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Phylogenetic relationships of Pestalotiopsis and allied genera inferredfrom ribosomal DNA sequences and morphological characters
Rajesh Jeewon,a,* Edward C.Y. Liew,b and Kevin D. Hydea
a Centre for Research in Fungal Diversity, Department of Ecology and Biodiversity, The University of Hong Kong, Pokfulam Rd,
Hong Kong, SAR, People�s Republic of Chinab School of Land, Water and Crop Sciences, McMillan Building A05, The University of Sydney, NSW 2006, Australia
Received 21 August 2001; received in revised form 8 November 2001
Abstract
The taxonomy of the coelomycetous fungus Pestalotiopsis and other closely related genera based on morphological characters
has been equivocal. To gain insight in the phylogenetic relationships of Pestalotiopsis and its allies, part of the large subunit (28S)
ribosomal DNA region was examined and compared with existing morphological information. Phylogenetic analyses were con-
ducted using parsimony, distance, and likelihood criteria. Results of the analyses showed that Bartalinia, Pestalotiopsis, Seima-
tosporium, and Seiridium represent distinct monophyletic groups with high bootstrap support. However, Truncatella species are
paraphyletic with Bartalinia, sharing a common ancestor. Pestalotia species sequenced clustered together with Pestalotiopsis. These
genera should be recognized as distinct natural groups except forMonochaetia and Discosia, which need to be further resolved. Tree
topologies are generally in concordance with previous morphological hypotheses, most notably the placement of all Pestalotia
species, except the type P. pezizoides, in Pestalotiopsis. Well-supported clades corresponding to groupings based on conidial
morphology were resolved and the relative importance of morphological characters for generic delimitation is discussed. Molecular
data also provide further evidence to support the association of these coelomycetes with the Amphisphaeriaceae.
� 2002 Elsevier Science (USA). All rights reserved.
Keywords: Pestalotia; Coelomycetes; Amphisphaeriaceae; Ribosomal DNA
1. Introduction
The genus Pestalotiopsis Steyaert is a heterogeneous
group of coelomycetous fungi where species are defined
primarily based on conidial characteristics including
size, septation, pigmentation, and presence or absence of
appendages (Nag Rag, 1993; Steyeart, 1949; Sutton,
1980). Other fungal genera such as Bartalinia Tassi,
Discosia Libert, Monochaetia (Saccardo) Allescher,Pestalotia de Notaris, Seimatosporium Corda, Seiridium
Nees: Fries, and Truncatella Steyeart, however, also
possess morphological characters very similar to those
of Pestalotiopsis, resulting in considerable ambiguity
and confusion in intergeneric classification of these
fungi. Most species in these genera have morphological
characters that overlap in many respects. These char-acters include number of median cells, which may or
may not be pigmented, presence of apical and basal
appendages, and hyaline apical or basal cells. The clas-
sification, validity, and delimitation of these genera have
been problematic and have been resolved differently by
various authors (Guba, 1961; Nag Rag, 1993; Steyeart,
1949; Sutton, 1980).
Guba (1929, 1961) adopted a broad generic conceptby synonymizing Pestalotiopsis and Truncatella with
Pestalotia. He divided the genus Pestalotia into three
sections designated Quadriloculatae, Quinqueloculatae,
and Sexloculatae for three-septate, four-septate, and
five-septate spores, respectively. The same subdivisions
were applied to Monochaetia (species having only one
apical appendage), which was treated as congeneric with
Seiridium. The main feature on which he relied for hissystem of classification was the number of apical ap-
pendages and he stated that the distinction between
Molecular Phylogenetics and Evolution 25 (2002) 378–392
MOLECULARPHYLOGENETICSANDEVOLUTION
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*Corresponding author. Fax: +852-251-760-82.
E-mail addresses: rjeewon@hkusua.hku.hk (R. Jeewon), kdhyde@
hkucc.hku.hk (K.D. Hyde).
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Pestalotia and Monochaetia rested primarily upon thenumber of apical appendages arising from the apical
cell.
This broad generic concept of Pestalotia was highly
criticized by Steyeart (1949) who erected two new gen-
era, Pestalotiopsis (for the group of fungi known as
Pestalotia section Quinqueloculatae) and Truncatella
(for the group of fungi known as Pestalotia section
Quadriloculatae). He also discarded the genus Mono-
chaetia and reassigned the species in Pestalotia into
these two new genera. Steyeart (1949) preferred to keep
the genus Pestalotia monotypic, which was represented
by Pestalotia pezizoides. This species is morphologically
distinct from all Pestalotiopsis species, as it possesses
cupulate conidiomata and distoseptate median cells. By
keeping Pestalotia monotypic, he divided the genus
Pestalotiopsis and Truncatella into different sectionsbased on the number of apical appendages. All the
species bearing single apical appendages (e.g., Mono-
chaetia) were not given generic status and were trans-
ferred to the section Monosetulatae of Pestalotiopsis
and Truncatella, thereby contradicting Guba�s treat-ment. Steyeart (1953a,b, 1961, 1963) published further
evidence in support of his contradictions despite major
criticisms by Guba (1955, 1956). Guba (1955) disagreedwith Steyeart (1949) and added his objections to Steye-
art�s treatment with substantial reasons and argued thatall of the new generic designations proposed by Steyeart
(1949) are related to synonymy. He further explained
that the presence of a single apical appendage in
Monochaetia species clearly sets them apart from Pest-
alotia and that Monochaetia merits generic status.
Steyeart (1956) pointed out that it was unwise to givegeneric status to Monochaetia based only on one mor-
phological character (i.e., the presence of a single apical
appendage), especially considering that Pestalotia, Pes-
talotiopsis, and Truncatella, which differ from each other
by a set of characters, were not given generic status.
However Guba (1961) and Steyeart (1949) did not
consider other genera such as Bartalinia, Discosia, and
Seimatosporium, which have close affinities to Pestal-
otiopsis.
Arx (1981) treated Bartalinia as congeneric to Se-
imatosporium but such a synonymy was not accepted by
Nag Rag (1993) who, on morphological grounds, ar-
gued that these two genera are distinct anamorphic
genera. The taxonomic history and complexity of Pes-
talotiopsis and its allies have been debated for over half a
century (Arx, 1981; Guba, 1955; Roberts and Swart,1980; Steyeart, 1949; Sutton, 1969, 1980) and more re-
cently by Nag Rag (1993). All previous studies on these
genera relied heavily on morphological characters as the
main criteria for generic delimitation without taking
into consideration phylogenetic relationships. Following
Steyeart�s concept, it has been necessary to reassign allfour-septate conidia (three median cells) to Pestalotiop-
sis, a view supported by Sutton (1969, 1980), but NagRag (1993) preferred to adopt a wider generic concept of
Pestalotiopsis by including three-septate (two median
cells) forms originally assigned to Truncatella. Sutton
(1969) discussed the validity of Pestalotiopsis and its
relationships with Pestalotia, Monochaetia, and Seiri-
dium. He favored Steyeart�s treatment but expanded thegeneric concepts of Monochaetia and Seiridium to in-
clude species with single apical appendages. The pres-ence of a single apical appendage was thought to be a
hallmark for Monochaetia and Seiridium, but the latter
merited generic status as the conidia are distoseptate.
whereas those of Monochaetia are euseptate (Nag Rag,
1993; Sutton, 1969). It has been recently shown that
conidium wall structure inMonochaetia and Seiridium is
different, the latter having thick-walled median cells,
whereas the former possesses a less elaborate structurewith thinner-walled and lighter-colored median cells
(Roberts and Swart, 1980).
Griffiths and Swart (1974a,b) investigated the devel-
opment of conidia in two species of Pestalotiopsis and in
P. pezizoides by means of electron microscopy in an
attempt to establish the affinities of these genera with
other members of Monochaetia and Seimatosporium.
Their results were congruent with that of Sutton (1969)that the difference in conidial wall structure with a more
elaborate zonation in P. pezizoides clearly distinguishes
it from Pestalotiopsis. Nag Rag (1993) suggested that
species accepted by Guba (1961) in Monochaetia and
Pestalotia section Sexloculatae may well belong to Se-
iridium and that a large number of taxa which properly
belong in Pestalotiopsis still remain in Pestalotia.
An important step toward a more natural classifica-tion of these fungi was taken by Sutton (1980) where
conidiogenesis was given more weighting as a taxonomic
character. However, all these genera exhibit similar ap-
pendage morphogenesis (Nag Rag, 1993) and conidio-
genesis (Sutton, 1961, 1980). Other developmental
studies including conidium ontogeny in Pestalotiopsis
neglecta (Jones, 1977) and conidiomatal development in
Pestalotiopsis (Watanabe et al., 1998) and Bartalinia
(Roux and Warmelo, 1990) have so far failed to eluci-
date relationships among these genera. Recently, Mor-
gan et al. (1998) explored the ability of artificial neural
networks to identify Monochaetia, Pestalotiopsis, and
Truncatella, but the results were not convincing enough
to assess the validity of these genera. Despite the fact
that these genera are currently being treated as distinct
taxa, the wide generic concept proposed by Guba (1961)has never been tested on other grounds except conidial
morphology.
Given the considerable taxonomic confusion of these
genera, this study was undertaken to address several
questions: (i) Do these genera represent natural groups?;
(ii) Which morphological characters are phylogeneti-
cally significant and are therefore useful for generic de-
R. Jeewon et al. / Molecular Phylogenetics and Evolution 25 (2002) 378–392 379
lineation?; and (iii) Are phylogenies based on molecularcharacters concordant with traditional morphology-
based classification schemes?
2. Materials and methods
2.1. Cultures and DNA extraction
Our taxonomic sampling included a total of 32 spe-
cies representing seven genera. Species names, accession
numbers, and geographical origin of the isolates are
listed in Table 1. For each isolate, single-spore cultures
were plated on potato dextrose agar and incubated at
25 �C for 10–20 days. Spores were examined under the
microscope to verify the authenticity of the fungi under
investigation. Some isolates were given different treat-ments to induce sporulation. Living cultures have been
deposited at The University of Hong Kong Culture
Collection. Genomic nucleic acids were extracted from
fresh fungal mycelia following a modified protocol of
Doyle and Doyle (1987). Briefly, mycelia were scraped
off from the surface of the plate, and ground with
200mg of sterilized quartz sand and 600 ll of 2� CTAB
extraction buffer (2% w/v CTAB, 100mM Tris–HCl,1.4M NaCl, 20mM EDTA, pH 8) in a 1.5-ml Eppen-
dorf tube. The whole contents were incubated at 60 �C ina water bath for 40min with occasional swirling. The
solution was then extracted two or three times with an
equal volume of phenol:chloroform (1:1) at 14,000g for
30min until no interface was visible. The upper aqueous
phase containing the DNA was precipitated by addition
of 2.5 volumes of absolute ethanol and kept at )20 �Covernight. The precipitated DNA was then washed with
70% ethanol, dried under vacuum, suspended in TE
buffer (1mM EDTA, 10mM Tris–HCl, pH 8), and
treated with RNase (1mg ml�1).
2.2. Amplification of genomic DNA
A fragment of DNA, spanning approximately 900 bpof the 50 end of the 28S ribosomal gene, was symmetricallyamplified using one pair of primers LROR (50-ACCCGCTGAACTTAAGC-30; Vilgalys and Hester, 1990)and LR5 (50-TCCTGAGGGAAACTTCG-30; Vilgalysand Hester, 1990). Amplification was performed using 2–
3 ll of genomic DNA in a standard 50-ll PCR mixture
(25mM MgCl2, 10� Mg-free buffer, 2.5 lM dNTPs,
1.5 lM primers, and 1 unit of Taq Polymerase) under thefollowing thermal conditions: 94 �C for 3min, 94 �C for50 s, 30 cycles of 94 �C for 50 s, 50 �C for 1min, and 72 �Cfor 1.5min, with a final extension step of 72 �C for 10min.Amplified products were visualized on 1% agarose gel
electrophoresis (stained with ethidium bromide) under
UV light to check for size and purity. Negative control
reactions omitting DNA were included in all sets of am-
plifications to monitor for potential contamination byexogenous DNA. PCR products were purified using mi-
nicolumns, purification resin, and buffer according to the
manufacturer�s protocol (Wizard PCR Preps DNA Pu-
rification System).
2.3. DNA sequencing and alignment
Both strands of the purified products were directly se-quenced in an automated sequencer (ALF Express,
Pharmacia-Biotech, Piscataway, NJ) following the man-
ufacturer�s protocol. Four sequencing primers were used:LROR (50-ACCCGCTGAACTTAAGC-30; Vilgalys andHester, 1990) and LR3R (50-GTCTTGAAACACGGACC30: Vilgalys and Hester, 1990) for sequences read-ing in the 50–30 direction and LR5 (50-TCCTGAGGGAAACTTCG: Vilgalys and Hester, 1990) and LR3 (50-CCGTGTTTCAAGACGGG: Vilgalys and Hester, 1990)
for sequences reading in the 30–50 direction. In addition tothe ingroup used, Pleospora herbarum var herbarum and
Dothidea sambuci were also sequenced and used as the
outgroup taxa to test potential monophyly and root the
cladograms. Reference sequences from different fungal
orders obtained from GenBank were also included in the
analysis. Sequences from each strain were assembled us-ing ALF software and SeqPup (Gilbert, 1996) to obtain
the entire sequence flanked by primers LR5 and LROR.
Initial alignments of the partial 28S rDNA from different
isolates were performed using the multiple alignment
program Clustal X (Thomson et al., 1997). Alignments
were checked and then manually edited where necessary.
2.4. Phylogenetic analysis
Maximum-parsimony (MP). Phylogenetic analyses
were performed using PAUP* 4.0b8 (Swofford, 2001).
Parsimony trees were obtained using heuristic searches
only because of the large data set. To increase the
probability of finding all most parsimonious trees,
searches were implemented using the random sequence
addition option and the tree bisection–reconnection(TBR) branch-swapping algorithm. Each search was
repeated 10 times from different random starting points
using the stepwise addition option. Single-position gaps
were treated as missing data. For phylogenetic analysis
each homologous sequence position was treated as a
discrete character with four possible unordered states
(A, G, C, or T), and equally weighted parsimony (with a
transition:transversion ratio of 1:1) was included in theparsimony analysis. A series of minor analyses under
different conditions (different transition:transversion
ratios and treating gaps as missing or fifth state) was
carried out to test the phylogenetic relationships among
the taxa and to determine the most reliable parameters
giving the best trees for subsequent analyses. Branch
support of the trees resulting from maximum-parsimony
380 R. Jeewon et al. / Molecular Phylogenetics and Evolution 25 (2002) 378–392
was assessed by bootstrapping (Felsenstein, 1985; San-
derson, 1989). The bootstrap analysis was performed
with 1000 replications using the heuristic search option
as described above to estimate the reliability of inferred
monophyletic groups. Consistency index (CI), retentionindex (RI), rescaled consistency index (RC), and ho-
moplasy index (HI) were calculated for all parsimony
trees.
In addition to MP with changes among character
states having unequal weights, alternative using analyses
were conducted based on the maximum-likelihood and
distance methods using PAUP*.
Maximum-likelihood (ML). For ML analyses, we
estimated parameters and trees using an iterative ap-
proach. A strict consensus tree was selected from an
earlier MP analysis as the starting tree for the maxi-
mum-likelihood analyses. A tree generated under thedistance criterion was also used as a starting tree for
maximum-likelihood. Two models of nucleotide substi-
tution, the HKY (Hasegawa et al., 1985) and F84
(Felsenstein, 1984) were used. The gamma model of site
rate variation was used with no enforcement of a mo-
lecular clock. Transition:transversion ratios and base
frequencies were estimated and initial branch lengths
Table 1
Fungal strains used in the study and their accession numbers, hosts, and locality
Taxon Source of culturesa Host/geographic origin GenBank Accession No. (LSU/ITS)
Ingroup
Bartalinia robillardoidesc BRIP 14180 Macrotyloma daltonii, Australia AF382366/AF405301
Bartalinia biscofiae HKUCC 6534 Unidentified dead leaf, Hong Kong AF382367
Bartalinia lateripes HKUCC 6654 Unidentified dead leaf, Hong Kong AF382368
Bartalinia laurinac HKUCC 6537 Unidentified dead leaf, Hong Kong AF382369/AF405302
Discosia sp.c HKUCC 6626 Unidentified dead leaf, Hong Kong AF382381/AF405303
Discostroma sp. HKUCC 1004 Unidentified terrestrial wood, Hong Kong AF382380
Lepteutypa cupressi IMI 052255 Cupressus forbesii, Kenya AF382379
Monochaetia monochaeta CBS 199.82 Quercus pubescens, Italy AF382370
Monochaetia karsteniic ICMP 10669 Camellia sp., New Zealand AF382371/AF405300
Pestalotia photiniae ICMP 10737 Photinia sp., New Zealand AF382363
Pestalotia sp. 1 ICMP 3062 Prunus domestica, New Zealand AF382364
Pestalotia vaccinii ICMP 5446 Vaccinium sp., New Zealand AF382362
Pestalotia sp. 2 ICMP 5476 Acca sellowiana, New Zealand AF382365
Pestalotia palmarumc ATCC 10085 Coconut Palm, India AF382361/AF009818
Pestalotiopsis maculansc CBS 322.76 Camellia sp., France AF382354/AF405296
Pestalotiopsis sp.c HKUCC 7982 Protea neriifolia, S. Africa AF382355/AF405297
Pestalotiopsis bilicia HKUCC 7983 Leucospermum sp., S. Africa AF382356
Pestalotiopsis versicolorc BRIP 14534 Psidium guajava, Australia AF382357/AF405298
Pestalotiopsis funeraec ICMP 7314 Cupressocyparis leylandii, New Zealand AF382358/AF405299
Pestalotiopsis sp. EN 8c HKUCC 7984 Scaevola hainanensis, Hong Kong AF382359/AF405294
Pestalotiopsis sp. EN 10c HKUCC 7985 Scaevola hainanensis, Hong Kong AF382360/AF405295
Seimalosporium grevillaec ICMP 10981 Protea sp., S. Africa AF382372/AF405304
Seimatosporium leptospermi ICMP 11845 Leptospermum scoparium, New Zealand AF382373
Seimatosporium vaccinii ICMP 7003 Vaccinium ashei Reade, New Zealand AF382374
Seimatosporium sp. HKUCC 7986 Leucospermum sp., S. Africa AF382375
Seiridium cardinale 1 CBS 172.56 NAb AF382376
Seiridium cardinale 2c ICMP 7323 Cupressocyparis leylandii, New Zealand AF382377/AF405305
Seiridium cupressi FABI, CMW 5596 Cupressus sempervirens, S. Africa AF382378
Truncatella angustatac ICMP 7062 Malus X domestica, New Zealand AF382383/AF405306
Truncatella conorum piceae ICMP 11213 Cedrus deodara, New Zealand AF382384
Truncatella laurocerasi ICMP 11214 Prunus persica, New Zealand AF382385
Truncatella sp. HKUCC 7987 Leucospermum sp., S. Africa AF382382
Outgroup
Xylaria hypoxylonc ATCC 42768 NA U47841/AF201711
Diaporthe phaseolorum NA U47830
Hypocrea schweinitzii NA U47833
Ophiostoma piliferum NA U47837
Pleospora herbarum var
herbarumc
CBS 191.86 Medicago saliva, India AF382386/AB026165
Dothidea sambuci CBS 198.58 Acer pseudoplatanus, Switzerland AF382387
aATCC, American Type Culture Collection; BRIP, Queensland Department of Primary Industries Plant Pathology Herbarium; CBS, Centra-
albureau voor Schimmelcultures; FABI, Forestry and Agricultural Biotechnology Institute; HKUCC, The University of Hong Kong Culture
Collection; ICMP, International Collection of Microorganisms from Plants.bNA, information not available.c Strains used in combined ITS/LSU analysis.
R. Jeewon et al. / Molecular Phylogenetics and Evolution 25 (2002) 378–392 381
were obtained using Rogers–Swofford approximationmethods. Using these initial estimates of substitution
rates and kinds a heuristic search with TBR branch
swapping was used to find a maximum-likelihood tree.
Neighbor-joining (NJ). In addition to MP and ML,
phenetic trees were constructed from distance matrix
values by the neighbor-joining method (Saitou and Nei,
1987) to reflect DNA sequence similarity. Based on the
assumptions about rates and nucleotide substitutionmodels, NJ trees were constructed under a variety of
distance measures including HKY (Hasegawa et al.,
1985), K2P (Kimura, 1980), JC (Jukes and Cantor,
1969), LogDet (Lockhart et al., 1994), and GTR (Lan-
ave et al., 1984; Rodriquez et al., 1990). One-thousand
neighbor-joining bootstrap replicates were performed
on the 28S data set. Gaps were treated as missing data
and all characters were given equal weight.Phylogenetic analysis and evaluating congruence of the
combined data set. Phylogenetic analyses (MP and ML)
based on a combined data set of the complete ITS 1,
5.8S, ITS 2, and partial LSU sequences were also con-
ducted. The combined data set consisted of 1575 bases
for 14 Pestalotiopsis isolates with Xylaria hypoxylon and
Pleospora herbarum as the outgroups. To find as many
equally parsimonious trees as possible, we used 10 rep-licated heuristic searches with random addition of taxa,
and support for clade stability was estimated using
nonparametric bootstrapping with 1000 replicates. The
partition homogeneity test (Cunningham, 1997; Farris
et al., 1995), as implemented in PAUP*, was used to
examine data for conflicting hierarchic signals and to
evaluate congruence of the combined data set. Most
parsimonious trees, using the same parameters as de-scribed above for MP analyses, were used to construct
trees for the combined data set. ML and NJ trees were
also generated by the same procedure as described
above and compared with MP trees.
Estimation of topological differences between trees.
Kishino–Hasegawa tests (Kishino and Hasegawa, 1989)
and Templeton tests (Templeton, 1983), as implemented
in PAUP*, were performed to determine whether thetrees inferred from the different tree-building methods
were significantly different. Trees were viewed in Tree-
view (Page, 1996). The nucleotide sequences reported in
this paper have been deposited in GenBank.
3. Results
3.1. Phylogenetic analyses of the large subunit (LSU)
data set
Maximum-parsimony (MP): The final LSU rDNA
alignment included 885 characters of which 178 (20.1%)
were parsimony-informative sites. The maximum-parsi-
mony analysis of the LSU data set with alignment gaps
treated as missing data with no differential weighting oftransitions against tranversions, and with random-addi-
tion sequence and TBR branch swapping yielded 10 most
parsimonious trees of tree length (TL) 573 steps with CI,
RI, RC, and HI of 0.682, 0.792, 0.541, and 0.318, re-
spectively. The strict consensus of the equally most par-
simonious tree is shown in Fig. 1. When a weighted
parsimony (transition:transversion ratios of 2:1 and 1:5)
was applied to the same data set, the tree topologywas thesame but with longer tree lengths of 810 and 619.5 steps,
respectively. Gaps treated as a fifth character with
weighted or unweighted parsimony had no effect on tree
topology (data not shown). Separate heuristic searches
implementing different addition sequences and swapping
strategies gave identical results. Bootstrapping with 1000
replicates provided good support for all the terminal
clades, and all the branches received more than 60%bootstrap support except for the node comprising
Monochaetia monochaeta (51%) (Fig. 1). All the genera
form distinct monophyletic clades with high bootstrap
values ð> 90%Þ except Truncatella, which appears to beparaphyletic. Pestalotiopsis and Pestalotia together form
a putative monophyletic group supported by a 94%
bootstrap value (Fig. 1).
To assess tree outputs, we divided the ingroup taxa inthe cladogram into seven clades (A–G). Generally, each
genus clustered separately from the rest except for Pest-
alotia and Discosia. Pestalotia clustered together and in-
termingled with Pestalotiopsis in one clade (clade A) with
a high bootstrap support (94%). Clade A can also be
further subdivided into two subclades, the top one ap-
pearing in 89% of the bootstrap replicates, whereas the
second appeared in 71%.M. monochaeta (Clade B) formsamonophyletic clade as the sister group to the preceeding
taxa of Pestalotiospsis but the confidence for the support
is quite low (51% bootstrap value). Five of the seven
clades have highbootstrap values, ð> 93%) but two clades
(B and G) have significantly lower bootstrap values (51%
and 88%, respectively). Clades C and D, also monophy-
letic, include Seiridium isolates together with Lepteutypa
cupressi (appears monophyletic in 93% of the 1000bootstrap replicates) and Bartalinia isolates (appears
monophyletic in 99% of the 1000 bootstrap replicates),
respectively. Conversely, clade E, which comprises only
Truncatella species, appears to be paraphyletic with re-
spect to the other ingroup. Interestingly, Truncatella
species appear to be very closely related to Bartalinia
species and this is supportedby abootstrap value of 100%.
Clade F contains Seimatosporium species with Discost-
roma and forms a distinct monophyletic group with a
bootstrap value of 97%. Sequential deletion of reference
taxa or use of different taxa as reference taxawas observed
to have no effect on the topology of the different phylog-
enies. By removing the loculoascomycetes (P. herbarum
var herbarum and D. sambuci) and members from the
other different orders (Diaporthe phaesolorum, Hypocrea
382 R. Jeewon et al. / Molecular Phylogenetics and Evolution 25 (2002) 378–392
schweinitzii, and Ophiostoma piliferum) except X. hypox-
ylon, it was found that the data set produced the same
topologies.
Maximum-likelihood (ML). Based on the likelihood
analyses under the HKY model with an estimated shape
parameter of 0.2487 and estimated transition:transver-
Fig. 1. Strict consensus of 10 equally most parsimonious trees generated from MP analysis of partial 28S rDNA gene sequences. Numbers above the
branches indicate bootstrap values from an anaylsis with 1000 replicates. Schematic representations on the righthand side show the significant
morphological characters traditionally used to distinguish these genera. Letters A–G represent each distinct genus. Designated outgroups are
Pleospora herbarum var herbarum and Dothidea sambuci.
R. Jeewon et al. / Molecular Phylogenetics and Evolution 25 (2002) 378–392 383
sion ratio of 1.6168, a ML tree of )loglikelihood4131.5538 was obtained (Fig. 2). Estimated base fre-
quencies were as follows: A ¼ 0:228, C ¼ 0:24,G ¼ 0:284, and T ¼ 0:248. The ML tree derived from
the HKY and F84 (Felsenstein, 1984) models were
identical and perfectly matched the MP tree topology.
Under the F84 model of nucleotide substitution, the
topology of the tree with respect to the ingroup was thesame except that X. hypoxylon did not form a separate
clade; instead, it clustered together with D. phaseolorum
and H. schweinitzii (results not shown).
Neighbor-joining (NJ). The dendograms generated
were similar under different models (HKY, K2P, JC,
and GTR) and the data confirmed the monophyly of the
Fig. 2. Cladogram generated from a ML search from partial 28S rDNA sequence data with the HKY substitution model (length¼ 573; CI¼ 0.682;Ln-likelihood¼)4141.1252). Designated outgroups are Pleospora herbarum var herbarum and Dothidea sambuci. Letters A–G represent each distinctgenus.
384 R. Jeewon et al. / Molecular Phylogenetics and Evolution 25 (2002) 378–392
different genera. Minimum-evolutionary distance anal-
ysis under the HKY model resulted in a single best tree
with slightly higher bootstrap values than those of the
other models and with a )loglikelihood of 4156.9816(Fig. 3). This tree is topologically identical to those
obtained in the MP and ML analyses except that M.
monochaeta positions itself as a sister group to the Se-
iridium clade. The estimate of phylogeny based on a
rooted tree indicates high levels of confidence ð> 80%Þ
for internal branches uniting species from the different
genera. NJ analysis confirmed the monophyly for the
different genera under investigation except for Mono-
chaetia. All models used for NJ analysis suggest that the
actual taxonomic status and the phylogenetic relation-
ships of M. monochaeta with its allies are doubtful. TheNJ trees positioned M. monochaeta as a basal, inter-
mediate lineage between Seiridium and Bartalinia but
this did not receive much bootstrap support (55%).
Fig. 3. Neighbor-joining tree generated from partial 28S rDNA gene sequences under the HKY substitution model. Values above branching nodes
indicate bootstrap support obtained from bootstrap analysis with 1000 replicates. Designated outgroups are Pleospora herbarum var herbarum and
Dothidea sambuci. Letters A–G represent clades for members of each genus.
R. Jeewon et al. / Molecular Phylogenetics and Evolution 25 (2002) 378–392 385
Contradictorily, the MP tree placedM. monochaeta as asister group of Pestalotiopsis with a bootstrap value of
51%.
3.2. Phylogenetic analyses and congruence of the com-
bined data set
The partition homogeneity test resulted in a P value
of 0.02, indicating that the combined data set was con-gruent and combinable. The phylogram generated by a
weighted MP analysis (transition:transversion ratio of
1.5:1) and treating gaps as missing data helps to resolve
intergeneric relationships. Two trees with 242 parsimo-
ny-informative sites were obtained (TL ¼ 1021:25,CI ¼ 0:786, RI ¼ 0:756, RC ¼ 0:590, HI ¼ 0:214, Lnlikelihood¼)6308.6580) (Fig. 4). This combined anal-ysis of the two rDNA regions generated a well-resolvedand strongly supported phylogeny that is topologically
congruent with the other trees. The results clearly sup-
port that all the genera are monophyletic as they form
distinct groupings with high levels of confidence (Fig. 4).
Discosia clusters together with Seimatosporium as in the
MP, ML, and NJ trees. An unweighted MP analysis
treating gaps as new states yielded one most parsimo-
nious tree with similar tree topology (results not shown).The ML tree obtained by analyzing the combined data
set turned out to be congruent with the MP tree (data
not shown).
3.3. Comparison between topologies of MP, ML, and NJ
trees
Both Kishino–Hasegawa and nonparametric tests
(Templeton test) used to evaluate trees showed that the
NJ tree is significantly different from the MP and ML
trees and is therefore rejected (Table 2).
4. Discussion
Steyeart (1949) established the genera Pestalotiopsis
and Truncatella to accommodate some species previ-
ously disposed of in Pestalotia and Monochaetia. He
retained Pestalotia as monotypic and discarded Mono-
chaetia. In contrast, Guba (1961) adopted a wide generic
concept of the genus Pestalotia by synonymizing Pes-
talotiopsis and Truncatella to Pestalotia and synony-
mizing Seiridium toMonochaetia. Arx (1981) treated the
genus Bartalinia as a synonym of Seimatosporium but,
based on morphology, Nag Rag (1993) disagreed be-
cause these two anamorphic genera are quite distinct.
Whether the proposed synonym of Arx (1981) is valid
was highly contentious. The main aim of this study was
to test the morphology-based hypotheses of Steyeart(1949) and Guba (1961) using molecular data.
Based on our current data, the wide generic concept
proposed by Guba (1961) and the treatment of Bartalinia
Fig. 4. Phylogenetic tree of 16 taxa estimated from a combined data set of partial nuclear LSU rDNA, ITS, and 5.8S rDNA sequences under the
maximum-parsimony optimality criterion. Pleospora herbarum is the designated outgroup. Bootstrap values generated from 1000 replicates are
shown above the branches.
386 R. Jeewon et al. / Molecular Phylogenetics and Evolution 25 (2002) 378–392
and Truncatella as synonyms of Seimatosporium and
Pestalotia, respectively (Arx, 1981), are rejected. How-
ever, findings are partially congruent with Steyeart�sview, which is in agreement with the commonly accepted
taxonomic classification (Nag Rag, 1993; Sutton, 1961,
1980). The traditional morphological characters used to
define the genera have been shown on the cladogram
inferred from analysis of DNA sequences (Fig. 1). Spe-cial emphasis has been placed on the different morpho-
logical characters that are typical of each genus. In
essence, topologies of all trees support recognizing
Bartalinia, Pestalotiopsis, Seimatosporium, Seiridium,
and Truncatella as natural groups. While our data do
not reject Discosia and Monochaetia as natural and
distinct genera, further studies are required to support
this.
4.1. Monophyly of Pestalotiopsis and Pestalotia
Our current results show that species of Pestalotiopsis
and Pestalotia under investigation are closely related as
they cluster together in a single monophyletic clade with
high bootstrap support. All these species are character-
ized by fusiform or slightly curved conidia bearing four-to five-euseptate median cells that are pigmented, with
apical appendages that arise as tubular extensions from
the apical cell, and a centric basal appendage arising
endogenously from the basal cell. The Pestalotia species
examined differ slightly in morphology from those of
Pestalotiopsis in that the spores possess median cells that
have slightly thicker walls and are doliform in shape,
mostly verrucose in ornamentation, and guttulate. Theyalso have a slower growth rate on synthetic media as
compared to Pestalotiopsis species. Most probably, the
fact that these median cells have thicker walls and ap-
pear somehow distoseptate might have confused some
mycologists during identification. As indicated in our
results and previous morphological hypotheses (Nag
Rag, 1993; Sutton, 1969, 1980) these species should be
Pestalotiopsis species as the median cells are not disto-septate as in Pestalotia. It is also clear that Pestalotia
species not having distoseptate conidia should be
transferred to Pestalotiopsis, as proposed by Steyeart
(1949), unless they possess other distinctive featurescharacteristics of other genera.
The taxonomic position of Pestalotiopsis has been
controversial (Guba, 1961; Steyeart, 1949). Steyeart
(1949) proposed that all Pestalotia species should be
transferred to Pestalotiopsis and that Pestalotia should
be monotypic with P. pezizoides, while Guba (1961)
advocated that Pestalotiopsis should be reduced to
synonymy. The molecular data are concordant withSteyeart�s treatment as all the Pestalotia species se-
quenced clustered together with Pestalotiopsis species.
P. pezizoides could not be included in our study as no
culture was available. Various attempts were made to
extract and amplify DNA from the dried specimens but
all our attempts were unsuccessful and yielded highly
damaged genomic DNA probably because the dried
material was too old. However the dried specimen wasexamined microscopically and it was found that it differs
from Pestalotiopsis in that it has got four-distoseptate
median cells with large lumens. Therefore it would not
be surprising that all Pestalotia species except P. pezi-
zoides properly belong to Pestalotiopsis.
One oddity in this monophyletic group is the clus-
tering ofMonochaetia karstenii within the Pestalotiopsis
clade. This culture was obtained from ICMP and mi-croscopic examination of the spores from the culture
revealed that this species actually produced spores with
a single apical appendage (characteristic of the genus
Monochaetia). Based on morphological similarities such
as apical appendage arising as a tubular extension from
the apical cell and median cells having thin walls, M.
karstenii was synonymized with Pestalotiopsis karstenii
by Nag Rag (1988, 1993). Based on molecular evidenceprovided here, we support this synonymy.
4.2. Monophyly of Seiridium
The presence of four-distoseptate median cells that
are dark brown and that of a single attenuated apical
appendage are believed to characterize species of Seiri-
dium. Guba (1961), Shoemaker et al. (1966), and Sutton(1969, 1975) have discussed the generic synonymy of
Seiridium. While Guba (1961) synonymized Seiridium
with Monochaetia by transferring all the Seiridium spe-
cies to the section Sexloculatae of Monochaetia, such
conservative treatment was not accepted by other
workers (Nag Rag, 1993; Roberts and Swart, 1980;
Sutton, 1969, 1980). The Seiridium isolates sequenced
form a monophyletic group in all our analyses. None ofthe trees obtained from our analyses support that Seir-
idium should be synonymized with Monochaetia. Even
though the bootstrap support for this relationship ap-
pears to be quite low, they are clearly distinct genera.
Therefore Guba�s synonymy of Seiridium is not vali-
dated. The presence of a single appendage is not a un-
ique feature characterizing Seiridium as a monophyletic
Table 2
Results of the Kishino–Hasegawa and templeton tests for the trees
generated by different optimality criteria
MP tree
(Fig. 1)
ML tree
(Fig. 2)
NJ tree
(Fig. 3)
Tree length (steps) 573 573 588
Consistency index 0.682 0.682 0.665
)Ln Likelihood 4141.2883 4131.5538 4156.9816
Kishino–Hasegawa test Pa¼ 0.1574 Best Pa¼ 0.0006Templeton test Pa¼ 0.5 Best Pa¼ 0.0005a Probability of getting a more extreme, t value under the null hy-
pothesis of no difference between the two trees (two-tailed test) with
significance at P < 0:05.
R. Jeewon et al. / Molecular Phylogenetics and Evolution 25 (2002) 378–392 387
group, as this character is also found inM. monochaeta,M. karstenii, and Seimatosporium species. Based on
morphological observations of the teleomorphs, Samu-
els et al. (1987) suggested a close affinity between Seir-
idium and Pestalotiopsis. This close relationship is
confirmed by our analyses as Seiridium forms the sister
group to Pestalotiopsis. The distoseptate feature of the
spore is, however, of particular phylogenetic relevance
in generic delimitation. It is noteworthy to point outhere that P. pezizoides is also characterized by disto-
septate conidia. Its affinities to Seiridium, however, are
yet to be resolved.
4.3. Monophyly of Bartalinia and Seimatosporium
Molecular results obtained here offer robust evidence
to support the distinct monophyletic status of Bartaliniaand Seimatosporium. There have been debates whether
to give Bartalinia generic status or treat it as congeneric
with Seimatosporium (Arx, 1981; Morgan-Jones et al.,
1972; Nag Rag, 1993) because Bartalinia species are
similar to Pestalotiopsis and Seimatosporium in having
euseptate median cells, two or three apical appendages,
and either exogenous or excentric basal appendages.
DNA analyses herein place the genus Bartalinia as amonophyletic group, distinct from Seimatosporium. This
finding does not corroborate the synonymy of Bartalinia
to Seimatosporium as proposed by Arx (1981), but is in
agreement with Nag Rag�s (1993) concept that Bartali-nia and Seimatosporium are distinct anamorphic genera.
Even though species from both genera possess basal
appendages that are excentric, this character is probably
evolutionarily convergent. Bartalinia is different fromSeimatosporium in that median cells are almost hyaline
or very pale brown (not pigmented in other genera),
with apical appendages arising from a particular locus
above the apical cell (not separated by a septum). Se-
imatosporium, on the other hand, is characterized by
having pigmented median cells, a mixture of appen-
daged and nonappendaged conidia with single apical
appendages, and excentric basal appendages.At the species level, Bartalinia laurina and B. biscofiae
cluster together in all trees as both of these species have
two hyaline median cells, whereas B. robillardoides and
B. lateripes have three hyaline median cells. This sug-
gests that the number of median cells may be phyloge-
netically important and a diagnostic character for the
segregation of species within Bartalinia but not for ge-
neric circumscription.
4.4. Paraphyly of Truncatella
Current results indicate that the genus Truncatella is
paraphyletic with Bartalinia sharing a common ances-
tor. Truncatella is characterized by having spores with
two brown or dark brown concolorous median cells
with thick walls and mostly irregularly branched apicalappendages. Although the results suggest that the
Truncatella species sampled are not monophyletic, they
do not support the previous morphological hypothesis
proposed by Guba (1961) that Truncatella is congeneric
with Pestalotiopsis.
Truncatella species appear to be very closely related
to Bartalinia species and this is inconsistent with the
morphology based hypotheses that Truncatella is relatedto Pestalotiopsis (Guba, 1961). The treatment proposed
by Arx (1981), that Bartalinia and Pestalotia should be
considered a synonym of Truncatella, is also refuted
here, as these two genera are quite distinct (based on our
study and on morphological characters; Nag Rag,
1993). Bartalinia is characterized by having only hyaline
median cells with axial apical appendages, which branch
once, whereas median cells are pigmented in Truncatellaand apical appendages arise usually irregularly from an
apical crest.
Our molecular results are also informative for clari-
fying relationships at the species level. Truncatella sp.
and T. conorum piceae clustered away from T. angustata
(type species) and T. laucocerasi. Truncatella sp. and T.
conorum piceae also possess two median cells but differ
from the other two species in terms of appendagecharacters. T. angustata and T. laucocerasi are charac-
terized by the presence of one or more than one irreg-
ularly and dichotomously branched apical appendages
at the apical cell with no basal appendages. Truncatella
sp. and T. conorum piceae possess only two unbranched
those similar to apical appendages found in Pestaloti-
opsis species. In addition, they possess basal append-
ages, which are absent in the other two species ofTruncatella studied. These unusual features separate
them from T. angustata and T. laucocerasi and are useful
in species delineation.
Another important conclusion from our molecular
analysis is that the wide generic concept of Pestalotiopsis
proposed by Nag Rag (1993) to accommodate species
with two and three median cells might be purely artificial.
Results herein do not support this hypothesis becauseTruncatella sp. and T. conorum piceae did not cluster with
Pestalotiopsis. However, our molecular study provides
good support for the classification established by Steyeart
(1949)who erected the genusTruncatella to accommodate
species with two median cells.
Interestingly, Truncatella sp. was originally classified
as a Seimatosporium species because of the presence of
excentric and eccentric basal appendages of the spore.However, our molecular study reveals that this species
should be a Truncatella species because the spores had
consistently two median cells with crenulated edges. The
presence of two median cells is most phylogenetically
significant at this taxonomic level and could be used to
distinguish between Truncatella species and other gen-
era.
388 R. Jeewon et al. / Molecular Phylogenetics and Evolution 25 (2002) 378–392
4.5. Phylogenetic status of Discosia
The grouping of Discosia as a sister taxon to Seima-
tosporium indicates close phylogenetic affinity but,
whether Discosia needs to be synonymized to Seima-
tosporium is uncertain. A larger sample size is required.
The single species used clustered separately from all of
the Seimatosporium species in ML and MP analyses and
this relationship is supported by a high bootstrap con-fidence. Nag Rag (1993) considered the genus Seima-
tosporium distinct from Discosia as it contains a mixture
of appendaged and nonappendaged conidia with ex-
centric basal appendages (not arising from the center)
and pigmented median cells. In contrast, Discosia is
characterized by basal appendages that are inserted at
the end cells on the concave side of the conidia and
median cells that are almost hyaline. Analyses of thecombined LSU and ITS data provide further evidence to
justify that Discosia is closely related to Seimatosporium.
Morphologically, there are some resemblances between
these genera and this raises the concern whether some of
the species in Discosia need to be reexamined and
transferred to Seimatosporium. The location of the co-
nidial septa and a rounded apical cell is quite a char-
acteristic morphological feature with a considerabletaxonomic value in delimiting these two genera (Vanev,
1991) but it would be premature to discuss a specific
taxonomic relationship given that only one species of
Discosia was included in this study. We are left with the
need to assess the phylogenetic relationships of Discosia
with Seimatosporium and their morphological synapor-
morphies as data available at present appear equivocal.
4.6. Phylogenetic status of Monochaetia
This genus shares similar characters with Pestaloti-
opsis except that the species possess only one apical
appendage arising from the apical cell. Results obtained
are incongruent with previous morphological hypothe-
ses on two major points. First, molecular analyses based
on MP and ML produced trees that place M. mono-
chaeta as the sister taxon to Pestalotiopsis and not
within Pestalotiopsis as proposed by Steyeart (1956). He
proposed that all the Monochaetia species should be
allocated in the section Monosetulae of Pestalotiopsis
but this is not supported herein. It has been observed
that M. monochaeta is structurally very similar to Pes-
talotiopsis (Griffiths and Swart, 1974a), emphasizing the
close relationships between these two genera. Sutton(1969) advocated that conidial wall structure should be
used as an additional morphological character to sup-
port Steyeart�s ideas, but our results indicate that such acharacter is not suitable for generic differentiation be-
tween Pestalotiopsis, Monochaetia, and Seiridium. Sec-
ond, the NJ analysis did not resolve the problem by
placing M. monochaeta as a sister taxon to Seiridium
instead of Pestalotiopsis. In any case,M. monochaeta didnot fit in either the Pestalotiopsis or the Seiridium clades.
This finding concurs with the morphological evidence
obtained by Roberts and Swart (1980) who observed
that conidial wall structures of Seiridium were structur-
ally different from those of M. monochaeta. We are not
surprised by the association between Monochaetia and
Seiridium (Fig. 3). Although Monochaetia was consid-
ered to be closely related to Pestalotiopsis (Steyeart,1949) due to the euseptate nature of the three median
cells, it has only a single apical appendage, unlike those
of Pestalotiopsis which have two to four apical ap-
pendages. Guba (1961) inferred a close relationship be-
tween Seiridium and Monochaetia based on
morphology, a connection supported by the results ob-
tained from the NJ analysis. However the presence of a
single appendage is also a common phenomenon in M.
karstenii, Seimatosporium, Seiridium, and Truncatella.
Our findings indicate that presence of a single append-
age may be a character that has evolved more than once
among these genera and therefore may not be phyloge-
netically significant nor useful for generic delimitation.
Steyeart (1956) postulated that Monochaetia should not
be given generic status based solely on the presence of a
single apical appendage, as opposed to Guba (1955) whostated that Monochaetia merits generic status. In an-
other study, it was observed that the acervulus mor-
phology of another species of Monochaetia, M. lutea,
has affinities with Seiridium. There are therefore mor-
phological grounds to suspect that the position of M.
monochaeta as revealed by molecular analysis in the NJ
analysis may reflect the correct phylogeny. Our study
supports neither Steyeart�s nor Guba�s views.Placement of M. monochaeta is problematic and lar-
gely unresolved in this study because of (i) an inconsis-
tency in the grouping pattern of M. monochaeta
obtained with the different methods of analysis and (ii)
the relatively low corresponding bootstrap values. The
phylogenetic relationships of Monochaetia among its
allies need to be established in future studies. Further-
more it remains unclear whether the presence of a singleapical appendage is significant enough to give Mono-
chaetia generic status as favored by Guba (1955, 1956,
1961). Our molecular data also do not favor Steyeart�streatment (1949) that the genus Monochaetia should be
discarded and all its species reallocated to the section
Monosetulae of Pestalotiopsis.
4.7. Affinities with the amphisphaeriaceae
These anamorphic coelomycetous fungi have been
shown to have close affinities with the ascomycetous
family Amphisphaeriaceae. Some have been frequently
reported to produce their sexual states in culture (Barr,
1975, 1990; Boesewinkel, 1983; M€uuller and Shoemaker,1965; Samuels et al., 1987; Shoemaker and M€uuller, 1964,
R. Jeewon et al. / Molecular Phylogenetics and Evolution 25 (2002) 378–392 389
1965; Shoemaker and Simpson, 1981; Swart, 1973; Zhuet al., 1991). Based on morphological studies, Kang et
al. (1999) considered confining the Amphisphaericeae to
those fungi producing Pestalotiopsis-like anamorphs. In
addition, molecular studies based on the ITS region of
the rDNA have revealed that Amphisphaeria umbrina,
Discostroma tosta, Lepteutypa cupressi, Pestalosphaeria
elaeidis (telemorph of Pestalotiopsis), and Pestalotia
palmarum are closely related (Kang et al., 1998). Anadditional yet equally important aspect addressed in this
study is the anamorph–teleomorph connections of
Seridium species with L. cupressi and of Seismatospo-
rium with Discostroma. These connections have so far
been based only on cultural characters (Boesewinkel,
1983; M€uuller and Shoemaker, 1965; Okane et al., 1995;Shoemaker and M€uuller, 1964, 1965; Swart, 1973). Theclustering of Discostroma sp. with Seimatosporium vac-
inii and of L. cupressi with Seridium cupressi in all trees
obtained clearly supports the previous anamorph–tele-
omorph connections and provides unambiguous evi-
dence that these anamorphic fungi have close affinities
with the Amphisphaericeae.
5. Conclusion
The current study does not support the taxonomic
treatment of Guba (1961). Phylogenetic analyses of the
rDNA sequences is generally in agreement with the
morphological hypotheses proposed by Steyeart (1949)
and Nag Rag (1993). The partial LSU sequences to-
gether with existing morphological data have provided
valuable insights in the understanding of the naturalrelationships at the intergeneric level among these coel-
omycetous fungi. Our results provide a better phyloge-
netic interpretation of morphological characters and
their utility in determining generic delineations. Analysis
of molecular data combined with morphological data
resolves many disputes not resolved by morphology
alone. Useful characters include pigmentation, septate
nature of median cells, and position of appendages withrespect to the apical and basal cells. Pestalotiopsis is
characterized by spores having mostly four-euseptate
and pigmented median cells with two to four apical
appendages arising as tubular extensions from the apical
cell and a centric basal appendage; Seridium spores
contain five- to six-distoseptate median cells; Bartalinia
is characterized by spores having almost hyaline median
cells with apical appendages arising from a particularlocus and not separated by a septum; Truncatella have
two pigmented median cells and Seimatosporium have
two or three pigmented median cells with a single apical
appendage and basal appendages that are excentric.
Morphological characters such as number of median
cells, number and presence of apical appendages, and
presence of excentric basal appendages have presumably
undergone convergent evolution and are of limited usein delineating these genera. A taxonomic key to sum-
marize the delineating morphological characters for
these genera is provided in Fig. 5.
From our study, there is little support to give Mono-
chaetia generic rank, although this genus may putatively
be characterized by the presence of a single apical ap-
pendage. The relationships between Seimatosporium and
Fig. 5. Dichotomous key to genera based on phylogenetically significant morphological characters.
390 R. Jeewon et al. / Molecular Phylogenetics and Evolution 25 (2002) 378–392
Discosia are also equivocal. However, Discosia may ten-tatively be delineated by having almost hyaline median
cells with appendages inserted at the concave side of the
conidium. Since the efficiencies of methods in recon-
structing accurate phytogenies are poor under conditions
of sparse taxonomic sampling, the relationships between
Monochaetia andDiscosiawith respect to the other genera
remain obscure. For a proper resolution of their system-
atic positions further taxon sampling involving the addi-tion of more diverse taxa and the inclusion of other
genomic loci is necessary. Furthermore, more morpho-
logical studies are required, especially those at the ultra-
structural level. It will also be beneficial to include other
related taxa in future studies, e.g., Bleptosporium, Dip-
loceras, Doliomyces, Labridella, and Pestalotia with
distoseptate conida, Pestalotiopsis species with three-
septate median cells, Sarcostroma, Zetiasplozna, and te-leomorphic amphisphaericeous species.
Acknowledgments
The University of Hong Kong is thanked for pro-
viding a Postgraduate scholarship to the first author.
Thanks are extended to the University of Hong Kong
Research Grants Council for funding this research. We
also thank several colleagues, A. Aptroot, R. Fogel,
E.H.C. Mckenzie. R.G. Shivas, J.E. Taylor, and M.J.
Wingfield, for providing cultures and specimens for this
study. J.A. Simpson is acknowledged for his discussionand suggestions during the course of this research. R.
Dulymamode and G.J.D. Smith are thanked for helpful
advice. Rajeeta Jeewon is thanked for additional sup-
port. Heidi Kong and Helen Leung are thanked for
laboratory assistance.
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