris
clustered within Pleosporaceae.Moleculardata,however,is
not available for Diadema and Diademosa.Graphyllium is
placed in Hysteriaceae because of its hysterothecium-like
ascomata with a slit like opening; this clearly diverges from
the lid like opening in Diademaceae.echaracteristic
feature of ascomata opening via a at circular lid is con-
sidered as an adaptation to the alpine habitat []. It is,
however, doubtful if this character is signicant and whether
Diademaceae is a separate family in the order Pleosporales.
Until further molecular data becomes available we maintain
Diademaceae with a single genus Diadema based on its large
transseptate ascospores surrounded by a distinct mucilage
sheath and ascomata with a circular, lid-like opening [].
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of the paper.
Acknowledgments
We are grateful to the Mushroom Research Foundation,
Chiang Rai, ailand, for supporting this research. MFLU
Grant no.  is thanked for supporting studies
on Dothideomycetes. Hiran A. Ariyawansa and Ji Chuan
Kang are grateful to the International collaboration plan of
Science and Technology at Guizhou Province (Contract no.
[] ) and the construction of innovation talent team
of Science and Technology at Guizhou Province (Contract
no. [] ). Hiran Ariyawansa is grateful to A. D
Ariyawansa and D. M. K Ariyawansa for their valuable
suggestions.
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