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Towards a natural classification of Astrosphaeriella-like species; introducing Astrosphaeriellaceae and Pseudoastrosphaeriellaceae fam. nov. and Astrosphaeriellopsis, gen. nov.

Authors:
  • Kunming Institute of Botany, Chinese Academy of Science, Kunming 650201, Yunnan, Chin
  • Qujing Normal University, Qujing, China

Abstract

Astrosphaeriella sensu lato is a common genus occurring on bamboo, palms and stout grasses. Species of Astrosphaeriella have been collected from various countries in tropical, subtropical or temperate regions. In Asia, species have been collected in Brunei, China, Indonesia, Japan, Philippines and Vietnam. There have been several morphological studies on Astrosphaeriella, but molecular work and phylogenetic analyses are generally lacking. Taxa included in Astrosphaeriella were characterized in three main groups 1) typical Astrosphaeriella species (sensu stricto) having carbonaceous, erumpent, conical ascostromata 2) atypical Astrosphaeriella species (sensu lato) having immersed, coriaceous ascostromata with short to long papilla and 3) lophiostoma-like species having immersed ascostromata with slit-like openings. Some of the latter Astrosphaeriella species, having slit-like openings, have been transferred to Fissuroma and Rimora in Aigialaceae. In this study five type specimens of Astrosphaeriella were loaned from herbaria worldwide and re-examined and are re-described and illustrated. Collections of Astrosphaeriella were also made in Thailand and morphologically examined. Pure cultures were obtained from single spores and used in molecular studies. The asexual morph was induced on sterile bamboo pieces placed on water agar. Phylogenetic analyses of combined LSU, SSU and TEF1 sequence data of astrosphaeriella-like species using Bayesian, Maximum parsimony (MP) and Randomized Accelerated Maximum Likelihood (RAxML) analyses were carried out. Phylogenetic analyses show that species of Astrosphaeriella can be distinguished in at least three families. Species of Astrosphaeriella sensu stricto with erumpent, carbonaceous ascostromata, form a strongly supported clade with Pteridiospora species and a new family, Astrosphaeriellaceae, is introduced to accommodate these taxa. The genera are revised and Astrosphaeriella bambusae, A. neofusispora, A. neostellata, A. thailandica, A. thysanolaenae and Pteridiospora chiangraiensis are introduced as new species. Astrosphaeriella exorrhiza is reported on a dead stem of Thysanolaena maxima and is the first record for Thailand. Reference specimens for A. fusispora and A. tornata are designated to stabilize the taxonomy of Astrosphaeriella. The coelomycetous asexual morph of A. bambusae is reported and forms hyaline, globose to subglobose, aseptate conidia. Species of Astrosphaeriella sensu lato with immersed, coriaceous ascostromata, with short to long papilla and striate ascospores, form a sister clade with Tetraplosphaeriaceae. The genus Pseudoastrosphaeriella is introduced to accommodate some of these taxa with three new species and three new combinations, viz. P. aequatoriensis, P. africana, P. bambusae, P. longicolla, P. papillata and P. thailandensis. A new family Pseudoastrosphaeriellaceae is introduced to accommodate this presently monotypic lineage comprising Pseudoastrosphaeriella. The asexual morph of P. thailandensis is described. Astrosphaeriella bakeriana forms a distinct clade basal to Aigialaceae. Astrosphaeriella bakeriana is excluded from Astrosphaeriella and a new genus Astrosphaeriellopsis, placed in Dothideomycetes genera incertae sedis, is introduced to accommodate this taxon. Fissuroma aggregata (Aigialaceae) is re-visited and is shown to be a cryptic species. Three new species of Fissuroma and a new combination are introduced based on morphology and phylogeny viz. F. bambusae, F. fissuristoma, F. neoaggregata and F. thailandicum. The asexual morph of Fissuroma bambusae is also reported.
Towards a natural classification of Astrosphaeriella-like species;
introducing Astrosphaeriellaceae and Pseudoastrosphaeriellaceae
fam. nov. and Astrosphaeriellopsis, gen. nov.
Rungtiwa Phookamsak
1,2,3,4
& Chada Norphanphoun
3,4
& Kazuaki Tanaka
9
&
Dong-Qin Dai
1,2,3,4
& Zong-Long Luo
3,4,8
& Jian-Kui Liu
7
& Hong-Yan Su
8
&
Darbhe J. Bhat
5,6
& Ali H. Bahkali
10
& Peter E. Mortimer
1,2
& Jian-Chu Xu
1,2,11
&
Kevin D. Hyde
1,2,3,4,10
Received: 22 September 2015 /Accepted: 22 October 2015
#
School of Science 2015
Abstract Astrosphaeriella sensu lato is a common genus oc-
curring on bamboo, palms and stout grasses. Species of
Astr osphaeriella have been collected from various countries in
tropical, subtropical or temperate regions. In Asia, species have
been collected in Brunei, China, Indonesia, Japan, Philippines
and V ietnam. There have been several morphological studies on
Astr osphaeriella, but molecular work and phylogenetic analy-
ses are generally lacking. Taxa included in Astrosphaeriella
were characterized in three main groups 1) typical
Astr osphaeriella species (sensu stricto) having carbonaceous,
erumpent, conical ascostromata 2) atypical Astrosphaeriella
species (sensu lato) having immersed, coriaceous ascostromata
with short to long papilla and 3) lophiostoma-like species hav-
ing immersed ascostromata with slit-like openings. Some of the
latter Astr osphaeriella species, having slit-like openings, have
been transferred to Fissur oma and Rimora in Aigialaceae.In
this study five type specimens of Astrosphaeriella were loaned
from herbaria worldwide and re-examined and are re-described
and illustrated. Collections of Astr osphaeriella were also made
in Thailand and morphologically examined. Pure cultures were
obtained from single spores and used in molecular studies. The
asexual morph was induced on sterile bamboo pieces placed on
water agar. Phylogenetic analyses of combined LSU, SSU and
TEF1 sequence data of astrosphaeriella-like species using
Bayesian, Maximum parsimony (MP) and Randomized Accel-
erated Maximum Likelihood (RAxML) analyses were carried
out. Phylogenetic analyses show that species of
Astr osphaeriella can be distinguished in at least three families.
Species of Astr osphaeriella sensu stricto with erumpent, carbo-
naceous ascostromata, form a strongly supported clade with
Pteridiospora species and a new family, Astrosphaeriellaceae,
is introduced to accommodate these taxa. The genera are revised
Electronic supplementary material The online version of this article
(doi:10.1007/s13225-015-0352-7) contains supplementary material,
which is available to authorized users.
* Kevin D. Hyde
kdhyde3@gmail.com
1
Key Laboratory for Plant Diversity and Biogeography of East Asia,
Kunming Institute of Botany, Chinese Academy of Science,
Kunming 650201, Yunnan, China
2
World Agroforestry Centre, East Asia Node, Heilongtan,
Kunming 650201, PeoplesRepublicofChina
3
School of Science, Mae Fah Luang University, Chiang Rai 57100,
Thailand
4
Center of Excellence in Fungal Research and School of Science, Mae
Fah Luang University, Chiang Rai 57100, Thailand
5
Formerly Department of Botany, Goa University, Goa, India
6
No. 128/1-J, Azad Housing Society, Curca, Goa 403108, India
7
Guizhou Key Laboratory of Agricultural Biotechnology, Guizhou,
Academy of Agricultural Sciences, Guiyang, Guizhou, China
8
College of Agriculture & Biology, Dali University,
Dali 671003, Yunnan, Peoples Republic of China
9
Faculty of Agriculture and Life Sciences, Hirosaki University, 3
Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan
10
Department of Botany and Microbiology, College of Science, King
Saud University, P.O. Box: 2455, Riyadh 1145, Saudi Arabia
11
CAS Key Laboratory of Genome Sciences and Information, Beijing
Institute of Genomics, Chinese Academy of Sciences, No. 1 Beichen
West Road, Chaoyang District, Beijing 100101, Peoples Republic of
China
Fungal Diversity
DOI 10.1007/s13225-015-0352-7
and Astr osphaeriella bambusae, A. neofusis pora, A. neostellata,
A. thailandica, A. thysanolaenae and Pteridiospora
chiangraiensis are introduced as new species. Astrosphaeriella
exorrhiza is reported on a dead stem of Thysanolaena maxima
and is the first record for Thailand. Reference specimens for
A. fusispora and A. tornata are designated to stabilize the tax-
onomy of Astrosphaeriella. The coelomycetous asexual morph
of A. bambusae is reported and forms hyaline, globose to
subglobose, aseptate conidia. Species of Astr osphaeriella sensu
lato with immersed, coriaceous ascostromata, with short to long
papilla and striate as cospores, for m a sister clade with
Tetraplosphaeriaceae. The genus Pseudoastrosphaeriella is in-
troduced to accommodate some of these taxa with three new
species and three new combinations, viz. P. aequatoriensis,
P. africana, P. bambusae, P. longicolla, P. papillata and
P. thailandensis. A new family Pseudoastrosphaeriellaceae is
introduced to accommodate this presently monotypic lineage
comprising Pseudoastrosphaeriella. The asexual morph of
P. thailandensis is described. Astrosphaeriella bakeriana forms
a distinct clade basal to Aigialaceae. Astrosphaeriella
bakeriana is excluded from Astr osphaeriella and a new genus
Astrosphaeriellopsis, place d in Dothideomycetes genera
incertae sedis, is introduced to accommodate this taxon.
Fissur oma aggregata (Aigialaceae) is re-visited and is shown
to be a cryptic species. Three new species of Fissuroma and a
new combination are introduced based on morphology and
phylogeny viz. F. bambusae, F. fissuristoma, F. neoaggregata
and F. thailandicum. The asexual m orph of Fissuroma
bambusae is also reported.
Keywords Asexual morph
.
Bamboo
.
Palm
.
Pseudoastrosphaeriella
.
Phylogeny
.
Pl
eosporales
.
Taxonomy
Introduction
Pleosporales is the largest order of Dothideomycetes and
comprises 40 families, 255 genera and more than 4700 species
(Kirk et al. 2008; Zhang et al. 2009a, 2012; Liu et al. 2011,
2015; Wijayawardene et al. 2014), while Liu et al. (2015)
added a new family, Paradictyoarthriniaceae in Pleosporales.
Some species of Pleosporales are important as they cause
disease on plants, animals or humans (Rinaldi et al. 1987;
Schoch et al. 2006, 2009;Hipolitoetal.2009;Manamgoda
et al. 2011, 2012; Zhang et al. 2009a, 2012; de Gruyter et al.
2012;Hydeetal.2013; Seyedmousavi et al. 2013). In addi-
tion, species of Pleosporales are also saprotrophs which de-
compos e organic matter (Kruys et al. 2006;Schochetal.
2006, 2009; Kodsueb et al. 2008; Zhang et al. 2009a, 2012;
Hyde et al. 2013), endophytes (Sánchez Márquez et al. 2008,
2010; Zhang et al. 2009a; Sakayaroj et al. 2010; Bhagat et al.
2012) or epiphytes on living plants (Zhang et al. 2009a, 2012;
Liu et al. 2011). Since 2009, various families in Pleosporales
have been re-visited and their taxonomy and phylogeny
reassessed (Tanaka et al. 2009; Manamgoda et al. 2011,
2012; de Gruyter et al. 2012; Zhang et al. 2012;Hydeetal.
2013;Quaedvliegetal.2013;
Woudenberg et al. 2013, 2015;
Ariyawansa et al. 2014a, b, c, d, e, 2015a, b; Phookamsak
et al. 2014b;Thambugalaetal.2014). However, several
groups could not be resolved by phylogenetic investigations
due to limited molecular data and thus their taxonomy
remained ambiguous (Zhang et al. 2012;Hydeetal.2013;
Liu et al. 2011, 2014; Phookamsak et al. 2014b).
Astrosphaeriella is a well-known, almost exclusively trop-
ical genus occurring on bamboo, palms and stout grasses, but
putative species are also known from subtropical or temperate
regions, such as northern Florida Japan and the southwest of
England (Barr 1990;Zhouetal.2003; Tanaka and Harada
2005;Hu2010; Zhang et al. 2012). The genus has been re-
ported as saprobic or parasitic from aquatic, submerged or
terrestrial habitats, mostly from dead parts of monocotyle-
dons. However, some Astrosphaeriella species ha ve been
found on dead parts of dicotyledons, such as Astrosphaeriella
applanata (Fr.) Scheinpflug, A. inaequalis (Fabre)
Scheinpflug and A. pinicola (Rehm) Scheinpflug (Yamamoto
et al. 1954; Scheinpflug 1958;HydeandFröhlich1998;Hu
2010; Tanaka and Harada 2005; Zhang et al. 2012)
Astrosphaeriella was introduced to accommodate a
dothideomycete species forming carbonaceous, conical to
mammiform ascostromata, with ruptured, reflexed, stellate,
host remnants around the base. The asci are bitunicate, cylin-
drical to cylindric-clavate, pseudoparaphyses trabeculate and
ascospores pale brown to brown and fusiform (Barr 1990;
Sydow and Sydow 1913; Fröhlich and Hyde 2000; Tanaka
and Harada 2005; Tanaka et al. 2009;Liuetal.2011).
Theissen (
1917)t
ransferredPemphidium erumpens (Berk. &
M.A. Curtis) Sacc. to Astrosphaeriella erumpens (Berk. &
M.A. Curtis) Theiss. von Höhnel (1920)introduced
A. bambusella Höhn. from Java and Saccardo (1928)com-
bined Amphisphaeria stellata Pat., which was morphological-
ly similar to A. fusispora Syd. & P. Syd., under
Astrosphaeriella stellata Sacc. There have been many mor-
phological based studies on Astrosphaeriella (Scheinpflug
1958;MüllerandvonArx1962; von Arx and Müller 1975;
Hawksworth 1981; Hawksworth and Boise 1985; Yue and
Eriksson 1986; Barr 1990;Hyde1992, 1994a, b; Aptroot
1995a; Fröhlich and Hyde 1995; Hyde and Fröhlich 1998;
San Martín and Lavín 1999;Zhouetal.2003; Chen and Hsieh
2004). However, there has been no detailed molecular inves-
tigation of this genus (Tanaka et al. 2009; Liu et al. 2011).
Other studies include Yamamoto et al. (1954), who intro-
duced a new taxon, Astrosphaeriella fuscomaculans W.
Yamam. causing fuscous speckles or BUnmon-chiku ^ on
Phyllostachys nigra (Lodd. ex Lindl.) Munro. The fungus
infected the culms and branches of living bamboo and formed
Fungal Diversity
irregularly elliptic or wholly irregular specks, which were dark
brown to blackish brown with white fine stripes (Yamamoto
et al. 1954). Hino and Katumoto (1956) had also reported
A. aosimensis I. Hino & Katum. from Livistona subglobosa
(Hassk.) Mart. These two species were reported from temper-
ate regions of Japan. Additionally, Scheinpflug (1958)intro-
duced three Astrosphaeriella species from dicotyledons from
temperate regions viz. A. applanata, A. inaequalis,and
A. pinicola. However, all species accepted by Scheinpflug
(1958) and another thr ee Astrosphaeriella species viz.
A. aosimensis, A. fuscomaculans and A. fusispora were syn-
onymized under Microthelia by Müller and von Arx (1962)
and this was followed by von Arx and ller (1975) and other
authors (Hawksworth 1981;Hu2010; Liu et al. 2011).
Hawksworth (1981) mentioned that Microthelia should
clearly be regarded as an earlier synonym of the lichenicolous
genus Anisomeridium (Hawksworth 1981;Hawksworthand
Sherwood 1981;Hu2010). Therefore, Hawksworth (1981)
reinstated Astrosphaeriella and designated A. stellata,anear-
lier name for A. fusispora, as the type. Hawksworth (1981)
accepted only four species in Astrosphaeriella viz.
A. aosimensis, A. stellata, A. trochus (Penzig & Sacc.) D.
Hawksw. and A. venezuelensis Barr & D. Hawsw.; whereas,
A. fusispora was treated as a synonym of A. stellata.
Hawksworth and Boise (1985) introduced six more species,
mostly with heavily pigmented and longitudinally striate
ascospores in Astrosphaeriella
. A key to the ten species of
Astr
osphaeriella was also provided in this paper. Yue and
Eriksson (1986) re-studied the g enus Rhynchostoma an d
found that R. lageniforme Teng. which was collected from
China, was similar to Astrosphaeriella. Therefore, Yue and
Eriksson (1986) transferred R. lageniforme to
Astrosphaeriella as A. lageniformis (Teng) J.Z. Yue & O.E.
Erikss. When Barr (1990)studiedtheorderMelanommatales
in North America, she also introduced a new species,
Astrosphaeriella floridana Barr from Sabal palmetto (Walt.)
Lodd. in Florida.
Hyde (1992, 1994a, b), Frö hlich and Hyd e (1995)and
Hyde and Fröhlich (1998) studied various Astrosphaeriella
species occurring on palms in tropical regions. Hyde (1992)
studied fungi on submerged decaying fronds of Nypa
fruticans Wurmb. from Brunei and introduced two new spe-
cies in Astrosphaeriella viz. A. nypae K.D. Hyde and
A. striataspora (K.D. Hyde) K.D. Hyde. Hyde (1994b)stud-
ied fungi on rachides of Livistona i n tropical Papua New
Guinea and reported A. aosimensis from rachides of Livistona.
In addition, he found a freshwater Astrosphaeriella species,
A. aquatica K.D. Hyde and this was reported as the first
Astrosphaeriella species from aquatic environments (Hyde
1994a). Fröhlich and Hyde (1995) had also introduced a
new species, Ast rosphaeriella fronsicola J. Fröhl. & K.D.
Hyde, associated with leaf spots of Oraniopsis and other
palms from Queensland.
Aptroot (1995a, b) re-visited the genus Didymosphaeria
and treated D. fusispora Penz. & Sacc as a synonym of
Astrosphaeriella minima Aptroot, while D. pustulata I. Hino
& Katum. was synonymized under
A. minoensis (H
ara) D.
Hawksw. Additionally, Aptroot (1995a)introduced
Astrosphaeriella papuana Aptroot on bamboo from Papua
New Guinea, a species with striate ascospores. Furthermore,
Aptroot (1995b) suggested that Didymosphaeria japonica I.
Hino & Katum., D. macrospora I. Hino & Katum. and
D. tosaensis I. Hino & Katum. should be accommodated in
Astrosphaeriella or Roussoella. Their large ascospores with a
distichous arrangement pointed to the genus Astrosphaeriella,
while the compound ascostromata were similar to the genus
Roussoella. These three species mentioned by Ap troot
(1995b) were currently accommodated in the genus
Roussoellopsis by Hino and Katumoto (1965) and the genus
was placed in the family Roussoellaceae based on molecular
data in Liu et al. (2014) and Phookamsak et al. (2014a).
A major morphological study of Astrosphaeriella was car-
ried out by Hyde and Fröhlich (1998). Hyde and Fröhlich
(1998) collected several Astrosphaeriella species from bam-
boo and palms in Australia, South East Asia and South Amer-
ica. In their study, 31 Astrosphaeriella species were reported,
with the key to species of Astrosphaeriella and these accepted
species have been re-described and discussed. Additionally,
Hyde and Fröhlich (1998)introducedtennewspecieswith
five new combinations viz. Astrosphaeriella aequatoriensis
K.D. Hyde & J. Fröhl., A. angustispora J. Fröhl. & K.D.
Hyde, A. australiensis K.D. Hyde & J. Fröhl., A. bakeriana
(Sacc.) K.D. Hyde & J. Fröhl., A. lenticularis K.D. Hyde & J.
Fröhl., A. livistonicola K.D. Hyde & J. Fröhl.,
A. lophiostomopsis K.D. Hyde & J. Fröhl., A. malayensis
K.D. Hyde & J. Fröhl., A. maquilingiana (Rehm) K.D. Hyde
& J. Fröhl., A. mauritiae K.D. Hyde & J. Fröhl., A. nipicola
(Cooke & Massee) K.D. Hyde & J. Fröhl., A. papillata K.D.
Hyde & J. Fröhl., A. samuelsii (Boise) K.D. Hyde & J. Fröhl.,
A. splendida K.D. Hyde & J. Fröhl. and A. uberina (Mont.)
K.D. Hyde & J. Fröhl.
Further As
trosphaeriella species have been introduced and
contributions to the genus have also been reported. San Martín
and Lavín (1999) added two new species, Astrosphaeriella
stellata var. palmicola F. San Martín & P. Lavín from palms
in Chiapas (currently as A. stellata)andA. vaginata F. San
Martín & P. Lavín from Bactris baculifera Karw. ex Mart. in
Tabasco, Mexico. They ha d also described and illustrated
some Astrosphaeriella species found in Mexico viz.
A. stellata, A. tornata (Berk. & M.A. Curtis) D. Hawksw. &
Boise and A. trochus.
The dothideomycete species characterized by ascostromata
with laterally compresse d- or crest-like ostioles occurring
mainly on wood and bark were previously accommodated in
the genus Lophiostoma Ces. & De Not. (Barr 1990;Yuanand
Zhao 1994; Holm and Holm 1988;Hydeetal.2000; Tanaka
Fungal Diversity
and Harada 2003; Zhang et al. 2009b). However, the crest-like
ostiole, could not always distinguish Lophiostoma from sim-
ilar Massarina species. Barr (1990)usedcellularversustra-
beculate pseudopa raphyses to sepa rate Lophiostoma in
Melanommatales from genera of Pleosporales and
Astrosphaeriella was placed in the former order. Liew et al.
(2000) however, has shown that this classification lacks sup-
port from molecular data.
Hyde et al. (2000) collected some Lophiostoma species
described from palms having trabeculate pseudoparaphyses,
which were not congeneric with Lophiostoma, but more sim-
ilar to Astrosphaeriella. Thus, Hyde et al. (2000)expandedthe
already broad generic concept of Astrosphaeriella to accom-
modate lophiostoma-like species having ascostromata with
slit-like ostioles (lacking crests) and trabeculate
pseudoparaphyses. Five species having slit-like ostioles were
included in Astrosphaeriella in Hyde et al. (2000)viz.
A. asiana (K.D. Hyde) Aptroot & K.D. Hyde,
A. daemonoropis J. Fröhl., K.D. Hyde & Aptroot,
A. fissuristoma J. Fröhl., K.D. Hyde & Aptroot, A. maculans
(Rehm) Aptroot et al. and A. mangrovis (Kohlm. & Vittal)
Aptroot & K.D. H yde. Astrosphaeriella immersa Taylor
et al. was also introduced in Hyde et al. (2000), but the species
had round ostioles.
Hsieh et al. (2000)reportedAstrosphaeriella stellata from
Taiwan, while this species was also found in Hong Kong by
Tsui et al. (2001). Zhou et al. (2003) surveyed the
bambusicolous fungi and reported six Astrosphaeriella spe-
cies from Hong Kong and Yunnan, China, including
A. bakeriana, A. fissuristoma, A. maculans, A. splendida and
A. stellata.
Rogers and Barr (2003) introduced a new s pecies,
Astrosphaeriella longispora J.D. Rogers & M.E. Barr from
Costa Rica for a species having carbonaceous, conical
ascostromata, and elongate fusiform ascospores. The species
had been collected from angiosperm wood.
Chen and Hsieh (2004
) described seven As
trosphaeriella
species from bamboo and grasses in Taiwan and introduced
two new species, A. macrospora Chi Y. Chen & W.H. Hsieh
and A. pallidipolaris Chi Y. Chen & W.H. Hsieh in their study,
both species having striate ascospores. Wang et al. (2004)also
introduced two new combi nations in Astrosphaeriella viz.
A. callicarpa (Penz. & Sacc.) Y.Z. Wang et al., and
A. polymorpha (Rehm & Fairm.) Y.Z. Wang et al. when they
revised the genus Amphisphaeria and transferred some
Amphisphaeria species to Astrosphaeriella (Hu 2010).
Tanaka and Harada (2005) transferred Melanopsamma
aggregata I. Hino & Katum. to Astrosphaeriella following
the concept of Hyde et al. (2000), while Jagadeesh Ram
et al. (2005) introduced Astrosphaeriella sundarbanensis
Jagadeesh & Ap troot on dry bark of Sonneratia apetala
Buch.-Ham. from India. Chen and Huang (2006) introduced
A. linguiformis Chi Y. Chen & J.W. Huang on bamboo from
Taiwan. Aptroot (2009)alsotransferredArthopyrenia picea
Shirley to Astrosphaeriella picea (Shirley) Aptroot.
It is reportedly difficult to obtain cultures from
Astrosphaeriella species (Liu et al. 2011), thus culture charac-
teristics and molecular data for the genus are generally lacking
(Tsui et al. 2001; Tanaka and Harada 2005; Liu et al. 2011).
The phylogenetic placement of Astrosphaeriella was first
shown by Tanaka et al. (2009) who introduced a new family
Tetraplosphaeriaceae from bambusicolous fungi in
Pleosporales and included three strains of Astrosphaeriella
aggregata (I. Hino & Katum.) Kaz. Tanaka & Y. Harada and
A. stellata in their analyses. The phylogenetic results showed
that Astrosphaeriella aggregata formed a di stinct clade
separate from A. stellata and Tanaka et al. (2009) concluded
that A. aggregata may not congeneric with A. stellata.This
was supported in the studies of Schoch et al. (2009) where
A. aggregata
clustered in the family Aig
ialaceae, whereas,
A. bakeriana formed a distinct clade at the base of this family.
Suetrong et al. (2009) introduced Rim ora in the family
Aigialaceae, based on molecular study to accommodate a
mangrove sp ecies, Rimora mangrovei (Kohlm. & Vittal)
Kohlm et al., which was previously described as
Astrosphaeriella mangrovis (Kohlm. & Vittal) Aptroot &
K.D. Hyde ( Lophiostoma mangrovei Kohlm. & Vittal) in
Hyde et al. (2000). Based on phylogenetic analyses, Liu et al.
(2011) introduced two new genera, Fissuroma and
Neoastrosphaeriella in Aigialaceae to accommodate species
with immersed ascostromata with slit-like ostioles, which
were previously accommodated in Astrosphaeriella.Ren
et al. (2013) introduced a new species Astrosphaeriella
thailandensis Ren et al. on submerged wood from a freshwater
stream in Thailand. Phylogenetic analyses of combined LSU
and SSU sequence data showed that A. thailandensis formed a
robust clade (97 % MP) with A. stellata (MFLUCC 10-0555)
and separated from A. stellata (strain KT 998). Whereas,
Pratibha and Prabhugaonkar (2015) reported the sexual morph
of Pithomyces flavus Berk. & Broome as Astrosphaeriella
vesuvius (berk. & broome) D. Hawksw & Boise. This species
was collected from a litter of Calamus thwaitesii in India.
Multigene phylogenetic analysis showed that Pithomyces
flavus clusters with Astrosphaeriella vesuvius and was closely
related to A. bakeriana in their analysis.
The natural placement of Astrosphaeriella is unresolved.
Hara (1913) intr oduced the family Astrocystidiaceae Hara
(asAsterocystidiaceae) and accommodated Astrosphaeriella
and Astrocystis in this family. Although, there is similarity in
the superficial ascostromata in some species of these genera,
Astrocystis species are unitunicate Xylariales in
Sordariomycetes (Maharachchikumbura et al. 2015;
Senanayake et al. 2015). Barr (1987
, 19
90) re-introduced the
family Platystomaceae and listed Astrosphaeriella, Javaria,
Platystomum, Pseudotrichia, Thyridaria and
Trematosphaeria in this family. Aptroot (1995a, b)followed
Fungal Diversity
Barr (1987, 1990) and placed Astrosphaeriella in
Platystomaceae, whereas, Hawksworth and Ainsworth
(1995) arranged Astrosphaeriella differently, and referred the
genus to Melanommataceae (Hu 2010; Liu et al. 2011). Sub-
sequently, Astrosphaeriella was accommodated in
Melanommataceae (Hyde et al 2000; Lumbsch and Huhndorf
2007;Kirketal.2008). Tanaka et al. (2009)however,sug-
gested that the placement of Astrosphaeriella in
Melanommataceae was not suitable and rejected the mono-
phyletic status based on their phylogenetic study (Tanaka et al.
2009; Liu et al. 2011). Lumbsch and Huhndorf (2010)treated
Astrosphaeriella in the Pleosporales family incertae sedis and
this is concurred in Liu et al. (2011).
There are presently 61 species epithets for
Astrosphaeriella in Index Fungorum (2015), while Kirk
et al. (2008) estimated that there were 51 species in the
genus; 104 individual sequences are available in
GenBank.
The genus Pteridiospora had been originally reported
as saprobic on bamboo in tropical regions (Penzig and
Saccardo 1897; Phookamsak et al. 2014a). Filer (1969)
introduced a new species, P. s pinosis por a Filer which
was isolated from mycorrhizae of sweetgum (Liquidambar
styraciflua L.) and green ash (Fraxinus pennsylvanica
Marsh.) in Northern Mississippi. Pteridiospora munkii
Subhedar & V.G. Rao was found on dead leaves of Phoe-
nix sylvestris (L.) Roxb. from India (Farr and Rossman
2015; Index Fungorum 2015). Guseinov (2000) had also
introduced P. chochrjakovii on Quercus pedunculiflora
K.Koch from Azerbaijan. This indicates that
Pteri diospora
ha
s a wide host range and is found in tropical and tem-
perate regions. Six epithets are listed for Pteridi ospora in
Index Fungorum (2015), with two species currently trans-
ferred to Splanchospora (Pleomassariaceae). Kirk et al.
(2008) estimated there were four species in Pteridiospora;
five sequences from putative species are available in
GenBank. Phookamsak et al. (2014a)collectedP. javanica
Penz. & Sacc. from Thailand and obtained molecular data
from this species. Pteridiospora javanica clustered with
Astrosphaeriella sensu stricto in their analyses.
Phookamsak et al. (2014a) suggested that Pteridiospora
is related to Astrosphaeriella and a new family was need-
ed to accommodate these genera. This was also suggested
in Liu et al. (2011).
Astrosphaeriella-like species appear to be numerous and
polyphyletic and are in need of study using molecular
data. In this study, we use 20 new strains of
astrosphaeriella-like taxa, three strains of Pteridiospora
and GenBank data to establish the phylogenetic relation-
ships of astrosphaeriella-like taxa from the basal lineages
of Pleosporales. The new family, Astrosphaeriellaceae is
introduced to accommodate species of Astrosphaeriella
sensu stricto and Pteridiospora species with erumpent,
carbonaceous ascostromata, while species of
Astrosphaeriella sensu lato with coriaceous, immersed
ascostromata and distinct necks are placed in a new family
Pseudoastrosphaeriellaceae. Two new genera,
Astrosphaeriellopsis and Pseudoastrosphaeriella are intro-
duced to accommodate species excluded from
Astrosphaeriella sensu stricto.
Material and methods
Isolation and identification
The dothideomycete species were collected from dead stems
or branches of bamboo and strout grasses in Chiang Mai and
Chiang Rai Provinces, Thailand. The s pecimens were
returned to the laboratory for observation, examination and
description following the methods described in Phookamsak
et al. (2014a, b). Single spore isolates were obtained as de-
scribed in Chomnunti et al. (2014). The ascostromata were
visualized under a stereo microscope (Motic series SMZ-
140) and the peridium removed from the apex towards the
sides by using a razor blade. The contents inside ascostromata
which comprise asci, ascospores and pseudoparaphyses were
picked off with a sterile needle and soaked in sterile water in a
glass container. The fungal contents were broken up further
mechanically until a spore suspension was obtained. The as-
cospore suspensions were dropped on water agar (WA; 15 g/l
sterile distilled water) and incubated at room temperature
overnight. Spore germination was observed and marked under
the Nikon ECLIPSE 80i compound microscope and germinat-
ing ascospores were transferred to malt extract agar (MEA;
33.6 g/l sterile distilled water, Difco malt extract) and cultivat-
ed in corn meal agar (CMA; 17 g/l sterile distilled water, Difco
corn meal agar) and potato dextrose agar (PDA; 39 g/l distilled
water, Difco potato dextrose) for recording growth rates and
culture characters.
Isolates are deposited in Mae Fah Luang University Cul-
ture Collection (MFLUCC) in Thailand, and duplicated in
BIOTEC Culture Collection (BCC), Bangkok, Thailand and
Kunming Culture Collection (KUMCC). A herbarium speci-
men was dried by using silica gel at room temperature for 7
10 days depending on the humidity and kept in an envelope or
a small box for d epositing in Mae Fah Luang University
(MFLU), Chiang Rai, Thailand and duplicated in Biotech
Bangkok Herbarium (BBH). Facesoffungi and Index
Fungorum numbers were obtained as described in Jayasiri
et al. (2015) and Index Fungorum (2015).
Type specimens of Astrosphaeriella species were requested
from FH, IFRD and S herbaria. Type species were examined
and re-described as in Hyde et al. (2013) and Phookamsak
et al. (2014a, b). Ascostromata were initially rehydrated in
water or adding 35 % KOH for 510 min. The ascostromata
Fungal Diversity
and peridium structures are illustrated from free hand sections
and the squash mounts were prepared to determine the micro-
morphological characters such as asci, ascospores and
hamathecium.
Micro-morphological characters were obtained under the
Nikon ECLIPSE 80i compound microscope and captured by
using a Cannon 550D digital camera with DIC microscopy. A
Sony DSC-T110 digital camera was used to capture macro-
morphological characters under a Motic (series SMZ-140)
stereo microscope. Squash mount preparations was obtained
for determining the micro-morphology, while free hand sec-
tions of ascostromata were made for illustrating ascostromata
and peridium structures. The asci were placed in Melzers
reagent for checking the reactions of the apical ring, whereas
ascospores were placed in Indian ink for checking mucilagi-
nous sheaths surrounding the ascospores. Photographic plates
were edited and combined using program Adobe Photoshop
version CS5 (Adobe Systems Inc., The United States) and
morphological characters measured in a Tarosoft (R) Image
Frame Work version 0.9.7 program. Permanent slides were
prepared by adding lactoglycerol and sealing with clear nail
polish (Phookamsak et al. 2014a, b).
Method to induce the asexual morph in culture
Sterile bamboo pieces (bamboo tooth picks) on water agar
(WA; 15 g/l sterile distilled water) were used to encourage
production of asexual morphs (Fig. 1). The bamboo can be
replaced with a different substrate, e.g. grass, rice straw, de-
pending on the host from which the taxon was isolated and
availability of material;
1.) Bamboo pieces were prepared in a media bottle (250 ml.)
and sterilized by autoclaving at least three times (auto-
clave at 121 °C with 15 psi for 15 min). While agar
powder was mixed with distilled water (15 g/l) and ster-
ilized using an autoclave.
2.) Warm water agar (WA) was poured in glass petri dishes
and surface dried in laminar flow cabinet.
3.) The sterilized bamboo pieces were placed on WA (45
pieces) and mycelia from the growing edge of the 1-
month-old cultures were cut in small pieces and placed
nearby the bamboo pieces (5 pieces) on WA. This step
was carried out in the laminar flow cabinet to avoid
contamination (Fig. 2).
4.) The petri dish was sealed by parafilm and left at room
temperature for 1 to 3 months or longer, depending on the
taxon. The culture was checked to establish if the asexual
morph had formed under the Motic (series SMZ-140)
stereo microscope and the morphological characters were
captured under Nikon ECLIPSE 80i compound micro-
scope with a Cannon 550D digital camera.
DNA extraction, PCR amplification and sequencing
DNA extraction was performed from fresh fungal mycelia
growing on MEA or PDA over 14 weeks at room tempera-
ture (2530 °C). The genomic DNA were obtained by a using
DNA extraction kit (A Biospin Fungus Genomic DNA Ex-
traction Kit, BioFlux®, China) following the protocols in the
manufacturers instructions (Hangzhou, P.R. China)
(Phookamsak et al. 2014a, b). The DNA products were kept
at 4 °C for use in DNA amplification and duplicated at -20 °C
for long term storage.
The DNA amplification was obtained by polymerase chain
reaction (PCR) using the respective gene primers (ITS, LSU,
SSU, RPB2,andTEF1) and amplified following the protocols
described in Phookamsak et al. (2014a, b). The quality of PCR
products were checked by using 1 % agarose gel electropho-
resis stained with ethidium bromide and sent to sequence at
Shanghai Sangon Biological Engineering Technology & Ser-
vices Co. (Shanghai, P.R. China) (Phookamsak et al. 2014a,
b).
Phylogenetic analyses
A BLAST search based on LSU sequence data was carried out
to reveal the closest matches with taxa in the order Pleosporales.
Additionally, the sequences datasets in Schoch et al. (2009),
Zhang et al. (20
12), Hyde et al. (2013), W ijayawardene et al.
(2014) and Ariyawansa et al. (2015 a)wereincludedinthisstudy
and are listed in Table 1. The newly generated sequence data of
thefungaltaxawereanalyzedwith related taxa and additional
taxa from selected families in Pleosporales viz. Aigialaceae,
Amnicolicolaceae, Anteagloniaceae, Astrosphaeriella,
Delitschiaceae, Lophiotremataceae, Lindgomyceta ceae,
Massariaceae, Salsugineaceae, Testudinaceae and
Tetraplosphaeriaceae obtained from GenBank (Table 1). The
LSU, SSU and TEF1 gene datasets were aligned singly by using
MAFFT: multiple sequence alignment software version 7.215
(Katoh and Standley, 2013: http://mafft.cbrc.jp/alignment/
server/) and improved manually where necessary in MEGA6
version 6.0 (Tamura et al. 2013). The combined sequence
alignments were obtained from MEGA6 version 6.0 (T amura
et al. 2013) and the se quence pairwise comparisons were per-
formed in BioEdit v. 7.2 (Hall 1999). The combined sequence
alignment was converted to NEXUS file for maximum parsi-
mony analysis using ClustalX2 v. 1.83 (Thompson et al. 1997)
and converted to PHYLIP file for RAxML using ALTER (align-
ment transformation environment: http://sing.ei.uvigo.es/
ALTER/; 2015). The NEXUS file deleting the symbol was
prepared for MrModeltest v. 2.2 (Nylander 2004) in PAUP v .
4.0b10 (Swofford 2002).
The Randomized Accelerated Maximum Likelihood
(RAxML) was obtained using RAxML version 7.3.0 (Silvestro
and Michalak 2011). One thousand non parametric bootstrap
Fungal Diversity
iterations were run with generalized time reversible (GTR) and a
discrete gamma distribution (Silvestro and Michalak 2011). A
discrete GAMMA (Yang 1994) was complemented for each
substitution model with four rate classes (Stamatakis et al.
2008). Rapid bootstrap analysis (Stamatakis et al. 2008)and
searches for the best-scoring ML tree (RAxML option B-f a^)
were applied (Silvestro and Michalak 2011).
Maximum parsimony (MP) analysis was obtained by
using PAUP v. 4.0b10 (Swofford 2002). The starting
tree(s) was obtained via stepwise addition with 100 repli-
cations of sequences. One thousand bootstrap replicates
with a heuristic search option and tree-bisection reconnec-
tion (TBR) of branch-swapping algorithms were obtained.
Maxtrees were setup at 5000 with a zero of maximum
branch length collapsed. Ambiguously aligned characters
were excluded. Gaps w ere treated as missing data. The
consistency index (CI), retention index (RI), rescaled con-
sistency index (RC) and homoplasy index (HI) were ap-
plied for generating trees under different optimality
criteria. Kishino-Hasegawa tests (KHT) (Kishino and
Hasegawa 1989) were performed to determine significant
parsimonious trees. Twenty-eight parsimonious trees were
saved and the best parsimonious tree was selected to rep-
resent the phylogenetic relationships among the taxa
(Ariyawansa et al. 2013; Phookamsak et al. 2014b).
Bayesian analysis was inferred from MrBayes v.
3.1.2 (Huelsenbeck and Ronquist 2001) using a uniform
[GTR+I+G] model (lset nst=6, rates = invgamma, Prset
statefreqpr = dirichlet,1,1,1,1). The models of nucleotide
substitution were performed by using MrModeltest 2.3
(Nylander 2004) and posterior probabil ities (PP) were
estimated from the Metropolis-Coupled Markov Chain
Monte Carlo (MCMC) sampling approach. Four simul-
taneous Markov chains were run for 5000000 genera-
tions and trees sampled every 100th generations. The
first 5000 trees representing the burn-in phase of con-
vergence of the four cha ins w ere discarde d. W hereas,
the r emaining trees were used to construct a 50 % ma-
jority rule consensus tree and calculating the Posterior
Probabilities (PP). Bayesian Posterior Probabilities
Fig. 2 Method to obtain asexual
morphs in culture. a
Representative culture on PDA
after 1 month (left)andWA
containing sterilize bamboo
pieces (right). b Culture cut in
small pieces and placed with
bamboo pieces on WA
Fig. 1 General materials needed
to induce the asexual morph.
Alcohol burner, 70 % ethanol and
lighter, extra fine forceps,
sterilized bamboo pieces
(bamboo tooth picks), water agar
and fungal culture grown on
MEA or PDA
Fungal Diversity
(BYPP) equal or greater than 0.95 are given below each
node (Fig. 3) (Suetrong et al. 2009;Ariyawansaetal.
2014e).
The phylograms are visualized in Treeview (Page 1996)
and made in Adobe Illustrator CS3 and Adobe Photoshop
version CS5 (Adobe Systems Inc., The United States). The
newly sequences generated in this study have been submitted
in GenBank.
Results and discussion
Phylogenetic analyses
A 50 % majority rule consensus of Bayesian phylogenetic recon-
struction was selected to clarify the phylogenetic relationships of
taxa in the families Astrosphaeriellaceae and
Pseudoastr osphaeriella ceae and the genus Astr osphaeriellopsis
Table 1 Isolates used in this study and their GenBank accession numbers. The ex-type strains are in bold and the newly generated sequences are
indicated in blue
Taxon Culture/Voucher No
1
GenBank Accession No.
2
LSU SSU TEF1
Aigialus grandis BCC 18419 GU479774 GU479738 GU479838
Aigialus grandis BCC 20000 GU479775 GU479739 GU479839
Aigialus mangrovis BCC 33563 GU479776 GU479741 GU479840
Aigialus mangrovis BCC 33564 GU479777 GU479742 GU479841
Aigialus parvus BCC 32558 GU479779 GU479743 GU479843
Aigialus parvus BCC 18403 GU479778 GU479744 GU479842
Aigialus rhizophorae BCC 33572 GU479780 GU479745 GU479844
Aigialus rhizophorae BCC 33573 GU479781 GU479746 GU479845
Amniculicola immersa CBS 123083 FJ795498 GU456295 GU456273
Amniculicola parva CBS 123092 FJ795497 GU296134 GU349065
Anteaglonium globosum SMH 5283 GQ221911 GQ221919
Anteaglonium parvulum SMH 5223 GQ221909 GQ221918
Anteaglonium abbreviatum ANM 925a GQ221877 GQ221924
Ascocratera manglicola HHUF 30032 GU479783 GU479748 GU479847
Ascocratera manglicola BCC 09270 GU479782 GU479747 GU479846
Astrosphaeriella bambusae MFLUCC 10-0095 JN846720 JN846741
Astrosphaeriella bambusae MFLUCC 13-0230 KT955461 KT955424
Astrosphaeriella fusispora MFLUCC 10-0555 KT955462 KT955443 KT955425
Astrosphaeriella neofusispora MFLUCC 11-0161 KT955463 KT955444 KT955426
Astrosphaeriella neostellata MFLUCC 11-0625
KT955464
Astrosphaeriella stellata MAFF 239487 AB524592 AB524451
Astrosphaeriella thailandica MFLUCC 11-0191 KT955465 KT955445 KT955427
Astrosphaeriella thysanolaenae MFLUCC 11-0186 KT955466 KT955446 KT955428
Astrosphaeriella tornata MFLUCC 11-0196 KT955467 KT955447 KT955429
Astrosphaeriella vesuvius MTCC 12224 KP814136 KP814137
Astrosphaeriellopsis bakeriana CBS 115556 GU301801 GU349015
Astrosphaeriellopsis bakeriana MFLUCC 11-0027 JN846730 JN846740
Clohesyomyces aquaticus MFLUCC 11 -0092 JX276950 JX276949
Delitschia chaetomioides SMH3253.2 GU390656 GU327753
Delitschia chaetomioides GKM1283 GU385172 GU327752
Delitschia didyma UME 31411 DQ384090 NG_016519
Delitschia winteri CBS 225.62 DQ678077 DQ678026 DQ677922
Fissuroma aggregata MAFF 239485 AB524590 AB524449
Fissuroma aggregata MAFF 239486 AB524591 AB524450 AB539105
Fissuroma bambusae MFLUCC 11-0160 KT955468 KT955448 KT955430
Fissuroma bambusae MFLUCC 11-0198 KT955469 KT955449 KT955431
Fissuroma maculans MFLUCC 10-0886 JN846724 JN846734
Fissuroma maculans MFLUCC 10-0887 JN846725 JN846736
Fissuroma neoaggregata MFLUCC 10-0554 KT955470 KT955450 KT955432
Fissuroma neoaggregata MFLUCC 13-0227 KT955471 KT955451 KT955433
Fissuroma thailandicum MFLUCC 11-0189 KT955472 KT955452 KT955434
Fungal Diversity
correlating with other selected families in the order Pleosporales
(Fig. 3). Combined LSU, SSU and TEF1 sequence data were
analyzed using the Randomized Accelerated Maximum Likeli-
hood (RAxML), maximum parsimony (MP) and Bayesian prob-
ability programmes.
The dataset comprises 81 taxa, with Hysterium
angustatum Alb. & Schwein. (CBS 123334, CBS
236.34) selected as an outgroup taxon. RAxML analysis
yielded a best scoring tree with a final ML optimization
likelihood value of 20693.077278. The matrix had 1143
distinct alignment patterns, with 23.25 % of undetermined
characters or gaps. Th e maximum par simonio us dataset
consists of 2874 total characters, 1979 characters were
constant, 715 characters were parsimony-informative and
Table 1 (continued)
Taxon Culture/Voucher
No
1
GenBank Accession No.
2
LSU SSU TEF1
Fissuroma thailandicum MFLUCC 11-0206 KT955473 KT955453 KT955435
Flammeascoma bambusae MFLUCC 10-0551 KP744485 KP753952
Hysterium angustatum CBS 123334 FJ161207 FJ161167 FJ161111
Hysterium angustatum CBS 236.34 FJ161180 GU397359 FJ161096
Lepidosphaeria nicotiae CBS 101341 DQ678067 DQ677910
Lindgomyces breviappendiculatus KT 1399 AB521749 AB521734
Lindgomyces cinctosporae R56-1 AB522431 AB522430
Lindgomyces ingoldianus ATCC 200398 AB521736 AB521719
Lophiotrema neoarundinaria KT 2200 AB524597 AB524456 AB539110
Lophiotrema nucula CBS 627.86 GU301837 GU296167 GU349073
Lophiotrema brunneosporum CBS 123095 GU301835 GU296165 GU349071
Lophiotrema lignicola CBS 122364 GU301836 GU296166 GU349072
Massaria campestris M28 HQ599385 HQ599449 HQ599325
Massaria inquinans M19 HQ599402 HQ599444 HQ599342
Massaria mediterranea M45 HQ599417 HQ599452 HQ599357
Massaria platanoidea M7 HQ599420 HQ599457 HQ599359
Massaria vomitoria M13 HQ599437 HQ599440 HQ59375
Neoastrosphaeriella krabiensis MFLUCC 11-0022 JN846727 JN846735
Neoastrosphaeriella krabiensis MFLUCC 11-0025 JN846729 JN846739
Neomassariosphaeria grandispora CBS 613.86 GU301842 GU296172 GU349036
Polyplosphaeria fusca MAFF 239685 AB524604 AB524463 AB524820
Pseudoastrosphaeriella africana MFLUCC 11-0176 KT955474 KT955454 KT955436
Pseudoastrosphaeriella bambusae MFLUCC 11-0205 KT955475 KT955455 KT955437
Pseudoastrosphaeriella longicolla MFLUCC 11-0171 KT955476 KT955456 KT955438
Pseudoastrosphaeriella thailandensis MFLUCC 10-0553 KT955477 KT955457 KT955439
Pseudoastrosphaeriella thailandensis MFLUCC 11-0144 KT955478 KT955458 KT955440
Pseudoastrosphaeriella thailandensis MFLUCC 14-0038 KT955479 KT955459 KT955441
Pseudotetraploa javanica MAFF 239498 AB524611 AB524470 AB524826
Pseudotrichia guatopoensis SMH 4535 GU385202 GU327774
Pteridiospora chiangraiensis MFLUCC 11-0162 KT955480 KT955460 KT955442
Pteridiospora javanica MFLUCC 11-0159
KJ742940 KJ739607 KJ739605
Pteridiospora javanica MFLUCC 11-0195 KJ742941 KJ739606
Repetophragma ontariense HKUCC 10830 DQ408575
Rimora mangrovei JK 5246A GU301868 GU296193
Salsuginea ramicola KT 2597.1 GU479800 GU479767 GU479861
Salsuginea ramicola KT 2597.2 GU479801 GU479768 GU479862
Tetraplosphaeria sasicola MAFF 239677 AB524631 AB524490 AB524838
Triplosphaeria maxima MAFF 239682 AB524637 AB524496 AB524843
Ulospora bilgramii CBS 110020 DQ678076 DQ678025 DQ677921
Verruculina enalia CBS 304.66 DQ678079 DQ678028 DQ677924
Verruculina enalia BCC 18401 GU479802 GU479770 GU479863
Fungal Diversity
180 available characters were parsimony-uninformative.
The heuristic search resulted in a best single tree from
28 parsimonious trees with a length of 3421 steps (CI=
0.365, RI=0.705, RC=0.257, HI: 0.635). Phylogenetic
trees derived from B ayesian, RAxML and MP analyses
gave similar overall topologies at the family relationships
and not significantly different (data not shown). The re-
lated families in Pleosporales showed similar results to
that in the studies of Liu et al. (2011), Zhang et al.
(2012), Hyde et al. (2013) and Wijayawardene et al.
(201 4). However, some taxa were resolved differently in
the internal node relationships among the Bayesian,
RAxML, and MP trees.
Strains of Aigialaceae formed a robust clade (Fig. 3)inall
analyses with each genus having clearly resolved relation-
ships. Six new morphologically similar strains of Fissuroma
are included in this study. However, they cluster in three dis-
tinct species, which are introduced as Fissur oma bambusae
Phookamsak & K.D. Hyde, F. neoaggregata Phookamsak &
K.D. Hyde and F. t ha i l a nd i c u m Phookamsak & K.D. Hyde in
this paper.
A new genus, Astrosphaeriellopsis is introduced to ac-
commodate a single species A. bakeriana which was pre-
viously included in Astrosphaeriella, but is not congeneric
in the combined gene tree. The two strains representing
A. bakeriana are a well resolved clade in our phylogenetic
analyses which is similar to the results in Liu et al.
(2011). Astrosphaeriellopsis bakeriana forms a single
clade basal to the family Aigialaceae in Bayesian and
MP trees, but clusters with Astrosp haeriellaceae in
RAxML analysis. Although, the two strains do not main-
tain a stable position in the phylogeny, they are always
distinct from other clades and we conclude that they rep-
resent a distinct genus which we introduce as
Astrosphaeriellopsis. The genus may need to be raised to
family rank with further populations of the tree.
Astrosphaeriellaceae is introduced to accommodate
Astrosphaeriella and Pteridiospora. The relationships among
taxa in this family have shown similar results in all analyses, with
Astr osphaeriellaceae forming a well-resolved clade, close to
Aigialaceae in Bayesian analysis, but are not supported in
RAxML and MP. Astrosphaeriella sensu stricto and
Pteridiospora forms a strongly supported clade (96 % MP,
96 % ML, 1.00 PP) in our analyses. Two Astrosphaerie lla spe-
cies (A. tornata and A. vesuvius) with reddish brown ascospores
do not cluster in Astr osphaeriella sensu stricto. These two species
form a sister clade at base of the family Astr osphaeriellaceae
with low bootstrap value. A new genus may need to be intro-
duced to accommodate the taxa having heavily, brown pigment,
ascospores when the phylogeny is better populated.
Pseudoastrosphaeriellaceae is also introduced in this study
to accommodate some species of As
trosphaeriella sensu lato.
Six new strains representing four distinct species are included
in the phylogenetic analyses and show similar results in
Bayesian, RAxML and MP trees.
Pseudoastrosphaeriellaceae forms a sister clade with
Tetraplosphaeriaceae, but relationships between these fami-
lies are not well-resolved, probably due to limited populations
in the analyses; thus more extensive sampling is needed.
Salsugineaceae is not stable in our analyses. The fam-
ily forms a sister clade with Lindgomycetacea e in the
Bayesian analysis, but forms a sing le clad e at base of
the fami ly Astrosphaeriellaceae in t he RAxML analysis,
while the family groups with Anteag loniaceae in MP
analysis. In this study, Pseudotrichia guatopoensis
(SMH 4535) clusters with the family Amnicolicolaceae
in Bayesian and MP analyses, but the species clusters
with Testudinaceae in RAxML analysis. This is similar
to the results of Zhang et al. (2012) and Hyde et al.
(2013). Ot her r espective families f orm well-defined clades
as basal lineages of the order Pleosporales.
Taxo nomy
Aigialaceae Suetrong et al., in Suetrong et al., Stud. Mycol.
64: 166 (2010)
Facesoffungi number: FoF 01214
Saprobic on submerged bark or wood of mangrove trees.
Sexual morphs: Ascostromata dark brown, deeply immersed,
or immersed beneath the host epidermis, subglobose to coni-
cal, or hemisphaerical, glabrous, coriaceous or carbonaceous,
ostiolate. Ostioles usually a slit-like opening. Hamathecium
comprising trabeculate pseudoparaphyses, embedded in a hy-
aline gelatinous matrix. Asci 8-spored, bitunicate,
fissitunicate, cylindrical, short-pedicellate, apically rounded,
with a non-amyloid ocular chamber. Ascospores overlapping
uni- to bi-seriate, hyaline to brown, ellipsoidal to fusiform,
septate to muriform, with a mucilaginous sheath or caps.
Asexual morph: Undetermined (from Suetrong et al. 2009
and Hyde et al. 2013).
Type genus: Aigialus Kohlm. & Schatz
Notes:Suetrongetal.(2009) introduced a new family
Aigialaceae to accommodate saprobic marine taxa, ini-
tially including t hree genera viz. Aigialus, Ascocratera
and Rimora (Suetrong et al. 2009; Liu et al. 2011;
Zhang et al. 2012; Hyde et al. 2013). Multigene
Fig. 3 A Bayesian 50 % majority rule consensus tree based on combined
dataset of LSU, SSU and TEF1 alignments. Bootstrap support values for
maximum likelihood (ML, blue) and maximum parsimony (MP, green)
equal or greater than 60 % are given above the nodes. Bayesian posterior
probabilities (BYPP, red) equal or higher than 0.95 are given below the
nodes. Hysterium angustatum (CBS 123334, CBS 236.34) was selected
as an ou tgroup taxon. Ex-type strains are in bold. Ne wly generated
sequences are in red and the type species are indicated in blue
Fungal Diversity
Fungal Diversity
analyses indicated that Astrosphaeriella aggregata was
not related t o th e Astrosphaeriella sensu stricto, but
clusteredwiththefamilyAigialaceae (Schoch et a l.
2009; Zhang et al . 2012).Thus,Liuetal.(2011)
established a new genus Fissuroma in Aigialaceae to
accommodate taxa with ascostromata having slit-like os-
tioles and hyaline, fusiform, didymosporous ascospores.
Fissuroma aggregata (I. Hino & Katum.) Phookamsak
et al . a nd F. m a c u l a n s (Rehm) J.K. Liu et al. were
transferred from Astrosphaeriella.
In this study, Aigialaceae forms a strongly supported clade
(89 % MP/100 % ML/1.00 PP) in Pleosporales as in Schoch
et al. (2009), Suetrong et al. (2009), Liu et al. (2011), Zhang
et al. (2012), Hyde et al. (2013) and Wijayawardene et al.
(2014). The genus Fissuroma is re-visited, as the phylogenetic
analyses ind icate t hat Fissuroma aggregata is a species
complex.
Fissuroma Liu et al, in Liu et a l., Fungal Divers 51(1): 145
(2012)
Facesoffungi number: FoF 01215
Saprobic on bambo o or palms appe aring as d ome-
shaped darkened areas on the host with slit-like ostioles.
Sexual morph: Ascostromata dark brown to black,
scattered to clustered, soli tary to gregarious, immersed
beneath host epidermis, becoming raised, hemisphaerical
domes, uni-loculate, rarely bi-loculate joined at the base,
glabrous, coriaceous or carbonaceous, ostiole a central,
slit-like o pening. Peridium thin- to thick-walled, of un-
equal thickness, poorly developed at the base, thick at
sides towards the apex, composed of dark brown pseudo-
parenchymatous cells, with host cells, plus fungal tissue,
arranged in a textura angularis to textura epidermoidea,
carbonaceous at slit-like opening. Hamathecium com-
posed of dense, anastomosing, trabeculate
pseudoparaphyses, embedded in a hyaline gelatinous ma-
trix. Asci 8-spored, bitunicate, fissitunicate, obclavate to
cylindrical, pedicellate, apically rounded, with an ocular
chamber. Ascosp ores overla pping uni-to tr i-seriate, h ya-
line, fusiform with narrowed ends, 1-septate, sli ghtly con-
stricted at the central septum, surrounded by a distinct
sheath. Asexual morph: Coelomycetous, reported as
pleurophomopsis-like (Tanaka and Harada
2005;L
iu
et al. 2011)
Type sp ecies: Fissuroma maculans (Rehm) J.K. Liu,
E.B.G. Jones & K.D. Hyde.
Notes: Liu et al. (2011)introducedFissuroma,typifiedby
F. maculans, to accommodate some lophiostoma-like species
having hemisphaerical ascostromata with slit-like ostioles, tra-
beculate pseudoparaphyses and hyaline, 1-septate ascospores,
with a second species, F. aggregata (Liu et al. 2011; Hyde
et al. 2013). These species had previously been placed in
Astr osphaeriella and Liu et al. (2011) had found that they were
not congeneric with Astr osphaeriella sensu stricto. Based on
phylogenetic analyses of combined LSU and SSU sequence data,
Fissuroma formed a robust clade in Aigialaceae.
In this study, several collections similar to Fissuroma
aggregata differed in ascospores size, and structure of
mucilaginous sheath. Phylogenetic analyses of combined
LSU, SSU and TEF1 sequence data (Fig. 3)indicatethat
Fissuroma agg regata comprises at l east three distinct spe-
cies. TEF1 gene pairwise comparison showed differences
in 26 base positions (Fig. 9). Six strains of Fissuroma
aggregata have been studied. Molecular analysis show
them to comprise at least three species and a morpholog-
ical comparison show that spore shape and sheath struc-
ture can be used to distinguish these species. Fissuroma
aggregata is re-described in this paper and three new spe-
cies with one combination are introduced.
Key to species of the genus Fissuroma
1. Ascostromata less than 800μm F. maculans
1. Ascostromata longer than 800μm 2
2. Ascospores with thick, distinctive sheath 3
2. Ascospores with thin sheath 4
3. Ascospores (40 )4346(52)×67(9) μm, with
spreading mucilaginous sheath surrounding the asco-
spores F. t h a i l a n d i c u m
3. Ascospores (40
)4
547(52)×68(9) μm,withmuci-
laginous sheath in envelop surrounding the ascospores
F. bambusae
4. Ascospores (43)4550(55)×79μm, with drawn out
terminal appendages F. fissuristoma
4. Ascospores without appendages 5
5. Ascospores (39 )(41 )47 50( 54)× 7 9 μ m ,
ascostromata depressed conical, with short slit-like
opening F. neoaggregata
5. Ascospores 4661(64)×79.5 μm, ascostromata
hemisphaerical, with long slit-like opening F. aggregata
Fissuroma aggregata (I. Hino & Katum.) Phookamsak
et al. in Liu et al., Fungal Divers 51(1): 145 (2011)
Melanopsamma aggregata I. Hino & Katum., Bull. Fac.
Agric. Yamaguchi Univ., 6: 53 (1955)
= Astrosphaeriella aggregata (I. Hino & Katum.) Kaz.
Tanaka & Y. Harada, Mycoscience 46: 115 (2005)
Facesoffungi number: FoF 01216, Fig. 4
Saprobic on bamboo. Sexual morph: Ascostromata 200
300μm high, 5001000μm diam., dark brown, scattered,
gregarious, immersed beneath host epidermis, appearing
as raised, dome-shaped regions, with, thick, rim-like osti-
oles on the host surface, hemisphaerical, with a flattened
base, uni-loculate, glabrous, wall slightly rough, coria-
ceous, ostiolate. Ostioles central, apapillate with long car-
bonaceous, and thick rim-like opening. Peridium thick of
unequal thickness, poorly developed at the base, thick at
Fungal Diversity
sides towards the apex, composed of dark brown to black
cells, carbonaceous at the apex, and membranacous at the
base. Hamathecium composed of dense, 1.52μm wide,
filiform, anastomosing, trabeculate pseudoparaphyses,
embedded in a hya line gelatinous matrix. Asci 143
185×1519.5μm (x =164.5×17.3μm, n=43), 8-spored,
bitunicate, obclavate, with a short furcate to truncate ped-
icel, apically rounded, with an indistinct ocular chamber.
Ascospores 4661(64)×79.5μm (
x =55.9×8.2μm, n=
30), overlapping bi- to tri-seriate at the base, uni-seriate
Fig. 4 Fissuroma aggregata (YAM 20365, holotype of Melanopsamma aggregata). a, b Appearance of ascostromata on host surface. cf Asci. gi
Ascospores. j Old ascospore. Scale bars cj=20μm
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Fungal Diversity
at the apex, hy aline, becoming brown at maturity, fusi-
form with acute ends, 1-septate, slightly constricted at
the septum, asymmetric, u pper cell shorter than lower
cell, smooth-walled, with guttules, surrounded by a thin,
distinct sheath. Asexual morph: Undetermined.
Material examined: JAPAN, Tokuyama, Yamaguchi, on
culms of Phyllostachys bambusoides Siebold & Zucc.
(Poaceae), 28 November 1954, I. Hino, YAM 20365
(holotype of Melanopsamma aggregata).
Notes: Fissuroma aggregata was described by Hino
and Katumoto (1 955)asM elanopsamma aggregata.
Tanaka and Harada (2005) found that Melanopsamma
was not a suitable genus for M. aggregata as it has
bitunicate asci with broadly fusiform ascospores, while
Melanopsamma species have unitunicate asci and ellipsoi-
dal a scospores ( Liu et al. 2011). Tan aka and Harada
( 2005) found that M. aggregata belonged in
Astrosphaeriella following the broad sense of Hyde
et al. (2000). Therefore, Tanaka and Harada (2005)trans-
ferred M. aggregata to Astrosphaeriella. Phylogenetic
analyses of combined LSU and SSU sequence data
showed that Astrosphaeriella aggregata was not related
to Astrosphaeriella sensu stricto (A. stellata,KT998)in
Tanaka et al. (2009). Astrosphaeriella aggregata formed a
single clade at the base of the family Testudinac eae in
Tanaka et al. (2009) and w as closely rela ted to the genus
Rimora in the family Aigialaceae in Schoch et al. (2009).
Therefore, Liu et al. (2011) transferred A. aggregata to
the genus Fissuroma.
Fissuroma bambusae Phookamsak & K.D. Hyde, sp. nov.
Index Fungorum number: IF551629, Facesoffungi num-
ber: FoF 01217, Fig. 5
Etymology: The epithet B
bambusae^ re
fers to the host, of
which the fungus was collected.
Holotype: MFLU 11-0196
Saprobic on bamboo. Sexual morph: Ascostromata 250
400μm high, 7501050μm diam., dark brown, gregarious,
immersed beneath host epidermis, visible as numerous,
raised, dome-shaped areas on the host surface,
hemisphaerical, flattened or wedge-shaped at the base,
uni-loculate, glabrous with rough walls, coriaceous,
ostiolate. Ostioles central, apapillate, with slightly long
carbonaceous, and thin, slit-like opening. Peridium 42
77(118) μm wide, of unequal thickness, poorly devel-
oped at the base, thick at sides towards the apex, com-
posed of several layers, of dark brown to black, pseudo-
parenchymatous cells, with host cells plus fungal tissue,
arranged in a textura angularis to textur a prism atica.
Hamathecium composed of dense, 0.51.5μm wide, fili-
form, trabeculate pseudoparaphyses, anastomosing among
the asci, embedded in a hyaline gelatinous matrix. Asci
(150)170187 ( 194)×(15)1719(22) μm (
x =178.
18.5μm, n=30), 8-spored, bitunicate, cylindric-clavate or
obclavate, with short furcate to truncate pedicel, apically
rounded with a truncate ocular chamber. Ascospores
(40)4547(52)×68(9) μm (
x =46.2×7.1μm, n=30),
overlapping bi- to tri- seriate at the base, uni-seriate at the
apex, hyaline, fusiform with acute ends, 1-septate, constrict-
ed at the septum, upper cell shorter and wider than lower
cell, smooth-walled, surrounded by a thick, distinct sheath.
Asexual mo rph: producing conidio