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Sequence data reveals phylogenetic affinities of fungal
anamorphs Bahusutrabeeja,Diplococcium,Natarajania,
Paliphora,Polyschema,Rattania and Spadicoides
Belle Damodara Shenoy &Rajesh Jeewon &Hongkai Wang &Kaur Amandeep &
Wellcome H. Ho &Darbhe Jayarama Bhat &Pedro W. Crous &Kevin D. Hyde
Received: 2 July 2010 / Accepted: 24 August 2010 / Published online: 11 September 2010
#Kevin D. Hyde 2010
Abstract Partial 28S rRNA gene sequence-data of the
strains of the anamorphic genera Bahusutrabeeja,Diplo-
coccium,Natarajania,Paliphora,Polyschema,Rattania
and Spadicoides were analysed to predict their phylogenetic
relationships and taxonomic placement within the Ascomy-
cota. Results indicate that Diplococcium and morphologi-
cally similar genera, i.e. Spadicoides,Paliphora and
Polyschema do not share a recent common ancestor. The
type species of Diplococcium,D. spicatum is referred to
Helotiales (Leotiomycetes). The placement of Spadicoides
bina, the type of the genus, is unresolved but it is shown to
be closely associated with Porosphaerella species, which
are sister taxa to Coniochaetales (Sordariomycetes). Three
Polyschema species analysed in this study represent a
monophyletic lineage and are related to Lentithecium
fluviatile and Leptosphaeria calvescens in Pleosporales
(Dothideomycetes). DNA sequence analysis also suggests
that Paliphora intermedia is a member of Chaetosphaer-
iaceae (Sordariomycetes). The type species of Bahusu-
trabeeja,B. dwaya, is phylogenetically related to
Neodeightonia (=Botryosphaeria)subglobosa in Botryos-
phaeriales (Dothideomycetes). Monotypic genera Natar-
ajania and Rattania are phylogenetically related to
members of Diaporthales and Chaetosphaeriales, respec-
tively. Future studies with extended gene datasets and type
strains are required to resolve many novel but morpholog-
ically unexplainable phylogenetic scenarios revealed from
this study. It is increasingly becoming evident that a fungal
B. D. Shenoy (*):K. Amandeep
Microbial Type Culture Collection and Gene Bank, Institute of
Microbial Technology (CSIR),
Sector 39-A,
Chandigarh 160 036, India
e-mail: shenoy@imtech.res.in
R. Jeewon (*)
Department of Health Sciences, Faculty of Science, University of
Mauritius,
Reduit, Mauritius
e-mail: rajeshjeewon@yahoo.com
H. Wang
Biotechnology Institute, Zhejiang University,
Hangzhou 310 029, People’s Republic of China
W. H. Ho
Plant Health and Environment Laboratory, Biosecurity New
Zealand, Ministry of Agriculture and Forestry,
231, Morrin Road, St. Johns, P.O. Box 2095, Auckland 1140,
New Zealand
D. J. Bhat
Department of Botany, Goa University,
Taleigao Plateau, Goa 403 206, India
P. W. Crous
CBS-KNAW Fungal Biodiversity Centre,
P.O. Box 85167, 3508 AD Utrecht, The Netherlands
K. D. Hyde
School of Science, Mae Fah Luang University,
Tasud,
Chiang Rai, Thailand
K. D. Hyde
Botany and Microbiology Department, College of Science, King
Saud University,
Riyadh, Saudi Arabia
Fungal Diversity (2010) 44:161–169
DOI 10.1007/s13225-010-0059-8
lineage may include a mosaic of anamorphs, teleomorphs
and pleomorphs whose morphologies may not always be
correlated. It is therefore suggested that where possible all
new species descriptions, whether teleomorphic, anamor-
phic or pleomorphic, should include DNA sequence-data to
facilitate amalgamation of anamorphic and pleomorphic
genera in a single phylogenetic classification system.
Keywords Ascomycetes .Asexual fungi .Hyphomycetes .
Molecular phylogeny .Taxonomy
Introduction
The phenomenon of pleomorphism in fungi, i.e. occurrence
of dual or multiple morphological forms of a fungal species
in different time and space has resulted in considerable
difficulties in developing a stable phylogenetic classifica-
tion system in kingdom Fungi (Shenoy et al. 2007). This
has partially resulted from the establishment of the dual
system of classification in which sexual and asexual fungal
forms are grouped separately. In this arrangement, more
than 20,000 anamorphs have been grouped in a pigeon-
hole-like system of genera, mainly based on anamorphic
characters (Shenoy et al. 2007). The practice of a dual
classification goes against the principle of natural classifi-
cation. Anamorphic genera are regularly described by
fungal taxonomists to facilitate practical purposes such as
identification, but with little consideration for the principle
of natural groupings in classification. Recent efforts in
fungal gene sequence-data analysis have shown that many
‘well-established’anamorphic genera are polyphyletic (e.g.
Seifert et al. 2000; Shenoy et al. 2006,2007; Crous 2009;
Crous et al. 2009b; Jones et al. 2009). There is a warranted
need to revisit the taxonomy of anamorphic genera and
refine their taxonomy based on a polyphasic approach as
demonstrated in recent studies (Groenewald et al. 2008;
Aveskamp et al. 2008,2009,2010). This paper presents our
recent studies on molecular phylogeny of seven anamorphic
genera that include members of the Diplococcium-complex
viz.Diplococcium Grove (1885), Spadicoides S. Hughes
(1958), Paliphora Sivanesan and B. Sutton (1985), and
Polyschema H.P. Upadhyay (1966) and three anamorphic
genera reported from India viz.Bahusutrabeeja Subrama-
nian and Bhat (1977), Natarajania Pratibha and Bhat
(2005) and Rattania Prabhugaonkar and Bhat (2009).
Diplococcium and Spadicoides have similar conidial
ontogeny. In both genera conidiogenous cells are terminal
or intercalary, and polytretic with several unthickened
conidiogenous loci (pores). Conidial ontogeny is holoblas-
tic and the conidiogenous pores are easily visible after
conidial secession. The conidia are acropleurogenous, dry,
dematiaceous, usually thick-walled, 0–5-septate and may
have thick, black or brown bands at the septa. The type
species of Diplococcium (i.e. D. spicatum Grove) and
Spadicoides (i.e. S. bina S. Hughes) differ mainly in
branching of conidiophores and catenation of conidia.
Diplococcium spicatum produces catenate conidia on
branched conidiophores, whereas Spadicoides bina produ-
ces solitary conidia on unbranched conidiophores (Hughes
1958; Ellis 1963; Sinclair et al. 1985; Goh and Hyde 1996).
The conidiophore branching was considered to be
taxonomically more important at the generic level than
conidial catenation by earlier mycologists (e.g. Ellis
1963,1971a,b,1972;Wang1976;WangandSutton
1982). Sinclair et al. (1985), however, stated that at the
generic level, conidiophore branching is taxonomically
less important than conidial catenation, and emended the
generic description of Spadicoides to include species that
produce solitary conidia from conidiophores that may be
simple or branched. This generic delineation based on a
single anamorphic character has been accepted by several
authors (e.g. Kuthubutheen and Nawawi 1991;Bhatand
Kendrick 1993;GohandHyde1996,1998;Hoetal.
2002).
Information on teleomorphs of Diplococcium and Spadi-
coides members is limited and ambiguous. Available taxo-
nomic information from morphological and culture-based
studies suggests that Diplococcium species might have
connections with phylogenetically divergent sexual morphol-
ogies. The suggested sexual forms include Helminthosphaeria
(Sordariomycetes)(Bisby1938; Ellis 1971b; Sutton 1973;
Subramanian 1983; Samuels et al. 1997; Goh and Hyde 1998;
Réblová 1999b)andOtthia (Dothideomycetes)(Subramanian
and Sekar 1989). There is one report that, based on
circumstantial evidence such as growth of the anamorph
on ascomata, suggested Tengiomyces as a possible sexual
form of Spadicoides (Réblová 1999a). These reports
indicate that members of Diplococcium-Spadicoides are
possibly derived from phylogenetically distant lineages.
Phylogenetic significance of conidial catenation in Diplococ-
cium-Spadicoides taxonomy is, therefore, doubtful and
questionable. DNA sequence-analysis is employed in this
study to determine the phylogenetic relationships and
possible taxonomic placement of these asexual fungi within
known ascomycetes.
Paliphora is similar to Spadicoides in conidial ontogeny
but produces hyaline, euseptate conidia (Sivanesan and
Sutton 1985), whereas Polyschema differs from Spadi-
coides mainly in having monotretic or polytretic conidiog-
enous cells borne on micronematous conidiophores
(Upadhyay 1966; Ellis 1971a;Hoetal.2002). Inclusion
of Paliphora and Polyschema in this study aims to examine
whether morphological similarities between these two
genera and Spadicoides correspond to the DNA-based
phylogenetic groupings. This paper also deals with phylo-
162 Fungal Diversity (2010) 44:161–169
genetic placement of three hyphomycetous genera, Bahusu-
trabeeja,Natarajania and Rattania reported from India.
Bahusutrabeeja was described to accommodate a hypho-
mycetous fungus that produces unicellular, hyaline, pear-
shaped to subglobose phialoconidia with several evenly
distributed appendages (Subramanian and Bhat 1977). This
species has been frequently reported from aquatic habitats
in Hong Kong and Guang Dong areas of China (Tsui et al.
2001; Wu and McKenzie 2003). Recently, this species has
been collected from Goa on leaves of Mallotus philippi-
nensis (family Euphorbiaceae). Natarajania is character-
ized by branched conidiophores with terminal, phialidic
conidiogenous cells bearing a distinct collar-canal and
producing slimy, dark-brown, smooth conidia (Pratibha
and Bhat 2005). Rattania includes a sporodochial fungus
with monoblastic conidiogenous cells that produce slimy,
fusiform, curved conidia bearing tiny setulae at both ends
(Prabhugaonkar and Bhat 2009). There are, however, no
known reports on teleomorphs of Bahusutrabeeja,Natar-
ajania and Rattania.
The present study, based on phylogenetic analyses of
partial 28S rRNA gene sequence-data, aims to facilitate
possible amalgamation of these seven anamorphic genera in
ascomycete taxonomy.
Materials and methods
Fungal isolates and DNA extraction
Taxa in this study along with their culture numbers, host/
substrate details, place of collection and NCBI-GenBank
accession numbers of partial 28S rRNA gene sequences are
listed in Table 1. The fungal isolates were grown on potato-
dextrose agar (PDA) and malt extract agar (MEA) for 2–
4 weeks. Genomic DNA from fungal mycelia was extracted
based on: 1) phenol-chloroform method as outlined in Cai
et al. (2005) or 2) Fungal/Bacterial DNA Kit following the
manufacturer’s protocols (Zymo Research, catalogue num-
ber D6005).
Polymerase Chain Reaction (PCR) amplification
and DNA Sequencing
DNA amplification was performed by PCR. The LROR-
LR5 (White et al. 1990) primer-pairs were used to
amplify partial 28S rRNA gene region. Amplification
reactions were performed in a 50 μl reaction volume as
outlined in Shenoy et al. (2006). The thermal cycle was
asfollows:95°Cfor3min,followedby34cyclesof
denaturation at 95°C for 1 min, annealing at 52°C for
30 s and elongation at 72°C for 1 min, with a final
extension step of 72°C for 10 min. The PCR products
spanning approximately 900 bp for partial 28S rRNA
gene were checked on 1% agarose electrophoresis gel
stained with ethidium bromide. PCR products were then
purified using commercial kits (Amersham Biosciences,
UK, catalogue number 27-09602-01; Qiagen, Valencia,
USA, catalogue number 28706). DNA sequencing was
performed using the above-mentioned primers in an
Applied Biosystem 3130/3730 analyzer at the Genome
Research Centre, The University of Hong Kong and at
the Central DNA sequencing facility of Institute of
Microbial Technology (CSIR), Chandigarh, India.
Sequence alignment and phylogenetic analyses
Sequences obtained from the respective primers were
aligned using Sequencher version 4.9 (Gene Codes Corpo-
ration, Ann Arbor, USA) and the consensus sequences were
deposited in NCBI-GenBank. Fifteen new sequences were
generated in this study (Table 1). Each consensus sequence
was subjected to a NCBI-BLAST search to verify its
identity. Additional sequences retrieved from NCBI-
GenBank and their accession numbers are listed in Fig. 1.
Sequences were assembled and aligned using the web
interface of MAFFT (Katoh et al. 2005) available at http://
mafft.cbrc.jp/alignment/server, optimised by eye and
manually corrected when necessary in MEGA4 (Tamura
et al. 2007; Kumar et al. 2008).
Ninety-nine ambiguously aligned characters and the
characters from intron regions were delimited and
excluded from all analyses. The likelihood model
parameters were estimated with MrModeltest version
2.1 (Nylander 2004). Maximum likelihood analyses were
performed using GARLI version 0.96 (Zwickl 2006)
with the default parameters except that the number of
independent search replicates was set to 5. The resultant
best-tree with a lowest likelihood ratio was chosen and
editedinMEGA4.Branchsupportwasestimatedby
performing 100 bootstrap replicates (Felsenstein 1985)in
GARLI. The resulting trees werefedintoPAUPversion
4b10 (Swofford 2002) to obtain a majority rule consensus
tree.
Bayesian posterior probabilities (PP) for each inter-
node were calculated with a Metropolis-coupled Markov
Chain Monte Carlo (MCMC) sampling method as
implemented in MrBayes version 3.1 (Huelsenbeck and
Ronquist 2001). Six simultaneous Markov chains were
run for one million generations (resulting 10K total trees).
The first 2,000 trees were discarded and the remaining
8,000wereusedforcalculatingPPinthemajorityrule
consensus rule tree. These analyses were repeated five
times starting from different random trees to ensure trees
from the same space were being sampled during each
analysis.
Fungal Diversity (2010) 44:161–169 163
Results
The 28S rRNA gene sequence dataset consisted of 83 taxa
including 15 newly generated sequences those belong to
seven anamorphic genera (Fig. 1and Table 1). The other
reference taxa included members of known families of
Dothideomycetes,Leotiomycetes and Sordariomycetes
(Fig. 1). After excluding 99 ambiguously aligned characters
(including a few characters from introns regions), the final
dataset comprised 902 characters. There were 494, 368 and
40 constant, parsimony-informative and autapomorphic
characters, respectively. Likelihood ratio test in MrModelt-
est suggested the best fit-model of evolution for this dataset
was GTR+I+G. The best-tree from ML analyses with a
lowest likelihood ratio of −9127.379 is shown in Fig. 1.
Bootstrap values (equal to or above 50%) based on 100
replicates are shown on the upper branches. Values from PP
(equal to or above 95%) from MCMC analyses are
represented as thickened branches on the tree.
Three strains of Diplococcium analysed in this study
showed phylogenetic affinities with members of Dothideo-
mycetes and Leotiomycetes (Fig. 1). Diplococcium spicatum
clustered with an uncultivable soil fungus (GenBank no.
EU861651), sister to an ericoid mycorrihizal fungus
(GenBank no. AY599241), a fungal endophyte (GenBank
no. DQ979447), Mollisea cinerea and Trimmatostroma
salicis. The Helotiales clade on the whole received strong
statistical support. The D. asperum strain, however,
clustered within Pleosporales (Dothideomycetes). It was
found to be basal to a clade containing Aquaticheirospora
lignicola,Dictyosporium sterilitziae,Phoma flavescens and
an uncultured ascomycetous strain (GenBank no.
EU490077). This clustering pattern, however, received less
than 50% bootstrap support.
Spadicoides species clustered with members of Dothi-
deomycetes and Sordariomycetes (Fig. 1). Spadicoides atra
formed a strongly supported monophyletic lineage with
Lentomitella species within the Sordariomycetes clade.
Table 1 Taxa in this study along with their strain numbers, host/substrate, place of collection and GenBank accession numbers of partial 28S
rRNA gene sequences
Taxon Strain no. Host/substrate
a
Place of collection GenBank
Acc. No.
Bahusutrabeeja
dwaya
MTCC 9680 (= GUFCC
4904)
Mallotus philippinensis, leaf India, Goa, Colem HM171320
Diplococcium
asperum
CBS 139.95 Apple leaf Italy EF204493
Diplococcium
spicatum
CBS 162.47 Alnus glutinosa, bark Info. not available EF204484
Diplococcium
spicatum
CBS 852.73 Wine cork France, Château Latour Labatut, Mtg
St. Emilion
EF204496
Natarajania
indica
MTCC 9659 (= GUFCC
5240)
b
Antiaris toxicaria, dead leaf India, Goa, Canacona, Netravali HM171321
Paliphora
intermedia
CBS 199.95 Buchenavia capitata, leaf Cuba EF204500
Paliphora
intermedia
CBS 896.97
c
Leaf litter Australia, Queensland; Lamington
National Park
EF204501
Polyschema
larviformis
CBS 463.88 Soil Turkey, Izmir EF204503
Polyschema
congolensis
CBS 542.73
b
Soil Zaire, Ndjili, Kinshasa EF204502
Polyschema
terricola
CBS 301.65
b
Soil under Saccharum officinarum,
25 cm depth
Brazil, Recife EF204504
Rattania
setulifera
MTCC 9698 (=GUFCC
15501)
b
Calamus thwaitesii, leaf India, Goa, Dhoodhsagar HM171322
Spadicoides atra CBS 489.77 Quercus petraea, branch Czech Republic, forest Lánská obora EF204506
Spadicoides bina CBS 113708 Picea abies Sweden, Uppland, Dalby par.,
Jerusalem
EF204507
Spadicoides
verrucosa
CBS 128.86
b
Bambusa sp., old fungi on leaf India, Andhra Pradesh; Adilabad EF204508
Spadicoides
xylogena
CBS 310.31 Agave sisal Info. not available EF204509
CBS Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; MTCC Microbial Type Culture Collection and Gene Bank (CSIR-
IMTECH), Chandigarh, India; GUFCC Fungus Culture Collection of Goa University, India
a
As per CBS Fungi Database;
b
Ex-type strain;
c
Isotype strain
164 Fungal Diversity (2010) 44:161–169
Rattania setulifera HM171322
Tainosphaeria crassiparies AF466089
Dictyochaeta simplex AF178559
Thozetella sp. EU825195
Thozetella nivea EU825200
Codinaeopsis gonytrichoides AF178556
Zignoella pulviscula AF466091
Chaetosphaeria luquillensis AF466074
Chaetosphaeria fuegiana EF063574
Chaetosphaeria callimorpha AF466062
Chaetosphaeria minuta AF466075
Paliphora intermedia EF204501
Paliphora intermedia EF204500
Chaetosphaeria caesariata AF466060
Chaetosphaeria chalaroides AY017372
Chaetosphaeriales
Helminthosphaeria clavariarum AY346283
Echinosphaeria canescens AY436404
Helminthosphaeria hyphodermae AY346284
Ruzenia spermoides AY436422
Helminthosphaeria carpathica AY346282
Helmintho-
sphaeriaceae
Coniochaetidium savoryi AY346276
Coniochaeta velutina FJ167402
Porosphaerella borinquensis EF063573
Spadicoides bina EF204507
Porosphaerella cordanophora AF178563
Coniochaetales
Myrmecridium schulzeri EU041834
Myrmecridium flexuosum EU041825
Dactylaria parvispora EU107296
Lentomitella sp. EU527994
Lentomitella crinigera AY761086
Spadicoides atra EF204506
Lentomitella sp. EF577060
Tectonidula hippocrepida FJ617557
Barbatosphaeria barbirostris EF577059
Ceratostomella pyrenaica DQ076323
Spadicoides verrucosa EF204508
Sordariomycetes incertae sedis
Phomopsis sp. AF439632
Diaporthe phaseolorum AY346279
Prosthecium acerophilum EU039994
Prosthecium opalus EU039990
Prosthecium ellipsosporum EU039986
Natarajania indica HM171321
Chromendothia citrina AF408335
Chrysoporthe cubensis AF408338
Microthia havanensis AF408339
Diaporthales
Sordariomycetes
Diplodia rosulata DQ377897
Botryosphaeria stevensii DQ377864
Phaeobotryosphaeria visci EU754216
Botryosphaeria sumachi DQ377865
Botryosphaeria corticola DQ377848
Sphaeropsis conspersa DQ377933
Diplodia pinea EU754157
Lasiodiplodia crassispora DQ377901
Bahusutrabeeja dwaya HM171320
Neodeightonia subglobosa DQ377866
Botryosphaeriales
Curvularia brachyspora AF279380
Spadicoides xylogena EF204509
Ascomycota sp. FJ971837
Setosphaeria monoceras AY016368
Cochliobolus heterostrophus AY544645
Dendryphiella arenaria DQ470971
Pleospora herbarum DQ678049
Phoma flavescens GU238075
Uncultured ascomycete EU490077
Dictyosporium strelitziae FJ839653
Aquaticheirospora lignicola AY736378
Diplococcium asperum EF204493
Lentithecium fluviatile FJ795451
Leptosphaeria calvescens AY849944
Polyschema sp. GU188573
Polyschema larviformis EF204503
Polyschema congolensis EF204502
Polyschema terricola EF204504
Pleosporales
Dothideomycetes
Vibrissea truncorum FJ176874
Vibrissea flavovirens AY789426
Diplococcium spicatum EF204496
Diplococcium spicatum EF204497
Uncultured soil fungus EU861651
Fungal endophyte DQ979447
Ericoid mycorrhizal fungus AY599241
Trimmatostroma salicis EU019300
Mollisia cinerea DQ470942
Helotiales
Peziza vesiculosa DQ470948
0.05
Leotiomycetes
97
73
88
100
100
66
56
100
64
85
64
97
90
100
86
99
54
71
70
54
82
100
94
100
100
69
100
93
100
63
80
84
55
100
54
100
100
64
86
100
70
93 95
100
100
51
62
98
97
99
100
56
88
Fig. 1 A maximum likelihood
(ML) tree generated based
on partial 28S rRNA gene
sequence-data (-lnL =
9127.379). Bootstrap values
(equal to or above 50%) based
on 100 replicates are shown
on the upper branches. Values
from PP (equal to or above
95%) from MCMC analyses are
represented as thickened
branches on the tree. The tree
is rooted with Peziza vesiculosa
Fungal Diversity (2010) 44:161–169 165
Spadicoides verrucosa clustered with Ceratostomella pyr-
enaica, sister to a clade containing Barbatosphaeria
barbirostris and Tectonidula hippocrepida. This cluster,
however, received less than 50% bootstrap support.
Spadicoides bina clustered with Porosphaerella borinquen-
sis and P. cordanophora with high support and this clade is
paraphyletic to Coniochaeta velutina and Coniochaetidium
savoryi.Spadicoides xylogena, however, belongs to the
Pleosporales clade (Fig. 1). It grouped with strong
statistical support with Curvularia brachyspora and an
unidentified ascomycete species (GenBank no. FJ971837).
Paliphora intermedia strains formed a distinct lineage
within Chaetosphaeriales but its position within the order
was not resolved (Fig. 1). Polyschema species from this
study formed a strongly supported monophyletic group
with Polyschema sp. (GenBank no. GU188573), and are
related to Lentithecium fluviatile and Leptosphaeria
calvescens within Pleosporales.Bahusutrabeeja dwaya
clustered with Neodeightonia (=Botryosphaeria) subglo-
bosa with 97% bootstrap and 100% PP support within
Botryosphaeriales (Dothideomycetes). Natarajania indica
clustered with Chromendothia citrina,Cryphonectria
cubensis and Microthia havanensis within Diaporthales
(Sordariomycetes). This clustering arrangement, however,
received only 69% bootstrap support. Rattania setulifera
is part of a moderately supported clade with Taniosphae-
ria crassiparies,Dictyochaeta simplex,andThozetella
species within Chaetosphaeriales.
Discussion
Diplococcium is one of the earliest described hyphomycetes
genera. The type species D. spicatum was originally
described from rotten wood in Sutton Coldfield, United
Kingdom. Unfortunately, no holotype was designated in the
protologue (Grove 1885). In the absence of any authenti-
cated type material or living culture of the fungus, it is
challenging to reconstruct the phylogeny of Diplococcium.
The two non-type strains of D. spicatum (CBS162.47 and
852.73) employed in this study are phylogenetically related
to an uncultured soil fungus (GenBank no. EU861651 and
deposited by Nemergut et al. 2008) within Helotiales
(Leotiomycetes) (Fig. 1). This association is ecologically
relevant as Diplococcium spicatum is known to inhabit
similar ecological niches such as decomposing plant
material in contact with soil (Goh and Hyde 1998).
Diplococcium spicatum has also been reported from the
indoor environment at a considerably higher level (avg.
CFU/g = 24850) (Scott 2001). Although Maximum
Likelihood analysis suggests a close evolutionary related-
ness between Diplococcium spicatum,Mollisia cinerea and
Trimmatostroma salicis, there are few morphological
characters to support this. Mollisia cinerea, the type species
of the genus, produces a phialidic anamorph (Crous et al.
2003), unlike Diplococcium. Though Diplococcium spica-
tum and Trimmatostroma salicis (a sporodochial anamorph)
are morphologically similar in producing catenate conidia,
T. salicis differs from the former in having meristematic,
arthric conidiogenous cells (Ellis 1971a).
Grouping of Diplococcium asperum within Pleospor-
ales (Dothideomycetes) proves that Diplococcium does not
represent a natural grouping. In this scenario, there is a
need to re-collect D. spicatum from its type locality and
redefine the species boundary based on DNA sequence-
data. Though the observed association of D. asperum
(CBS 139.95; isolated from apple leaf) with Aquaticheir-
ospora lignicola,Dictyosporium sterilitziae,Phoma fla-
vescens andanunculturedascomycetesstrain(GenBank
no. EU490077) was not statistically supported (Fig. 1), D.
asperum has 96% sequence identity with these taxa and
shares similar ecological niches (decomposing plant
materials/soil) (Kodsueb et al. 2007;Hollister2008;Crous
et al. 2009a;Aveskampetal.2010).
Diplococcium may also have phylogenetic associations
with other fungal orders/families. Several Helminthosphae-
ria species have been associated with Diplococcium
anamorphs but these associations are yet to be confirmed
through culture/ DNA-based methods (e.g. Goh and Hyde
1998; Samuels et al. 1997; Réblová 1999b; Huhndorf et al.
2004). Though we have involved members of Helminthos-
phaeriaceae (including H. clavariarum, the presumed
teleomorph of D. clavariarum) in our analysis, D. spicatum
and D. asperum did not show a close phylogenetic
relationship with this family (Fig. 1). It still remains to be
probed whether Diplococcium and Helminthosphaeria are
two spore stages of a single fungus or if Diplococcium
leads a fungicolous lifestyle on the latter. The culture-based
teleomorphic connection of D. pulneyense with Otthia
pulneyensis (Dothideomycetes) (Subramanian and Sekar
1989) has to be re-examined. The type material of Otthia,
based on which Diplococcium pulneyense was connected to
Otthia, has been found to actually represent a Botryos-
phaeria-like species (Phillips et al. 2005; Crous et al.
2006). It is subject to future studies whether Diplococcium
pulneyense belongs to Botryosphaeriales or not. There is
one report on the Selenosporella synanamorph of Diplo-
coccium hughesii (Wang and Sutton 1998). Selenosporella
is known to be polyphyletic (Seifert et al. 2000) and as on
17 June 2010, there are no DNA sequence-data available
under this generic name in NCBI-GenBank to verify any
phylogenetic connection between the two.
This study reveals some novel phylogenetic scenarios
with respect to placement of Spadicoides species (Fig. 1).
The placement of the type species of Spadicoides,S. bina
with Porosphaerella species near Coniochaetales is
166 Fungal Diversity (2010) 44:161–169
intriguing. Porosphaerella species are known to produce
Cordana and morphologically similar Pseudobotrys ana-
morphs. These anamorphs produce conidiogenous loci
resembling spinules, unlike S. bina that produces con-
idiogenous pores (Hughes 1958; Fernández and Huhndorf
2004). There are few comparable morpho-taxonomic
characters that can explain the relationship between the
known Porosphaerella anamorphs and Spadicoides.This
is the first report of a Spadicoides anamorph in the
Porosphaerella lineage. It is clear from this study that
Spadicoides atra is a part of the Lentomitella lineage
(Sordariomycetes) that is known to produce Phaeoisaria-
like anamorphs in culture. Spadicoides and Phaeoisaria
anamorphs are morphologically similar in having solitary,
acropleurogenous conidia, but the latter is known to
produce denticulate conidiogenous cells (Réblová 2006;
Huhndorf et al. 2008), unlike Spadicoides. The phyloge-
netic placement of S. verrucosa needs to be resolved in
future studies as its association with Ceratostomella
pyrenaica received low statistical support (Fig. 1). Phylo-
genetic association of Spadicoides species with the
presumed teleomorph Tengiomyces (Réblová 1999b)could
not be tested due to lack of DNA sequences of
Tengiomyces in public DNA databases.
The placement of Spadicoides xylogena within the
Pleosporales can be explained with the available morpho-
logical data. Spadicoides shares similar conidial ontogeny
with the known anamorphic Pleosporales such as Alter-
naria,Bipolaris,Curvularia,Dendrophyion,Dendro-
phyiopsis,Dendryphiella,Drechslera,Exserohilum,
Helminthosporium,Scolecobasidium and Ulocladium
(Sivanesan 1984; Schoch et al. 2006). Spadicoides is
morphologically similar to these anamorphs in having
tretic/polytretic conidiogenous cells. In this study, Spadi-
coides xylogena shares a close phylogenetic relationship
with Curvularia brachyspora. Both of them have solitary
acropleurogenous conidia produced on polytretic conidiog-
enous cells but C. brachyspora differs in having conidia
produced on sympodial conidiogenous cells (Ellis 1971a).
The Spadicoides-like anamorphs Paliphora and Poly-
schema were not related to the four Spadicoides species
included in this study. Members of Polyschema appear to
represent a distinct lineage within Pleosporales (Fig. 1).
Their close affinities with Lentithecium fluviatile and
Leptosphaeria calvescens and correct placement within
Pleosporales (Fig. 1) should be tested using more strains
from diverse ecological habitats as well as anamorphs of L.
fluviatile and L. calvescens.Paliphora intermedia constitutes
a distinct lineage within Chaetosphaeriales (Fig. 1). This
result represents a novel phylogenetic scenario in which a
polytretic anamorph is reported within Chaetosphaeriales
that has been known to include mainly phialidic (Réblová
and Winka 2001; Huhndorf and Fernández 2005; Fernández
et al. 2006) and a few Sporidesmium-like anamorphs
(Réblová 1999b; Shenoy et al. 2006). We are also reporting
Rattania as a new introduction in the anamorph-rich
Chaetosphaeriales clade. Rattania setulifera shows a close
phylogenetic relationship with phialidic anamorphs such as
Codinaea (i.e. anamorphic Tanios p h a e r i a)andDictyochaeta.
Both Rattania and Codinaea produce conidia with tiny
setulae at both ends but the former produces monoblastic
conidiogenous cells, unlike Codinaea and Dictyochaeta
(Fernández and Huhndorf 2005).
A close phylogenetic relationship of Bahusutrabeeja
dwaya with Neodeightonia (=Botryosphaeria)subglobosa
is interesting. The latter is known to produce a Diplodia-
like coelomycetous anamorph (Crous et al. 2006). Bahusu-
trabeeja is morphologically different from Sphaeriopsis in
having phialidic conidiogenous cells that produce unicellu-
lar conidia with 8–12 slender appendages (Subramanian
and Bhat 1977). The monotypic genus Natarajania is
related to members of the Cryphonectriaceae in Diapor-
thales that is known to include mainly coelomycetous
anamorphs (Gryzenhout et al. 2006). Natarajania is one of
the few known hyphomycetes in Diaporthales and its
correct placement warrants further investigation.
Conclusion
Many of the novel phylogenetic scenarios revealed from
this study cannot easily be explained with available
morphological data. Recent studies have revealed the
presence of fungal lineages which are mosaic of ana-
morphs, teleomorphs and pleomorphs and with little
morphological-data in support of their phylogenetic prox-
imity (Shenoy et al. 2006,2007). Fungal taxonomists
should, therefore, voluntarily include (where possible)
DNA sequence-data in all new species description, whether
teleomorphic, anamorphic or pleomorphic. This would
facilitate a taxonomic link of anamorphic genera in a single
phylogenetic classification system.
The GenBank sequence data must also be used with
caution. Cai et al. (2009) and Hyde et al. (2009)showedthat
more than 86% of names used for C. gloeosporioides
sequences were wrongly applied. We also wonder how
many of the generic names may be wrong? We presently
have few options but to accept GenBank names as being
probably correct, but in future we hope that more and more
taxa will be epitypified so that conclusions of the type made
from studies of this sort can be confidently made.
Acknowledgements BDS and KA would like to thank Institute of
Microbial Technology (Council of Scientific and Industrial Research,
India) for support and encouragement. BDS would like to thank the
University of Hong Kong for the award of a postgraduate studentship
(2003–2007). Helen Leung and Heidi Kong from the University of
Fungal Diversity (2010) 44:161–169 167
Hong Kong are thanked for technical assistance during 2003–2007.
DJB is thankful to the Indian University Grants Commission for a
Special Assistance Programme support. RJ would like to acknowledge
the research support by the Department of Health Sciences, Faculty of
Science, University of Mauritius.
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