m y c o l o g i c a l r e s e a r c h 1 1 0 ( 2 0 0 6 ) 359 – 368
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Molecular Systematics of Zopfiella and allied genera:
evidence from multi-gene sequence analyses
Lei CAI*, Rajesh JEEWON, Kevin D. HYDE
Centre for Research in Fungal Diversity, Department of Ecology and Biodiversity, The University of Hong Kong,
Pokfulam Rd, Hong Kong SAR, PR China
article info
abstract
Article history:
This study aims to reveal the phylogenetic relationships of Zopfiella and allied genera in the
Received 22 June 2005
Sordariales. Multiple gene sequences (partial 28 S rDNA, ITS/5.8 S rDNA and partial b-tubu-
Received in revised form
lin) were analysed using MP and Bayesian analyses. Analyses of different gene datasets
10 December 2005
were performed individually and then combined to infer phylogenies. Phylogenetic analy-
Accepted 4 January 2006
ses show that currently recognised Zopfiella species are polyphyletic. Based on sequence
Published online 20 March 2006
analyses and morphology, it appears that Zopfiella should be restricted to species having as-
Corresponding Editor:
cospores with a septum in the dark cell. Our molecular analysis also shows that Zopfiella
David L. Hawksworth
should be placed in Lasiosphaeriaceae rather than Chaetomiaceae. Cercophora and Podospora
are also polyphyletic, which is in agreement with previous studies. Our analyses show
Keywords:
that species possessing a Cladorrhinum anamorph are phylogenetically closely related. In
Ascomycota
addition, there are several strongly supported clades, characterised by species possessing
Lasiosphaeriaceae
divergent morphological characters. It is difficult to predict which characters are phyloge-
Molecular phylogenetics
netically informative for delimiting these clades.
Sordariales
ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
b-Tubulin
Introduction
Zopfiella, a teleomorphic genus in the family Lasiosphaeriaceae
(Sordariales, Ascomycota), was first established by Winter
(1884) to accommodate two species, Z. tabulata and Z. curvata.
Zopfiella species have been characterised by non-ostiolate
ascomata, clavate to cylindrical, usually evanescent asci lacking an apical ring. Ascospores are ellipsoidal, dark brown,
transversely septate, with a hyaline pedicel which often collapses (Guarro et al. 1991). The anamorphs of most Zopfiella
species are unknown, with Z. latipes forming a Humicola-like
anamorph in culture (Guarro et al. 1991). There have been considerable confusion and controversies about the phylogenetic
relationships of Zopfiella and other morphologically similar
genera such as Podospora and Cercophora (Guarro et al. 1991).
Podospora species generally differ from those of Zopfiella in
that their ascospores are larger, with gelatinous appendages,
and pedicels that are longer and non-collapsed (Guarro et al.
1991). The immature ascospores of Podospora are usually clavate rather than ellipsoidal as found in Zopfiella. Zopfiella has
also traditionally been distinguished from Podospora by the
presence of non-ostiolate ascomata. This morphological character, however, has been regarded as unreliable based on cultural studies (e.g. von Arx 1973). Although the above
distinctions have been widely used in the classification of
this group of fungi, the division between Podospora and Zopfiella is still problematical. Khan and Krug (1990) have suggested that Z. tabulata, the type species of Zopfiella belongs in
Podospora. Guarro et al. (1991) was of the opinion that many
species of Zopfiella should be transferred to Podospora.
* Corresponding author. Fax: þ852-25176082.
E-mail address: leicai@hkusua.hku.hk
0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.mycres.2006.01.007
360
Tripterospora is another genus bearing morphological similarities to Zopfiella and Podospora. This genus was established
by Cain (1956) based on cleistothecial ascomata and lack of gelatinous appendages. Tripterospora can be distinguished from
Zopfiella by the lack of septum in the dark cell of ascospores
(Cain 1956). The taxonomic relationships between Tripterospora and Zopfiella has been widely debated. Malloch and
Cain (1971) and Khan and Krug (1990) preferred to separate
the two genera. Lundqvist (1969, 1972) however, suggested
that Tripterospora and Zopfiella should be better treated as congeneric. Guarro et al. (1991) agreed with Lundqvist that the
separation of the two genera was artificial. Similar taxonomic
views were shared by von Arx (1973) and Udagawa and Furuya
(1974). Most recently, Tripterospora has been treated as a synonym of Zopfiella (Guarro et al. 1991; Kirk et al. 2001). Nevertheless, whether Tripterospora and Zopfiella are distinct or
congeneric is a matter of personal opinion.
Cercophora is also similar to Zopfiella and Podospora in many
morphological features. Cercophora resembles Podospora in
having gelatinous appendages attached to either the dark
cell and/or the pedicel (Lundqvist 1972). There are only some
minor distinctions in ascospore shape currently applied in
their taxonomy. The anamorph of Cercophora is similar to
that of Podospora, being Cladorrhinum or Phialophora (Kendrick
& Dicosmo 1979; Udagawa & Muroi 1979; Mouchacca & Gams
1993). Cercophora has been suggested as being polyphyletic
(Lundqvist 1972; Miller & Huhndorf 2001), and its relationships
with other genera such as Bombardia, Lasiosphaeria, Podospora
and Zopfiella are also unclear (Lundqvist 1972).
In order to investigate the phylogenetic relationships of
Zopfiella and other allied genera, a number of fungi that exhibit
a broad range of ascomatal and ascospore morphologies were
sampled. Phylogenetic analyses were conducted based on partial 28 S rDNA, ITS/5.8 S rDNA and partial b-tubulin sequences
using MP and bayesian analyses. The objectives of this study
were: (1) to examine the phylogenetic relationships of Zopfiella
and its allies; and (2) to provide an overview of the phylogenetic significance of morphologies in the delineation of these
closely related genera.
Materials and methods
DNA extraction, PCR, and sequencing
Cultures were obtained from culture collections CBS (Utrecht)
and NITE (Tsukuba) (Table 1). Isolates were grown on potato
dextrose agar (PDA) for two to four weeks and total genomic
DNA was extracted from fresh mycelium using the protocol
as outlined by Jeewon et al. (2003) and Lacap et al. (2003). Partial
28 S rDNA, complete ITS/5.8 S rDNA and partial b-tubulin were
amplified using fungal specific primers LROR and LR5 (Vilgalys
& Hester 1990), ITS5 and ITS4 (White et al. 1990) and Bt2A and
Bt2B (Glass & Donaldson 1995), respectively. The PCR thermal
cycle were same as that of Cai et al. (2006). PCR products were
checked on 1 % agarose electrophoresis gels stained with
ethidium bromide.
PCR products were then purified using minicolumns, purification resin and buffer according to the manufacturer’s protocols (Amersham Bioscience, UK, product code 27-9602-01).
L. Cai et al.
DNA sequencing was performed using the primers mentioned
above in an Applied Biosystem 3730 DNA Analyzer at the
Genome Research Centre (University of Hong Kong).
Sequence alignment and phylogenetic analysis
For each fungal strain, sequences obtained from pair primers
were aligned to obtain an assembled sequence using Bioedit
(Hall 1999). In total four datasets were analysed: 28 S rDNA
dataset, ITS/5.8 S rDNA dataset, b-tubulin dataset, and a combined dataset. Novel sequences generated from this study
were submitted to GenBank (Table 1). Sequences for each
strain, together with reference sequences obtained from GenBank (Table 2), were aligned using Clustal X (Thomson et al.
1997). Alignment was manually adjusted to allow maximum
alignment and minimise gaps.
Phylogenetic analyses were performed by using PAUP*
4.0b10 (Swofford 2002). Ambiguously aligned regions were
excluded from all analyses. Unweighted parsimony (UP) and
weighted parsimony (WP) analyses were performed with
gaps treated as missing data. WP analyses were performed using a symmetric step matrix generated with the program
STMatrix version 2.2 (François Lutzoni & Stefan Zoller, Department of Biology, Duke University), by which the relative frequencies of nucleotide substitutions were calculated and
converted into costs of changes. Trees were inferred using
the heuristic search option with tree bisection reconnection
(TBR) branch swapping and 1000 random sequence additions.
Maxtrees were unlimited, branches of zero length were collapsed and all parsimonious trees were saved. Descriptive
tree statistics such as tree length (TL), consistency index (CI),
retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were calculated for trees generated under
different optimality criteria. Clade stability was assessed in
a bootstrap (BS) analysis with 1000 replicates, each with ten
replicates of random stepwise addition of taxa. Kishino–
Hasegawa tests (KH Test) (Kishino & Hasegawa 1989) and
Templeton test (Templeton 1983) were performed in order
to determine whether trees were significantly different. Trees
were viewed in Treeview (Page 1996).
A model of evolution was estimated by using Modeltest 3.06
(Posada & Crandall 1998). Posterior probabilities (PP) (Rannala &
Yang 1996; Zhaxybayeva & Gogarten 2002) were determined by
MCMC sampling in MrBayes 3.0b4 (Huelsenbeck & Ronquist
2001), using above estimated model of evolution. Six simultaneous Markov chains were run for 1,000,000 generations and
trees were sampled every 100th generations (resulting in 10,000
total trees). The first 2000 trees that represented the burn-in
phase of the analyses were discarded. The remaining 8000 trees
were used for calculating PP in the majority rule consensus tree.
Results
The 28 S rDNA dataset comprised 868 sites, of which four ambiguous regions were excluded in the analysis. There were 228
parsimony informative characters (PIC) in those included regions. Two hundred and eighty trees were generated from
UP, while WP resulted in a single tree. KH test
(0.3784 P 0.8900) and Templeton tests (0.2351 P 0.7230)
Molecular systematics of Zopfiella
361
Table 1 – Sequences generated from this study
Species
Isolate codea
Achaetomium strumarium
Apodus deciduus
A. oryzae
Cercophora ambigua
C. caudata
C. coprophila
C. samala
Diplogelasinospora inaequalis
CBS 333.67
CBS 506.70
CBS 376.74
CBS 215.60
CBS 606.72
NITE 32091
CBS 109.93
CBS 436.74
D. grovesii
Gelasinospora calospora
G. tetrasperma
Lasiosphaeris hispida
Neurospora tetrasperma
Podospora cupiformis
P. didyma
P. appendiculata
P. austroamericana
P. cochleariformis
P. curvicolla
P. intestinacea
Schizothecium aloides
S. curvisporum
S. fimbriatum
S. glutinans
Sordaria fimicola
S. lappae
Zopfiella karachiensis
Z. latipes
Z. longicaudata
Z. tabulata
Z. tetraspora
Z. erostrata
CBS 340.73
NITE 32008
CBS 178.33
CBS 955.72
NITE 32011
CBS 246.71
CBS 232.78
NITE 8549
CBS 724.68
CBS 249.71
NITE 8548
CBS 113106
CBS 879.72
CBS 507.50
CBS 144.54
CBS 134.83
CBS 508.50
CBS 154.97
NITE 32902
NITE 9826
NITE 30296
CBS 230.78
NITE 32904
CBS 255.71
Origins
Soil, India, isotype
Dung, USA, type
Straw, Italy, type
Twig, Canada
Soil, Netherlands
Burnt soil, Japan
Dung, Japan
Soil, Papua
New Guinea, type
Soil, Japan, type
Burnt soil
Dung, Canada, type
Wood, Germany
Burnt soil
Dung, Africa, type
Dung, Canada
Dung, Japan
Flower, India, isotype
Dung, Africa
Dung, Japan
Dung, New Zealand
Soil, Netherlands
Carrot Canada
Dung
Bearberry, Switzerland
Dung, Canada
Soil, Hungary
Soil, Japan
Soil, Japan
Soil, Japan
Dung, Canada
Soil
Dung, Africa
GenBank accessing No.
28 S rDNA
ITS rDNA
b-tubulin
AY681170
AY681165
AY681166
AY999114
AY999113
AY999112
AY999111
AY681167
AY681204
AY681199
AY681200
AY999137
AY999135
AY999136
AY999134
AY681201
AY681238
AY681233
AY681234
AY999147
AY999151
AY999141
AY999140
AY681235
AY681168
AY681155
AY681144
AY681169
AY681159
AY999102
AY999100
AY999103
AY999101
AY999098
AY999099
AY999104
AY999097
AY999096
AY999092
AY999093
AY681160
AY681137
AY999106
AY999107
AY999109
AY999105
AY999108
AY999110
AY681202
AY681190
AY681178
AY681203
AY681194
AY999125
AY999127
AY999126
AY999124
AY999123
AY999122
AY999121
AY999120
AY999119
AY999115
AY999116
AY681188
AY681171
AY999128
AY999129
AY999131
AY999132
AY999130
AY999133
AY681236
AY681223
AY681212
AY681237
AY681227
AY999149
AY999142
AY999144
AY999138
AY999145
AY999148
AY999152
AY999159
AY999155
AY999156
AY999157
AY681228
AY681205
AY999153
AY999146
d
AY999143
AY999139
AY999150
a Abbreviations: CBS, Centraalbureau voor Schimmelcultures, Utrecht, NITE, National Institute of Technology and Evaluation, Tsukuba, Japan.
showed that these trees were not significantly different. The
single tree generated from WP (TL ¼ 1956.61, CI ¼ 0.399,
RI ¼ 0.662, RC ¼ 0.264, HI ¼ 0.601) is shown in Fig 1.
ITS/5.8 S dataset had 48 taxa with 715 characters, of which
12 ambiguous regions were excluded in the analysis. There
were 103 PIC in this dataset. UP generated 261 trees, while
WP resulted in three trees. These 264 trees were not significantly different based on KH test (0.2904 P 1.0000) and
Templeton tests (0.1797 P 1.0000). One of the three trees
generated from the WP (TL ¼ 756.00, CI ¼ 0.523, RI ¼ 0.717,
RC ¼ 0.375, HI ¼ 0.477) is shown in Fig 2.
The b-tubulin dataset comprised 31 taxa, and 566 sites. Five
ambiguous regions were excluded in all analyses. There
were 74 PIC in this dataset. Two trees and one tree, respectively, were generated from UP and WP. KH test
(0.6923 P 0.7020) and Templeton tests (0.8271 P 0.9903)
revealed no significant difference among these trees. The single tree generated from the WP (TL ¼ 438.39, CI ¼ 0.442,
RI ¼ 0.583, RC ¼ 0.258, HI ¼ 0.558) is shown in Fig 3.
The combined dataset with 31 taxa, has 2039 characters.
Sixteen ambiguous regions were excluded in all analyses,
and there were 239 PIC in those included regions. Ten trees
and one tree, respectively, were generated from UP and
WP. KH test (0.6024 P 1.0000) and Templeton tests
(0.3227 P 1.0000) revealed that these trees were not significantly different. The single tree generated from the WP
(TL ¼ 1511.22, CI ¼ 0.517, RI ¼ 0.680, RC ¼ 0.352, HI ¼ 0.483) is
shown in Fig 4.
Although there were different taxon sampling between
datasets, phylogenies generated were essentially similar in
species groupings (Figs 1–4). To discuss tree outputs, phylograms were divided into several clades (A, B, C, Figs 1–4).
Among them, clades A and C are strongly supported by 92 %
BS/ 99 % PP and 92 % BS/ 100 % PP respectively in the 28 S
rDNA tree (Fig 1). In the combined gene tree, the support for
these two clades was even higher (Fig 4). Clade B received
moderate support (e.g. 85 % BS in 28 S rDNA tree; Fig 1). In addition, clade stabilities are relatively low in the trees generated from ITS/5.8 S rDNA and b-tubulin dataset (Figs 2–3),
owing to the lower number of PIC in these datasets. The btubulin dataset and the combined dataset, consisting of the
same 31 taxa, generated trees that are essentially similar in
the main groupings (Figs 3–4). However, the tree generated
from the combined dataset is much better resolved. For instance, in the combined gene tree, 21 subclades are supported
by >50 % BS and 21 subclades are supported by >95 % PP
362
L. Cai et al.
Table 2 – Additional sequences used in analyses
(obtained from GenBank)
Species
Amphisphaeria umbrina
Apiosordaria verruculosa
Aporothielavia leptoderma
Barrina polyspora
Bombardia bombarda
Bombardioidea anartia
Cercophora appalachianensis
C. areolata
C. areolata
C. atropurpurea
C. costaricensis
C. mirabilis
C. newfieldiana
C. septentrionalis
C. scortea
C. striata
C. sulphurella
C. sulphurella
Chaetomium globosum
C. globosum
Chaetosphaeria innumera
Coniochaetidium savoryi
Corynascus kuwaitiensis
Diaporthe pustulata
Halosphaeria appendiculata
Immersiella caudata
I. immersa
Jugulospora rotula
Lasiosphaeria ovina
L. rugulosa
Lasiosphaeris hirsuta
Lignincola laevis
Melanochaeta hemipsila
Pestalotiopsis versicolor
Podospora anserina
P. austrohemisphaerica
P. bicolor
P. comata
P. comata
P. decidua
P. decipiens
P. decipiens
P. ellisiana
P. fibrinocaudata
P. fimiseda
P. fimiseda
P. petrogale
P. pleiospora
P. setosa
Strattonia carbonaria
Thielavia cephalothecoides
T. coactilis
T. fraqilis
Triangularia mangenotii
Valsella salicis
Xylaria hypoxylon
Zygopleurage zygospora
b 28 S rDNA.
c ITS/5.8 S rDNA.
GenBank no.
AF452029b
AY346258b
AF096186b
AY346261b
AY346263b
AY346264b
AF132328b
AY587936b
AY587911c
AY780057b
AY780059b
AY346271b
AF064642b
U47823b
AY780063b
AY780065b
AY587938b
AY587913c
AY545729b
AY429056c
AY017375b
AY346276b
AJ715483c
AF408358b
U46885b
AY436407b
AY436408b
AY346287b
AY436413b
AY436414b
AY436417b
U46890b
AY346292b
AF409993c
AY525771c
AY026939c
AF443848c
AY780072b
AF443849c
AF443851c
AY780073b
AY515359c
AY515360c
AY780074b
AY346296b
AY515361c
AY071831c
AY515364c
AF443852c
AY346302b
AF286413b
AJ271585c
AJ271578c
AY346303b
AF408389b
U47841b
AY346306b
(Fig 4). Conversely, there were only 13 and ten subclades supported by BS and PP, respectively, in the b-tubulin tree (Fig 3).
The inclusion of more genes thus, resulted in a better statistical support for most of the clades.
Results generated from this study showed: (1) Zopfiella is
polyphyletic, with six different species clustered in five different clades as shown in Fig 1. Zopfiella species are interspersed
in the trees and show association with a variety of genera such
as Apiosordaria, Apodus, Cercophora, Podospora and Triangularia;
(2) Zopfiella species clustered with lasiosphaeriaceous species
rather than chaetomiaceous species; (3) Cercophora and Podospora are also polyphyletic, which is in agreement with previous studies (Miller & Huhndorf 2005; Cai et al. 2005); (4) Species
possessing a Cladorrhinum anamorph grouped in clade A, indicating their close phylogenetic relationships; and (5) Other
highly supported clades, such as clades A and C, include species having diverse morphological characters.
Discussion
Phylogenetic relationships of Zopfiella
Phylogenetic analyses indicate that Zopfiella species, as currently circumscribed, do not constitute a monophyletic group
(Figs 1–4). The non-ostiolate ascomata, and the absence of gelatinous appendages have been considered important in distinguishing Zopfiella from Podospora (Guarro et al. 1991).
However, both of these characters are shown to be unreliable
in understanding phylogenetic relationships. That Non-ostiolate Zopfiella species interspersed in different clades in the
trees (Figs 1–4) suggests multiple origins of this morphological
character, and this conclusion is in agreement with previous
studies (e.g. Berbee & Taylor 1992; Rehner & Samuels 1995;
Suh & Blackwell 1999; Cai et al. 2006). In addition, the presence
or absence of gelatinous appendages, as shown here, are phylogenetically less informative. As shown in the phylogenetic
trees, Zopfiella species grouped in different clades that also include many species possessing elaborate gelatinous appendages. Similar results pertaining to Schizothecium have been
reported (Cai et al. 2005), in which strongly supported Schizothecium clade included species with or without gelatinous appendages. Although the presence of the gelatinous
appendages have been suggested to be an adaptation for those
coprophilous species, this morphology appears to have arisen
independently in several different lineages.
Tripterospora has been treated as a synonym of Zopfiella
(Kirk et al. 2001; Eriksson et al. 2004). So-called Tripterospora
species are morphologically similar to other Zopfiella species
but differ in lacking a septum in the brown cell of the ascospores. Molecular analyses in our study showed that these
species did not cluster together. Z. erostrata, Z. tetraspora, Z.
longicaudata and Z. latipes have ascospores lacking a septum
in the dark cell and had been placed in Tripterospora. However,
these species are found to cluster in different distantly related
clades. (Figs 1–4). Tripterospora is therefore, an artificial taxonomic arrangement. Cain (1956) established a new family Tripterosporaceae based on several species of Tripterospora, but this
familial circumscription clearly does not reflect a natural
grouping.
Molecular systematics of Zopfiella
363
82/100
Zopfiella tetraspora
*/67
Podospora comata
*/71
Cercophora striata
*/51
Cercophora samala
Zopfiella longicaudata
98/100
Clade A
Apiosordaria verruculosa
Podospora austroamericana
74/100
100/100
Podospora fimiseda
Cercophora costaricensis
Cercophora
coprophila
*/58
Chaetomium globosum
*/77
Aporothielavia leptoderma
Chaetomiaceae
*/55 54/96
Thielavia cephalothecoides
Achaetomium strumarium
Diplogelasinospora inaequalis
95/100
Diplogelasinospora grovesii
Triangularia mangenotii
64/*
Zopfiella latipes
70/90
Apodus oryzae
*/52
Cercophora mirabilis
Cercophora appalachianensis
*/93
Cercophora septentrionalis
Podospora
cochleariformis
97/100
Podospora decipiens
80/79 Gelasinospora calospora
Gelasinospora tetrasperma
Sordariaceae
100/100
Neurospora tetrasperma
100/100 Sordaria lappae
Sordaria fimicola
99/100
Bombardia bombarda
Bombardioidea anartia
Podospora didyma
85/88
Cercophora sulphurella
Clade B
Zopfiella tabulata
Cercophora ambigua
82/56
Cercophora areolata
100/100
Lasiosphaeria rugulosa
Lasiosphaeria ovina
89/88 Cercophora scortea
*/100
Podospora appendiculata
Podospora fibrinocaudata
76/100
Schizothecium fimbriatum
63/*
Schizothecium
Schizothecium curvisporum
100/100
Schizothecium glutinans
species
Schizothecium aloides
Zopfiella karachiensis
Cercophora atropurpurea
98/98 Jugulospora rotula
Strattonia carbonaria
96/100 Immersiella immersa
70/100
Immersiella caudata
Cercophora macrocarpa
100/100 Apodus deciduus
53/62
Cercophora newfieldiana
70/73
Zopfiella erostrata
70/53
Cercophora caudata
Clade C
Podospora intestinacea
92/100
Podospora curvicolla
92/100 65/100
Podospora cupiformis
Zygopleurage zygospora
Lasiosphaeris hispida
89/100
Lasiosphaeris hirsuta
100/100
Chaetosphaeria innumera
100/100
Melanochaeta hemipsila
Coniochaetidium savoryi
93/100
Barrina polyspora
Xylaria hypoxylon
100/100
Amphisphaeria umbrina
51/*
Diaporthe pustulata
100/100
Valsella salicis
Lignincola laevis
Halosphaeriales appendiculata
92/99
69/99
Sordariales
Chaetosphaeriales
Coniochaetales
Xylariales
Diaporthales
Halosphaeriales
10
Fig 1 – Phylogram of single tree generated from parsimony analysis based on 28 S rDNA sequences (TL [ 1956.61, CI [ 0.399,
RI [ 0.662, RC [ 0.264, HI [ 0.601). Data were analysed with random addition sequence, WP and treating gaps as
missing data. Values before the backslash are parsimony BS (above 50 %) while after are Bayesian posterior probabilities
(above 50 %). The tree is rooted with Lignincola laevis and Halosphaeria appendiculata. *Clades that receive less than 50 % support.
As the current concept of Zopfiella does not reflect natural
relationships, taxonomic changes will be necessary. Typical
Zopfiella species, such as the type species Z. tabulata, have ascospores with a septum in the dark cell. In the present study,
Z. tabulata nested in clade B in all the trees (Figs 1–4). Other
species grouped in clade B include Podospora didyma and Cercophora sulphurella. It is interesting that both P. didyma and C. sulphurella also produce one septum in the dark cell (Mirza & Cain
1969; Hilber & Hilber 1979). A possible explanation for this is
that these species are united by their morphological similarity
(ascospores with septate dark cell), and this character, can be
phylogenetically informative. In addition, C. ambigua and C.
areolata appear close to the above species in clade B (resolved
as a sister clade to clade B, Fig 1). In these two species, a septum in the dark cell of the ascospore has also been observed
(Hilber & Hilber 1979; Udagawa & Muroi 1979). Cain (1956)
364
L. Cai et al.
Podospora didyma
Zopfiella tabulata
57/*
68/*
Clade B
Cercophora sulphurella
Podospora appendiculata
Cercophora ambigua
73/89
Cercophora areolata
70/98 Zopfiella latipes
Apodus oryzae
Podospora cochleariformis
87/95
Podospora bicolor
*/64
100/100 Podospora decipiens
Podospora pleiospora
Podospora austrohemisphaerica
Diplogelasinospora inaequalis
Diplogelasinospora grovesii
99/100
Podospora curvicolla
Podospora decidua
Podospora cupiformis
Podospora intestinacea
100/100
99/100
*/74
100/100
Clade C
Apodus deciduus
Zopfiella erostrata
Cercophora caudata
Zopfiella karachiensis
Podospora ellisiana
Schizothecium aloides
*/80
Schizothecium glutinans
97/100
Schizothecium species
Schizothecium curvisporum
*/54
Schizothecium fimbriatum
Podospora petrogale
57/99
*/98
Chaetomium globosum
Corynascus kuwaitiensis
*/98
Thielavia fragilis
Thielavia coactilis
Podospora anserina
87/100
77/97
Podospora comata
85/98
*/89
*/71
99/99
66/99
Chaetomiaceae
Cercophora samala
Podospora austroamericana
Zopfiella tetraspora
Podospora setosa
Zopfiella longicaudata
Cercophora coprophila
Podospora fimiseda
Gelasinospora tetrasperma
Gelasinospora calospora
Neurospora tetrasperma
Clade A
Sordariaceae
Sordaria lappae
Sordaria fimicola
Pestalotiopsis versicolor
10
Fig 2 – Phylogram of one of three trees generated from parsimony analysis based on ITS/5.8 S rDNA sequences (TL [ 756.00,
CI [ 0.523, RI [ 0.717, RC [ 0.375, HI [ 0.477). Data were analysed with random addition sequence, WP and treating gaps
as newstate. Values before the backslash are parsimony BS (above 50 %) while after are Bayesian posterior probabilities
(above 50 %). The tree is rooted with Pestalotiopsis versicolor.*Clades that receive less than 50 % support.
suggested that the presence of a septum in the dark cell might
represent side branches in the evolution in lasiosphaeriaceous species. A similar view was shared by Mirza & Cain
(1969). Our molecular results are generally congruent with
Cain’s (1956) postulate as these species are phylogenetically
closely related. The present study is in agreement with Cai
et al. (2006), who found that the spore septum is useful in understanding the phylogenetic relationship amongst the
families Lasiosphaeriaceae and Sordariaceae. We would therefore suggest that Zopfiella should be restricted to species
with a septum in the dark cell as Zopfiella species that lack
a spore septum are not related. It appears highly plausible
that P. didyma and C. sulphurella in clade B should be transferred to Zopfiella, but there are some indications that do not
favour this amendment. In particular, the septum in the
dark cell of the ascospores seems has arisen independently
Molecular systematics of Zopfiella
365
90/97
Zopfiella latipes
Apodus oryzae
Cercophora ambigua
Podospora appendiculata
Zopfiella karachiensis
Podospora cochleariformis
Apodus deciduus
Diplogelasinospora inaequalis
87/100
Diplogelasinospora grovesii
99/100
Podospora didyma
Clade B
Zopfiella tabulata
Lasiosphaeris hispida
Zopfiella erostrata
71/94
Cercophora caudata
Podospora intestinacea
Clade C
Podospora curvicolla
64/95
Podospora cupiformis
Gelasinospora calospora
100/*
85/59
52/*
Neurospora tetrasperma
Gelasinospora tetrasperma
98/100
Sordariaceae
Sordaria lappae
Sordaria fimicola
Schizothecium curvisporum
59/88
*/61
Schizothecium fimbriatum
82/100
*/99
Schizothecium aloides
Schizothecium species
Schizothecium glutinans
Podospora austroamericana
73/85
Cercophora samala
82/97
*/96
Clade A
Zopfiella tetraspora
Cercophora coprophila
Achaetomium strumarium
10
Fig 3 – Phylogram of the single tree generated from parsimony analysis based on b-tubulin sequences (TL [ 438.39,
CI [ 0.442, RI [ 0.583, RC [ 0.258, HI [ 0.558). Data were analysed with random addition sequence, WP and treating gaps as
missing data. Values before the backslash are parsimony BS (above 50 %) while after are Bayesian posterior probabilities
(above 50 %). The tree is rooted with Achaetomium strumarium. *Clades that receive less than 50 % support.
in a few more distantly related fungi. For example, C. newfieldiana is also characterised by a similar spore septum in the dark
cell of the ascospore (Hilber & Hilber 1979), but it is more
closely related to Apodus deciduus, Z. erostrata, and C. caudata
(clade C).
Huhndorf et al. (2004) found that Z. ebriosa clustered with
chaetomiaceous species rather than lasiosphaeriaceous species and based on this they suggested the transfer of Zopfiella
from Lasiosphaeriaceae into Chaetomiaceae. The inclusion of
six different Zopfiella species in this study reveals that Zopfiella
is more closely related to other lasiosphaeriaceous species
rather than chaetomiaceous species. The affinity of Z. ebriosa
(AY346305) was also tested and as reported by Huhndorf
et al. (2004), it clustered with chaetomiaceous species (result
not shown). However, Z. ebriosa as used by Huhndorf et al. is
an atypical species of Zopfiella. As first described by Guarro
et al. (1991), Z. ebriosa is markedly different from all other Zopfiella species in lacking a spore germ pore. Furthermore, the
cylindrical asci and the ostiolate tendency of this fungus is
also different from other Zopfiella species. Therefore, Huhndorf et al.’s familial replacement of Zopfiella in the family Chaetomiaceae based on only one and an atypical species is not
accepted here.
The association of Cercophora and Podospora with a variety
of genera in our phylogenetic trees shows that the current circumscription of them does not accurately reflect natural relationships. It is therefore, inappropriate to discuss and make
any conclusive taxonomic statement of Cercophora and Podospora. Nomenclatural changes to classify them into natural
groupings are left for future studies. The present study is in
agreement with previous studies that the traditionally used
ascospore morphology is highly homoplasious.
366
L. Cai et al.
100/100
Zopfiella erostrata
Cercophora caudata
52/72
Podospora intestinacea
60/99
99/100
89/100
Clade C
Apodus deciduus
Podospora curvicolla
98/100
Podospora cupiformis
Zopfiella karachiensis
63/99
Schizothecium fimbriatum
80/100
Schizothecium curvisporum
*/98
100/100
Schizothecium aloides
Schizothecium species
Schizothecium glutinans
Lasiosphaeris hispida
99/100
74/99
*/88
Zopfiella latipes
Apodus oryzae
*/100
Podospora cochleariformis
Cercophora ambigua
*/54
100/100
Podospora didyma
Clade B
*/98
Zopfiella tabulata
100/100
Diplogelasinospora inaequalis
*/97
Podospora appendiculata
*/99
Diplogelasinospora grovesii
86/*
75/*
100/*
100/100
Gelasinospora tetrasperma
Gelasinospora calospora
Neurospora tetrasperma
100/100
Sordariaceae
Sordaria lappae
Sordaria fimicola
53/51
100/100
Podospora austroamericana
Zopfiella tetraspora
84/100
Clade A
Cercophora samala
Cercophora coprophila
Achaetomium strumarium
10
Fig 4 – Phylogram of one of the single tree generated from parsimony analysis based on combined b-tubulin, ITS rDNA
and 28 S rDNA sequences (TL [ 1511.22, CI [ 0.517, RI [ 0.680, RC [ 0.352, HI [ 0.483). Data were analysed with random
addition sequence, WP and treating gaps as missing data. Values before the backslash are parsimony BS (above 50 %)
while after are Bayesian posterior probabilities (above 50 %). The tree is rooted with Achaetomium strumarium. *Clades that
receive less than 50 % support.
Phylogenetic relationships of species possessing a
Cladorrhinum anamorph
Phylogenies based on sequence analyses have been shown to
be important in the classification of many anamorphs and
their integration with teleomorphs (Taylor 1995; Rehner &
Samuels 1995; Jacobs & Rehner 1998). Most of the known anamorphs of Podospora species have been reported as Phialophora,
except Podospora fimiseda which produces a Cladorrhinum anamorph (Bell & Mahoney 1997; Lundqvist et al. 1999). The anamorphs of Cercophora are similar, mostly Cladorrhinum and
Phialophora (Mouchacca & Gams 1993; Udagawa & Muroi
1979; Miller & Huhndorf 2001). Phialophora has been shown to
be a heterogeneous assemblage of anamorphs of unrelated ascomycetes in various studies (Domsch et al. 1980; Gams 2000).
Cladorrhinum species, however, have been reported to be anamorphs of several species, including Apiosordaria verruculosa
(von Arx & Gams 1967), Cercophora samala (Udagawa & Muroi
1979), Cercophora striata (Miller & Huhndorf 2001) and Podospora
fimiseda (Bell & Mahoney 1997). Interestingly, all of the above
species clustered together in clade A (Fig 1). Podospora austroamericana (syn. P. castorinospora), another species clustered in
clade A, produces an anamorph characterised by very short,
peg-like phialides scattered on the mycelium (Cain 1962; Mirza
& Cain 1969). Although a proper anamorphic connection has
not been established by above authors, these anamorphic
characters are mostly restricted to Cladorrhinum. Our results
therefore, indicate that species possessing a Cladorrhinum anamorph are phylogenetically closely related, despite the anamorphs of several other species in clade A not being known
and requiring further investigation.
Implications of several supported clades
In our phylogenetic trees, there are several strongly supported
clades, such as clades A and C. However, species in these
Molecular systematics of Zopfiella
groupings have diverse morphological characters. For instance, in clade A, although they have similar anamorphs,
their teleomorph morphologies are quite divergent. Besides
the highly diverse ascospore morphology, taxa in clade A are
also diverse in characters, such as ascomata being non-ostiolate or ostiolate, glabrous or adorned with hairs, asci being 4or 8-spored, cylindrical or clavate, and peridia of different
types. As these morphological characters also occur in several
other taxa throughout the phylogram, it is presently unclear
which characters are phylogenetically the most informative
for delimiting these clades. Even for the ascomatal morphology, which has been regarded as a more reliable phylogenetic
predictor (Miller & Huhndorf 2005), our study suggests a
conservative application of it. For instance, P. fimiseda and
C. costaricensis clustered in clade A (Fig 1). These two species
are characterised by a typical pseudo-bombardioid peridium
(a kind of non-stromatic peridium wall with a gelatinized
layer consisting of interwoven, thin-walled hyphae) which
has been regarded as phylogenetically important (Miller &
Huhndorf 2001, 2005). However, with a broader taxonomic
sampling in our study, species in clade A is shown to have various types of peridia. For instance, C. striata has peridium consisting of flattened, elongate or polygonal cells (Miller &
Huhndorf 2001); C. samala has a peridium of ‘textura angularis’,
consisting brown, polygonal cells (Udagawa & Muroi 1979);
Z. tetraspora and Z. longicaudata have ascomata that are cleistothecial and consist of compressed, elongated cells, and they
are polygonal or irregular in surface view (Cain 1956; Rai
et al. 1963).
Clade C, with high bootstrap and posterior probabilities
support contained a group of species from Apodus, Cercophora,
Zopfiella, Podospora and Zygopleurage. All sampled species in
clade C possess ascomata that are covered by flexuous, long
or short, brown hairs. Nevertheless, these hairs show variations among different species. For example, the ascomata of
A. deciduus and Zopfiella erostrata are covered with very long,
flexuous, somewhat rigid, septate, brown hairs (Malloch &
Cain 1970; Guarro et al. 1991); P. intestinacea has ascomata covered with short, flexuous, soft, septate, ramified hairs
(Lundqvist 1972); P. curvicolla has ascomata covered by short,
flexuous, light brown, septate hairs and on the neck covered
by long, slender, tapering tufts of rigid, cylindrical, septate
hairs (Mirza & Cain 1969).
Zygopleurage is morphologically interesting as it produces
ascospores that are elongate, biseptate and provided with
swollen, pigmented end cells and a long, vermiform, hyaline
intercalary middle cell. This is abnormal in lasiosphaeriaceous species and is not stable in some cases. For instance,
in P. trichomanes, some ascospores may become biseptate
with a hyaline middle cell similar to those of Zygopleurage species. This may indicate a close affinity between some Podospora species and Zygopleurage species. Lundqvist (1972) has
suggested that Zygopleurage may share a common ancestry
and evolved in parallel with some Podospora species. Zygopleurage might be closely related to several species such as P. curvicolla, P. cupiformis, and P. intestinacea, which also clustered in
clade C.
In this study, several well-supported clades in which species are characterised by divergent morphological characters
are conservatively treated. It may be that a combination of
367
morphological characters unites these clades. Based on our
present understanding of this group of fungi, it is not possible
to establish which character or character combination unites
above supported monophyletic clades. Other authors may
have different notion on the taxonomic treatment of these
taxa. Regardless of the subjectivity in taxonomic practice,
the important implication of this study are that phylogenetically defined groups of individuals exist, and the relationships
among them can and should be considered when these species are included in further studies of evolutionary biology.
Acknowledgements
This study was funded by the Hong Kong Research Grants
Council (HKU 7320/02M), National Natural Science Foundation
of China (NSFC3026002) and International Cooperation Research Foundation, Yunnan Province (2000C002). The University of Hong Kong is acknowledged for providing L.C. with
a postgraduate scholarship. We are grateful to CBS, NITE
and ICMP for providing cultures. Helen Leung, Keith Cheung
and Heidi Kong are thanked for technical assistance.
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