MOLECULAR
PHYLOGENETICS
AND
EVOLUTION
Molecular Phylogenetics and Evolution 27 (2003) 372–383
www.elsevier.com/locate/ympev
Phylogenetic significance of morphological characters
in the taxonomy of Pestalotiopsis species
Rajesh Jeewon,a,* Edward C.Y. Liew,b Jack A. Simpson,c I. John Hodgkiss,d
and Kevin D. Hyded
a
School of Biological Sciences, King Henry Building, University of Portsmouth, Portsmouth PO1 2DY, UK
School of Land, Water and Crop Sciences, McMillan Building A05, The University of Sydney, NSW 2006, Australia
c
Research Division, State Forests of New South Wales, P.O. Box 100, Beecroft, NSW, Australia
Centre for Research in Fungal Diversity, Department of Ecology and Biodiversity, The University of Hong Kong, Pokfulam Rd,
Hong Kong, SAR, PeopleÕs Republic of China
b
d
Received 19 December 2001; revised 12 November 2002
Abstract
There has been considerable disagreement regarding the relationships among Pestalotiopsis species and their delimitations. A
molecular phylogenetic analysis was conducted on 32 species of Pestalotiopsis in order to evaluate the utility of morphological
characters currently used in their taxonomy. Phylogenetic relationships were inferred from nucleotide sequences in the ITS regions
and 5.8S gene of the rDNA under four optimality criteria: maximum parsimony, weighted parsimony, maximum likelihood, and
neighbor joining. Phylogenies estimated from all analyses yielded trees of essentially similar topology and revealed 3 major groups
that correspond with morphology-based classification systems. Molecular data indicated that the genus contains two distinct lineages based on pigmentation of median cells and four distinct groupings based on morphology of apical appendages. The analyses
did not support reliability of other phenotypic characters of this genus, such as spore dimensions. Characters with particular
phylogenetic significance are discussed in relation to the taxonomy of Pestalotiopsis.
Ó 2003 Elsevier Science (USA). All rights reserved.
1. Introduction
Pestalotiopsis Steyaert consists of approximately 205
described species that are easily identified by the presence of relatively fusiform conidia formed within compact acervuli (CABI Bioscience database, 2001). The
conidia are usually 5-celled, with 3 brown median cells
and hyaline end cells, and with two or more apical appendages arising from the apical cell. Pestalotiopsis
species are ubiquitous in distribution, occurring on a
wide range of substrata. Many are saprobes (Wu et al.,
1982) while others are pathogenic or endophytic on
living plant leaves and twigs (Bissett, 1982; Brown et al.,
1998; Howard and Albregs, 1973; Hyde and Fr€
ohlich,
1995; Karaca and Erper, 2001; Rivera and Wright, 2000;
Taylor et al., 2001; Tuset et al., 1999). Some of these
*
Corresponding author. Fax: 852-2517-6082.
E-mail address: rajeshjeewon@yahoo.com (R. Jeewon).
Pestalotiopsis species have gained much attention in
recent years as they have been found to produce many
important secondary metabolites (Li et al., 2001; Li and
Strobel, 2001; Ogawa et al., 1995; Pulici et al., 1997;
Strobel et al., 1996). Pestalotiopsis species are anamorphic members of the family Amphisphaeriaceae (Barr,
1975, 1990; Kang et al., 1998, 1999).
Pestalotiopsis is a complex genus and consists of
members difficult to classify at the species level. At
present, inter-specific delineation of this genus is based
on morphology of the conidia (Guba, 1961; Nag Rag,
1993), conidiogenesis (Sutton, 1980) and teleomorph
association, which has been described for only a few
species (Barr, 1975, 1990; Metz et al., 2000; Zhu et al.,
1991). Since the establishment of the genus (Steyaert,
1949), numerous taxonomic studies have been conducted in an attempt to devise a suitable classification
scheme for the different species (Guba, 1961; Nag Rag,
1993; Suto and Kobayashi, 1993; Sutton, 1980).
1055-7903/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S1055-7903(03)00010-1
R. Jeewon et al. / Molecular Phylogenetics and Evolution 27 (2003) 372–383
Steyaert (1949) recognized Pestalotiopsis as a distinct
genus, which is not congeneric to Pestalotia as proposed
by Guba (1929). This was supported by a recent molecular study based on rDNA sequences (Jeewon et al.,
2002). Steyaert (1949) divided the genus Pestalotiopsis
into different sections based on the number of apical
appendages: Monosetulatae, Bisetulatae, Trisetulatae,
and Multisetulatae for species bearing 1, 2, 3, and more
than 3 apical appendages, respectively. Each section was
further divided into subsections based on differences in
conidial shape, pigmentation of median cells, and presence or absence of spatulated apical appendages. An
alternative arrangement was proposed by Guba (1961)
who grouped Pestalotiopsis species into 3 major sections
based on differences in pigmentation of the median cells:
concolorous (for those possessing equally colored median cells), versicolorous: umber olivaceous (two upper
median cells umber and lowest median cell yellow
brown), versicolorous: fuliginous olivaceous (two upper
median cells fuliginous, usually opaque, often swollen
with a dark central band, and lowest median cell light
brown). However, the main features that he relied on
were morphometry of the conidia, and number and
characteristics of the appendages. A total number of 258
species were described in his monograph.
Pigmentation is the result of the deposition of melanin granules within the cell matrix but the origin of such
pigmentation has not been established in all species except Pestalotiopsis funerea and Pestalotiopsis triseta
(Griffiths and Swart, 1974). Griffiths and Swart (1974)
recognized that differences in pigmentation of median
cells were of some taxonomic value. This corroborated
with the results of Sutton (1961), who investigated cultural differences on different media and the relative
abundance of different spore types of Pestalotiopsis
sydowiana present in the conidial life cycle. However, in
another study carried out by Satya and Saksena (1984),
pigmentation of the median cells was shown to be unreliable for differentiating certain Pestalotiopsis species.
They observed that Pestalotiopsis glandicola and Pestalotiopsis versicolor var. polygoni produced spores of
different color intensities in culture and on different
hosts and argued that color contrast of median cells is
not a dependable character. Individual species were also
found to produce different spore shapes (claviform and
fusiform) and were thus incongruent with SteyaertÕs
system. Another difficulty in the classification of Pestalotiopsis species stems from the various degrees of
cultural variation seen within a species, for such characters as growth rate, conidial morphology and fruiting
structure charateristics. Dube and Bilgrami (1965) observed morphological variations in the shape, number
and orientation of appendages in cultures of Pestalotiopsis darjeelingensis. A similar phenomenon was also
reported by Purohit and Bilgrami (1969) who examined
more than 100 pathogenic isolates of Pestalotiopsis.
373
Hughes (1953) and Kendrick (1979) pointed out that
developmental features of conidia and conidiophores
should be given more importance in taxonomic studies.
This concept was also advocated by Sutton (1980), who
suggested that a more rationale and natural classification of coelomycetous fungi would be one based on
conidiogenesis. This approach however has been aimed
mainly at suprageneric classification and in most cases
conidiogenous structures have been very difficult to interpret (Morgan-Jones et al., 1972). Watanabe et al.
(1998) investigated the conidiomatal development of
Pestalotiopsis guepinii and Pestalotiospis neglecta, and
found that the two species possess the same type of
acervulus development, which is similar to those of
Phoma richardiae and Phyllosticta harai. Morphological
and developmental studies have been inadequate in establishing evolutionary relationships in Pestalotiopsis.
Recently, the taxonomy of this genus was reviewed
by Morgan et al. (1998) who explored the utility of artificial neural networks to identify Pestalotiopsis species.
These networks have been demonstrated to be quite
informative as they revealed that some morphological
characters are not good either individually or in combination, and that some species are not sufficiently different to warrant species designation. These studies,
however, were restricted in taxonomic sampling and did
not explicitly test phylogenetic hypotheses. The systematic relationships of Pestalotiopsis species are difficult to establish as many of them have characters that
overlap in many respects. While all species possess appendages, pigmented median cells and spores of similar
shape, the major delimiting characters at the species level have been spore and appendage sizes in a broad
range of taxa (Guba, 1961; Nag Rag, 1993; Steyaert,
1949). In addition many species have been described,
renamed and synonymized based on slight differences in
spore morphology from culture and host (Mordue,
1985, 1986; Nag Rag, 1985, 1986; Pal and Purkayastha,
1992; Venkatasubbaiah et al., 1991). The taxonomic
affinities of Pestalotiopsis species have been equivocal,
confused and hampered by differences of opinions regarding the basic criteria used in segregating species.
Molecular studies have shown that Pestalotiopsis is a
monophyletic genus (Jeewon et al., 2002) but relationships at the species level were not addressed. The purpose of the current study is to investigate phylogenetic
relationships among Pestalotiopsis species by analysis of
sequence data derived from the rDNA. The region targeted is the ITS and the 5.8S gene. The specific objectives are to elucidate how morphologically different
species are phylogenetically related; to determine whether morphological-based classification schemes are
congruent with phylogenies derived from molecular
characters; and to test the validity of morphological
characters currently used for differentiating Pestalotiopsis species.
374
R. Jeewon et al. / Molecular Phylogenetics and Evolution 27 (2003) 372–383
2. Materials and methods
2.2. DNA extraction, amplification, and sequencing
2.1. Sources of fungal strains
For each culture, mycelia were scraped from the
surface of the agar and used for DNA extraction following a modified protocol of Doyle and Doyle (1987).
Target regions of the rDNA 5.8S gene + ITS regions
were amplified symmetrically using primers ITS 4 and
ITS 5 (White et al., 1990). Taq polymerase was used in
the PCR to amplify approximately 650 base pairs with
the following thermal cycling profile: initial denaturation of the double stranded DNA for 3 min at 94 °C,
followed by 30 cycles of 1 min denaturation at 94 °C,
primer annealing at 54 °C for 50 s, 1.5 min extension at
72 °C, and a final extension for 10 min at 72 °C. A
small sample of each amplified product was size-verified by gel electrophoresis. PCR products were purified
using the Wizard Preps DNA purification system
(Promega, Madison, WI, USA). Primers ITS 2, ITS 3,
ITS 4, and ITS 5 (White et al., 1990) were used to
Thirty-two strains of Pestalotiopsis were selected
for this study on the basis of their morphological
characters. Representatives exhibiting a broad range
of varying morphological characters were included.
The sources of these cultures and specimens are listed
in Table 1. For each strain, conidia were isolated,
and single spore cultures were grown on PDA at 25 °C
for 7–10 days. Strain identity was verified by microscopically examining the fruiting bodies and spores.
The morphological characters (ornamentation and
pigmentation of the median cells as well as length
and width of the conidia and appendages) for each
strain were recorded. Species identification was based
on the keys provided by Guba (1961) and Steyaert
(1949).
Table 1
Representative strains of Pestalotiopsis used in this study, their accession numbers, hosts, and geographical origins
Species
Source of culturea
Host/geographic origin
GenBank Accession No.
Pestalotiopsis adusta
Pestalotiopsis aquatica
Pestalotiopsis bilicia
Pestalotiopsis disseminata
Pestalotiopsis funerea
Pestalotiopsis gracilis
Pestalotiopsis karstenii
Pestalotiopsis leucotho€es
Pestalotiopsis maculans
Pestalotiopsis microspora
Pestalotiopsis neglecta
Pestalotiopsis palmarum
Pestalotiopsis pauciseta
Pestalotiopsis rhododendri
Pestalotiopsis sydowiana
Pestalotiopsis theae
Pestalotiopsis uvicola
Pestalotiopsis versicolor 1
Pestalotiopsis versicolor 2
Pestalotiopsis vismiae
Pestalotiopsis sp. 1
Pestalotiopsis sp. 2
Pestalotiopsis sp. 3
Pestalotiopsis sp. 4
Pestalotiopsis sp. 5
Pestalotiopsis sp. 6
Pestalotiopsis sp. 7
Pestalotiopsis sp. EN8
Pestalotiopsis sp. 8
Pestalotiopsis sp. EN9
Pestalotiopsis sp. 9
Pestalotiopsis sp. EN12
Seiridium cardinale
ICMP 5434
HKUCC 8311
BRIP 25718
HKUCC 255
ICMP 7314
HKUCC 8320
ICMP 10669
HKUCC 8315
CBS 322.76
HKUCC 8316
HKUM 996
BRIP 25618
ICMP 11874
BRIP 25628
HKUCC 8326
HKUCC 7982
BRIP 25613
BRIP 25468
BRIP 14534
HKUCC 8328
BRIP 25446
HKUCC 8323
BRIP 25640
BRIP 25624
HKUCC 8322
BRIP 25619
HKUCC 8324
HKUCC 7984
STE-U 1755
HKUCC 8319
HKUCC 8325
HKUCC 8321
ICMP 7323
Digitalis purpurea, New Zealand
Leucospermum sp., S. Africa
Xanthorrhoea sp., Australia
Sonneratia alba, The Philippines
Cupressocyparis leylandii, New Zealand
Scaevola hainanensis, Hong Kong, China
Camellia sp., New Zealand
Telopea sp., Hawaii, USA
Camellia sp., France
Aegiceras cornilatum, Hong Kong
Calamus sp., Australia
Palm, Australia
Ulex europaeus, New Zealand
Antidesma ghaesembilla, Australia
Protea mellifera, S. Africa
Protea mellifera, S. Africa
Verticordia sp., Australia
Garcia mangostana, Australia
Psidium guajava, Australia
Leucospermum sp., Hawaii, USA
Garcia mangostana, Australia
Saccharum officinarum, Hong Kong, China
Callistemon sp., Australia
Nepenthes khasiana, Australia
Unidentified leaf, Hong Kong, China
Nepenthes truncata, The Philippines
Leucospermum sp., S. Africa
Scaevola hainanensis, Hong Kong, China
Leucospermum sp., S. Africa
Scaevola hainanensis, Hong Kong, China
Leucospermum sp., S. Africa
Scaevola hainanensis, Hong Kong, China
Cupressocyparis leylandii, New Zealand
AF409955
AF409956
AF409973
AF409976
AF405299
AF409962
AF405300
AF409969
AF405296
AF409958
AF409975
AF409990
AF409972
AF409986
AF409970
AF405297
AF409994
AF409993
AF405298
AF409977
AF409984
AF409968
AF409985
AF409989
AF409992
AF409991
AF409961
AF405294
AF409980
AF409963
AF409979
AF409994
AF409995
a
BRIP, Queensland Department of Primary Industries Plant Pathology Herbarium; CBS, Centraalbureau voor Schimmelcultures; HKUCC, The
University of Hong Kong Culture Collection; ICMP, International Collection of Microorganisms from Plants; STE-U, University of Stellenbosch
Culture Collection.
R. Jeewon et al. / Molecular Phylogenetics and Evolution 27 (2003) 372–383
sequence both strands of the DNA molecule in an
automated sequencer (ALF Express, Pharmacia-Biotech, Piscataway, NJ, USA) following the manufacturerÕs protocols. Nucleotide sequences reported in this
paper have been deposited in GenBank and are listed
in Table 1.
2.3. Sequence assembly and alignment
DNA sequences obtained for each strain from each
primer were inspected individually for quality and then
spliced together using the ALF software. Both strands
of the DNA were then assembled to produce a consensus sequence for each strain using SeqPup (Gilbert,
1996). All sequences were aligned using Clustal X with
default settings (Thomson et al., 1997). Gaps were introduced to improve alignments, which were finally
optimized manually. Trees were viewed in Treeview
(Page, 1996).
2.4. Phylogenetic analyses
Phylogenies based on the ITS and 5.8S gene data
were constructed by performing heuristic searches under
four optimality criteria: maximum parsimony (MP),
weigthed parsimony (WP), maximum likelihood (ML),
and neighbor joining (NJ). Searches were carried out
using PAUP* 4.0b8 (Swofford, 2001).
2.4.1. Maximum parsimony
MP analysis was performed using the heuristic search
option, simple, and random addition stepwise of taxa.
All characters for the datasets were coded as unordered
and branch swapping was performed using the tree bisection-reconnection (TBR) swapping algorithm. The
entire dataset was analyzed by treating gaps as missing
data as well as fifth state and with transitions–transversions weighted equally. The dataset consisted of 531
sites of which 79 were parsimony informative. Nonparametric bootstrapping (Felsenstein, 1985; Sanderson,
1989) with 1000 replications was used to assess the
confidence associated with the various clades. A maxtrees limit of 1000 trees and simple sequence addition
were used in the bootstrap analyses. The outgroup taxa
selected for rooting the trees were a sister group to
Pestalotiopsis based on previous phylogenetic analyses
(Jeewon et al., 2002). Outgroups of more distantly related genera were attempted, but these created excessive
ambiguous alignment in the highly variable ITS regions.
Consistency index (CI) and other tree indices were calculated for each consensus tree to give an indication of
the amount of homoplasy present.
2.4.2. Weighted parsimony
A weighted parsimony analysis was carried out with
transitions weighted 1.5 and 2 times over transver-
375
sions. Gaps were treated as missing data or fifth state.
Insertions/deletions (indels) were included in the analysis because in most of the analyzed sequences, indels
were short (1–3 nucleotides) except for two regions in
the ITS 1, which were 16 and 15 nucleotides, respectively. These two large indel regions were excluded
from the analysis. Support for the inferred trees topologies was evaluated using 1000 bootstrap replications, as implemented in PAUP* 4.0b8 (Swofford,
2001).
2.4.3. Maximum likelihood
Analyses were conducted under the likelihood criterion following an iterative search strategy. A single
most parsimonious tree was used as starting tree in the
ML search. The HKY and F84 models of nucleotide
substitution were used with rates assumed to follow a
gamma distribution with no enforcement of a molecular clock. Analyses were performed by firstly estimating
the transition–transversion ratios, shape parameter of
the gamma distribution and base frequencies. These
estimated parameters were used in subsequent ML
searches.
2.4.4. Neighbor joining
For distance analysis, the dataset was analyzed under
a variety of assumptions and under different nucleotide
substitution models including HKY85 (Hasegawa et al.,
1985) and K2P (Kimura, 1980). All characters were
treated as unordered and were weighted equally. Bootstrap values were obtained from 1000 replicates.
To evaluate the statistical significance of all the topologies inferred from the different optimality criteria,
the Kishino and Hasegawa (1989) and Templeton (1983)
tests as implemented in PAUP* 4.0b8 (Swofford, 2001)
were conducted. Trees were viewed in Treeview (Page,
1996).
3. Results
MP analysis with gaps treated as missing data yielded
78 equally most parsimonious trees, the strict consensus
of which was 135 steps in length (CI ¼ 0.852, RI ¼ 0.970,
)log likelihood ¼ 1506.4127). All MP analyses treating
gaps as missing data essentially yielded trees of similar
topologies and same )log likelihoods (Table 2). This
dataset could not be bootstrapped as the procedure was
computationally too demanding and had to be aborted
after 20 replicates. Cladistic analysis employing the criterion of weighted parsimony (transitions weighted 2
times over transversions, as related to the estimated
values) and treating gaps as fifth state yielded 9 trees, the
strict consensus tree of which is shown in Fig. 1
(TL ¼ 214 steps, C ¼ 0.813, RI ¼ 0.969, )log likelihood ¼ 1490.6493). The likelihoods of the 3 consensus
376
R. Jeewon et al. / Molecular Phylogenetics and Evolution 27 (2003) 372–383
Table 2
Summary of the Kishino–Hasegawa and Templeton tests on the topologies obtained from parsimony analyses with various transition–transversion
differential weightings
TT ratio
TL
CI
RI
HI
)ln L
KH/Templeton testa
Gapmode ¼ missing
1:1
1.5:1
2:1
135
159.5
184
0.852
0.862
0.870
0.970
0.972
0.974
0.148
0.138
0.130
1506.4127
1506.4127
1506.4127
P ¼ 0:3178=P ¼ 1:0
P ¼ 0:3178=P ¼ 1:0
P ¼ 0:3178=P ¼ 1:0
Gapmode ¼ newstate
1:1
1.5:1
2:1
203
222
241
0.847
0.829
0.813
0.975
0.972
0.969
0.153
0.171
0.187
1510.5714
1496.0495
1492.6901
P ¼ 0:1025=P ¼ 0:25
P ¼ 0:1575=P ¼ 0:5
Best
TT, transition–transversion; TL, tree length; CI, consistency index; RI, retention index; HI, homoplasy index; )ln L ¼ )log likelihood.
Probability of getting a more extreme T value under the null hypothesis of no difference between the two trees (two tailed test) with significance
at P < 0:05.
a
trees obtained with gaps treated as newstate and under
different transitions:transversions, however were not
significantly worse (P < 0:05, Table 2). Consensus trees
generated by both MP and WP analyses (transitions
weighted 2 times over transversions) had essentially
similar tree topologies except that one of the clades was
partially unresolved when gaps were treated as missing
data and transitions:transversions weighted equally.
The maximum likelihood analysis using either a MP
or NJ tree as starting trees and under different models of
evolution gave trees of similar topologies. The HKY
model of substitution with an estimated shape parameter yielded 2 trees, the consensus of which is shown in
Fig. 2. This tree was identical in topology with that
obtained by unweighted parsimony and treating gaps as
missing data. Two ML trees similar in topology were
obtained, which had the same )log likelihood score of
1490.6493. Tree length was 135, CI ¼ 0.852, RI ¼ 0.970,
and HI ¼ 0.148. Estimated transition:transversion ratio
was 2.0059 and base frequencies were as follows:
A ¼ 0.2446, C ¼ 0.2274, G ¼ 0.2, and T ¼ 0.3287.
A phylogram constructed under the Neighbor Joining
criterion yielded a tree, which was essentially the same as
the unweighted MP tree but less resolved (data not
shown). Bootstrap support was strong for all the branches (>65%). Tree length was 155, CI ¼ 0.742, RI ¼
0.941, HI ¼ 0.258, and )log likelihood ¼ 1543.1011.
Table 3 shows the results of the Kishino–Hasegawa
and Templeton tests between all alternative topologies
obtained by MP, WP, ML, and NJ criteria. The
ML tree was identified as the best. Results indicate
that trees produced by MP, WP (treating gaps as
newstate) and ML are not significantly different,
whereas the NJ tree is significantly worse and is
therefore rejected.
Although trees from MP and WP analyses are very
similar in topology, the WP tree generated by treating
gaps as newstate has been selected for the purpose
of inferring phylogenies among Pestalotiopsis species
because the confidence of its topology could be sta-
tistically assessed by bootstrapping and it shows relationships which are more resolved among the species
in Subclade b (Fig. 1). All clades are supported by
high bootstrap values. As shown in Figs. 1 and 2, the
partition of the genus Pestalotiopsis falls into three
distinct lineages (Clades X, Y, and Z). These three
groups of Pestalotiopsis are all monophyletic and are
supported with high bootstrap confidence of 100%.
Clade X consists of species, which possess versicolorous median cells. Clade Y and Clade Z group species that characterized by brown concolorous median
cells. Characters of conidial morphology are shown in
Fig. 1.
3.1. Pigmentation of median cells
Two clades are observed for pigmentation of versicolorous and concolorous. The versicolorous clade
(Clade X) is monophyletic, while the concolorous clades
(Clade Y and Z) are paraphyletic (Fig. 1). All phylogenetic analyses support the separation of species
possessing versicolorous median cells from species possessing concolorous median cells as recognized by Guba
(1961) and Steyaert (1949). However the grouping of
Pestalotiopsis species with versicolorous median cells
into two categories (umber olivaceous and fuliginous
olivaceous) is not supported by our data as species
possessing both types of pigmentation cluster together.
For instance, Pestalotiopsis sp. 3, characterized by umber olivaceous median cells, groups together with other
members in the same Subclade possessing fuliginous
median cells (Subclade a). Another interesting group in
the tree are the representatives of Subclade b, which are
characterized by umber olivaceous median, with the
exception of Pestalotiopsis sp. EN8, Pestalotiopsis sp. 4,
and Pestalotiopsis pauciseta, which also possess a small
number of fuliginous olivaceous median cells. Members
of Subclade b are closely related to those of Subclade a
but are distinguished by having umber olivaceous median cells with no dark septa separating the two upper
R. Jeewon et al. / Molecular Phylogenetics and Evolution 27 (2003) 372–383
377
Fig. 1. Strict consensus of 9 most parsimonious trees based on the ITS and 5.8S dataset of 33 taxa (TL ¼ 241 steps, CI ¼ 0.813, RI ¼ 0.969, and
)log likelihood ¼ 1492.6901). Tree was obtained by treating gaps as a fifth character and weighting transitions 2 times over transversions. Groups
labeled X–Z are the same as in Fig. 1 and the letters (a–g) indicate the different monophyletic subclades. Morphological characters distinguishing
each group pertaining to each clade are shown on the right of the cladogram. Numbers above the nodes represent the proportion of 1000 bootstrap
replications. The designated outgroup was Seiridium cardinale. The asterisk (*) indicates the clades which receives less than 50% bootstrap support.
378
R. Jeewon et al. / Molecular Phylogenetics and Evolution 27 (2003) 372–383
Fig. 2. Strict consensus of 2 trees generated from a maximum likelihood analysis of the ITS and 5.8S dataset (TL ¼ 135, CI ¼ 0.852, RI ¼ 0.970, and
)log likelihood ¼ 1490.6493). Letters X–Z above the branches represent the different monophyletic groups having distinct morphological characters.
The asterisk (*) indicates the clade which is more resolved in Fig. 1. Designated outgroup was Seiridium cardinale.
379
R. Jeewon et al. / Molecular Phylogenetics and Evolution 27 (2003) 372–383
Table 3
Results of the Kishino–Hasegawa (KH) and Templeton tests for the trees generated by different optimality criteria
Consistency index
)Ln likelihood
KH testc
Templeton Testc
MP treea
WP treeb (Fig. 1)
ML tree (Fig. 2)
NJ tree
0.852
1506.4127
P ¼ 0:3178
P ¼ 0:3178
0.813
1492.6901
P ¼ 1:0
P ¼ 1:0
0.852
1490.6493
Best
Best
0.742
1543.1011
P ¼ 0:0001
P ¼ 0:0001
a
MP tree treating gaps as missing data and equal weighting.
WP tree treating gaps as fifth state with transition weighted 2 times over transversions.
c
Probability of getting a more extreme T value under the null hypothesis of no difference between the two trees (two tailed test) with significance
at P < 0:05.
b
median cells. During the course of our study it was
found that P. pauciseta, P. sydowiana, and Pestalotiopsis
leucotho€es produced a small number of spores possessing umber and fuliginous olivaceous median cells as
well. Clade Y, which forms the sister group to Clade X,
is characterized by species with concolorous median
cells. The same applies to Clade Z, which is supported
by a 100% bootstrap confidence.
those possessing 3 appendages as well. In addition, we
observed that P. adusta, Pestalotiopsis microspora,
Pestalotiopsis sp. 7, and Pestalotiopsis sp. 9 possess
mostly 2 apical appendages, but some species produced an equal number of conidia with 3 apical appendages.
3.2. Appendage morphology
Clade Z forms a distinct monophyletic clade (100%
bootstrap) and the relationships within this clade are
consistent in all phylogenies inferred. This clade consists
of species characterized by brown concolorous median
cells and diverse spore sizes. Species in Group X, Y, and
those in Subclades c, d, and g (Clade Z) are characterized by conidial length greater than 20 lm whereas
species in Subclades e and f possess spore length of less
than 20 lm. The only ambiguity is P. adusta, which
possesses a conidial length of less than 20 lm but clusters with other species possessing conidial length of
greater than 20 lm.
It is also worth mentioning that species grouping
based on conidial width is supported by the sequence
data. Species having conidial width of less than 6 lm are
present in Clades Y and Z only whereas Clade X includes species characterized by a conidial width of
greater than 6 lm. However, conidial form did not
correspond to any particular grouping in the trees.
Species possessing conidia that are fusiform, fusiformelliptical, clavate, and reniform intermingle in all the
clades. Different types of ornamentation of the median
cells can be seen in different groups. Members of Clade
X are characterized by having the two upper median
cells with verruculose and verrucose ornamentation.
Species of Clade Y and Z possess median cells, which
have smooth ornamentation with the exception of species in Subclade g that possess spinose and foveate ornamentation.
Another well-defined clade includes Pestalotiopsis
theae, Pestalotiopsis sp. 5, and Pestalotiopsis sp. 6 (Clade
Y), which is supported by 100% bootstrap confidence.
Morphologically this monophyletic group contains taxa
that are characterized by brown concolorous median
cells, long fusiform conidia (greater than 25 lm in length),
apical appendages with a length ranging from 25 to 40 lm
and which is knobbed at the tip. All species in other clades
possess apical appendages that are not knobbed.
Results also show that species form distinct groupings
based on the length of the apical appendages. Within
Clade X, two Subclades are evident based on dimensions
of apical appendages. Subclade a groups species having
apical appendages of greater than 25 lm in length
whereas Subclade b groups species having apical appendages of 20–25 lm in length. There are also other welldefined Subclades in Clade Z based on the dimension of
apical appendages. Apical appendage length of 15–19 lm
can be found in Subclades c, f, and g (except Pestalotiopsis adusta which has a length of less than 15 lm)
whereas species characterized by apical appendages of
less than 15 lm can be found only in Subclades d and e.
There are, however no clades that correspond to
particular groupings based on the dimension of basal
appendages or of the presence of 2 or 3 apical appendages. Species possessing basal appendages less
than or greater than 5 lm appear to intermingle in all
clades. Such is also the case regarding the number of
apical appendages. Species of Subclades c, d, e, and f
examined in this study possess 2 as well as 3 apical
appendages. Those species possessing mostly 2 appendages did not cluster with others sharing the same
number of apical appendages; they intermingled with
3.3. Conidial size and shape
4. Discussion
Like most coelomycetes, classification of Pestalotiopsis species based on morphological characters has
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R. Jeewon et al. / Molecular Phylogenetics and Evolution 27 (2003) 372–383
been equivocal because of overlapping variation in size
and shape of homologous structures. Major emphasis
has been placed on the pigmentation of median cells and
the size of appendages. In this study, the ITS regions
and 5.8S gene of the rDNA of 32 isolates were analyzed
to elucidate relationships within Pestalotiopsis. This
molecular based cladistic analysis provides a context for
reexamining the key characters that have been used in
the classification of this genus.
4.1. Pigmentation of median cells
Pigmentation of the median cells has been considered to be of great taxonomic importance at the species level. Sequences from the ITS and 5.8S gene
analyzed in this study demonstrated strong bootstrap
support for a close phylogenetic relationship among
species possessing versicolorous median cells as well as
those species characterized by concolorous median
cells. The data are also generally concordant with
species relationships proposed by Guba (1961) and
Steyaert (1949) who classified all species producing
versicolorous median cells under one section (Versicolores) and species characterized by concolorous median cells in another section (Concolorae). However,
morphological subgroups of versicolorae as defined by
Guba (1961), who organized species possessing versicolorous median cells into 2 sections according different color intensities (umber olivaceous and fuliginous
olivaceous) is doubtful because species relationships
between the two different color intensities were not
resolved confidently.
By examining conidia of P. glandicola and P. versicolor var. polygoni on the host and in culture, Purohit
and Bilgrami (1969) and Satya and Saksena (1984)
showed that the conidia exhibited different color intensities (concolorous as well as versicolorous) on different substrates, thus demonstrating the difficulty in
using degree of pigmentation as a reliable character.
They pointed out that the color of median cells is an
unstable feature used in the systematics of Pestalotiopsis. Molecular data, however, is inconsistent with
their findings as species producing spores with concolorous median cells and versicolorous median cells
form distinct groups supported by high bootstrap values. Therefore, despite arguments in the morphological
approach over the reliability of pigmentation as a
taxonomic character, results here indicate that pigmentation is a sound diagnostic character for species
differentiation.
Current results are in agreement with Sutton (1961),
who investigated cultural differences of P. sydowania on
different media and observed that the species produced
umber as well as fuliginous median cells. Sutton (1961)
inferred that spores either: (i) undergo a natural sequence of maturation, developing from one spore type
to another, or (ii) produce morphologically distinct
spore types. The former conclusion was refuted by
Purohit and Bilgrami (1969). Based on the results obtained herein, it can be hypothesized that some species
may produce umber as well as fuliginous median cells at
different stages of maturity. Pigmentation is the result of
deposition of melanin granules within the cell matrix but
the origin of such pigmentation has not been established
except in P. funerea and P. triseta (Griffiths and Swart,
1974). Probably those species possessing versicolorous
median cells contain similar pigments but this has not
been investigated.
4.2. Appendage morphology
Species possessing apical appendages that are knobbed clearly fall into a monophyletic clade (Clade Y).
Although these species possess other characters more
commonly found in Group Z, they did not cluster in
that group as expected. This group, characterized by
concolorous median cells forms the sister group to Clade
X, which possesses versicolorous median cells. Presumably these species have lost the ability to produce versicolorous median cells and have gained the ability to
produce knobbed apical appendages. The presence of a
knobbed apical appendage seems to have evolved once
during the evolution of Pestalotiopsis species, and hence
is a phylogenetically reliable taxonomic character.
SteyaertÕs and GubaÕs system that grouped all species
possessing spathulate appendages in one section
(spathulate) is therefore valid.
The mean lengths of apical and basal appendages
have been used as important taxonomic characters to
delineate species. From the phylogenies generated here it
can be seen that species sharing similar apical appendage
length are closely related to each other (Fig. 1). Sequence data suggest the possibility that there may be
four phylogenetic groups within the current morphological concept of Pestalotiopsis. These include species
possessing apical appendage length: (i) less than
15 lm, (ii) 15–19 lm, (iii) 20–25 lm, and (iv) greater than
25 lm.
Current molecular analyses also resolved longstanding confusion as to whether species characterized
by different basal appendage dimensions are closely related. Species with basal appendages greater or less than
5 lm appear to overlap in all the clades and there is not a
clear relationship among species sharing similar length
of basal appendages. Therefore, length of the basal appendage as a taxonomic criterion to segregate species
can be misleading. On the other hand, absence of basal
appendages may be quite important for species delimitation. In this study, only 2 species (Pestalotiopsis karstenii and Pestalotiopsis maculans) are characterized by
the absence of basal appendages and they cluster together with a high bootstrap support.
R. Jeewon et al. / Molecular Phylogenetics and Evolution 27 (2003) 372–383
Based on our morphological observations and molecular data, it can also be inferred that the number of
apical appendages is not a reliable taxonomic character
to separate species within the genus Pestalotiopsis. There
is no clear separation of species possessing 2 or 3 apical
appendages. Many of them, for instance, Pestalotiopsis
disseminata, Pestalotiopsis uvicola, and Pestalotiopsis
palmarum, examined in this study possess 2 as well as 3
apical appendages. Moreover those species possessing
mostly 2 appendages (for example, P. adusta, P. microspora, Pestalotiopsis sp. 7, and Pestalotiopsis sp. 9)
are not phylogenetically closely related to other species
sharing the same number of apical appendages. Instead
they cluster according to spore and apical appendage
length. It is worth mentioning that, during microscopic
examination of spores, some species produced an equal
number of conidia with 2 and 3 apical appendages.
Results presented here corroborate the findings of Suto
and Kobayashi (1993) who pointed out that it was impossible to group species based on the number of apical
appendages. Furthermore, the bisetulae section in the
system advocated by Steyaert (1949) to accommodate
for species with 2 apical appendages is doubtful.
4.3. Conidial size and shape
Traditionally, taxonomists placed much emphasis on
length and width of the conidia (length–width ratio),
length of appendages and ornamentation of median
cells. Spore length has been used as a key character and
many new species have been named based on slight
differences in spore sizes (Mordue, 1985, 1986; Nag Rag,
1985, 1986; Pal and Purkayastha, 1992; Venkatasubbaiah et al., 1991). The results here, however, indicate that
spore size is homoplasious. Species sharing similar conidial length did not cluster together and are therefore
not closely related to each other. Similar conclusions
were made by Dube and Bilgrami (1965), whose work
indicated that conidial length is not a reliable taxonomic
character to define Pestalotiopsis species.
On the other hand, spore width can be phylogenetically informative and given high taxonomic weighting
since all species in Clade X are characterized by conidial
width of greater than 6 lm, whereas those in Clade Y
and Z possess a conidial width of less than 6 lm.
Pestalotiopsis microspora and Pestalotiopsis sp. EN12
clustered together and are closely related to P. karstenii
and P. maculans. These four species share the same conidium length (less than 20 lm) whereas members of
other clades are characterized by spores greater than
20 lm in length. Spore length is the only morphological
difference separating species in Subclades e and f from
species from other Subclades in Clade Z. Comparison of
P. microspora and Pestalotiopsis sp. EN12 ITS sequences revealed a high degree of similarity (96%) and a
98% nucleotide similarity among species in Subclade f.
381
Species of Subclade d, which share 100% sequence
similarity (Pestalotiopsis bicilia, Pestalotiopsis sp. 7, and
Pestalotiopsis vismiae) and Subclade e (P. microspora
and Pestalotiopsis sp. EN12) are phylogenetically closely
related. This is consistent with the fact that these species
produce spores that possess very short apical appendages, their lengths ranging from 8–15 lm. The principal
separation between species in Subclade d and those in
Subclade e (P. microspora and Pestalotiopsis sp. EN12)
is based on spore sizes. The latter group is characterized
by having spore lengths of less than 20 lm, whereas
those in Subclade d possess spore lengths of greater than
20 lm. Their placement as different species is supported
by the molecular data even though these species share
the same appendage length. These results generally lead
to the assumption that classification of Pestalotiopsis
species based on conidium sizes and conidium length–
width ratio as proposed by Guba (1961) and Nag Rag
(1993) might be artificial. On the basis of tremendous
variation in spore sizes and in light of the rDNA phylogeny, it can be inferred that conidium sizes can be
taxonomically useful when given lower weighting.
Molecular data does not reveal a close relationship
among species characterized by similar conidial form.
This suggests that conidial form is uninformative for
species differentiation and results cast doubt on the
grouping of Pestalotiopsis species into different groups
based on the form of the conidia as outlined by Steyaert
(1949). Presumably these species are dimorphic or polymorphic and exhibit different forms under different cultural conditions as suggested by Dube and Bilgrami
(1965). On the other hand, it appears that ornamentation
is closely related to the degree of pigmentation of median
cells. The presence of verruculose ornamentation is more
common in species possessing fuliginous median cells,
whereas smooth ornamentation is mostly present in
species characterized by concolorous median cells.
The phylogenetic affinities of P. adusta (Subclade c
remain ambiguous and are not dealt with in detail as it
groups with members of Subclade d in the NJ tree and
because of a lack of bootstrap support. The diagnostic
morphological features that separate members of
Subclade g (basal to the other species in group Z) are
obscure, as this group possesses divergent morphologies.
They possibly form a genetic species complex indistinguishable using the molecular approach here and its
phylogenetic relationships remain uncertain.
Another point of interest is the clustering of P. maculans and P. karstenii, which agrees with morphologybased systems. The type species of the genus, P. maculans
is characterized by a spore size of 15–20 lm; 1–3 apical
appendages, which are at times branched, and basal appendages that are usually absent. Similar morphologies
are shared by P. karstenii except that the latter possesses
only 1 apical appendage that usually forms 2–3 branches.
Nag Rag (1993) and Guba (1961) recognized a close re-
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R. Jeewon et al. / Molecular Phylogenetics and Evolution 27 (2003) 372–383
lationship between these two species on morphological
grounds and host association (Camellia). Guba (1961)
considered P. maculans and P. karstenii as synonyms.
Such synonymy was not accepted by Nag Rag (1993) and
Sutton (1980), who treated these as two distinct species.
When these two species were examined under the microscope, the main difference observed was the number of
appendages arising from the apical cell. Our sequencebased analyses also demonstrate that these two species
are closely related (98% sequence similarity and 90%
bootstrap support). It is possible that P. maculans and P.
karstenii are synonyms, as the characters used to support
their distinction can be interpreted as extreme reductions.
5. Conclusion
Based on rDNA evidence, pigmentation of the median cells appears to be an important phylogenetic
marker for species delineation within Pestalotiopsis as
species possessing versicolorous median cells are distinct
from those characterized by concolorous median cells. A
close relationship among pigmented median cells has
been suggested by numerous taxonomists (Guba, 1961;
Steyaert, 1949; Sutton, 1980) and this is supported
herein by molecular evidence. However, GubaÕs subsections of species possessing versicolorous median cells
(umber versicolorous and fuliginous versicolorous) are
doubtful. Appendage tip morphology and pigmentation
of median cells are phylogenetically significant and
should be given high weighting in species delineation.
Conidium length, length of basal appendages, ornamentation of median cells, and spore form appear to be
highly plastic morphologies. A word of caution is necessary when grouping species based on spore sizes. This
character has been given too much emphasis in the past
when describing new species but our results stimulate a
serious reconsideration. Nevertheless, spore length
could be useful when given lower taxonomic weighting.
Distinction of Pestalotiopsis species based on spore sizes
can be more reliable for delineating species groups rather than individual taxa. Contrarily conidial form and
ornamentation of median cells appear to have undergone convergent evolution within Pestalotiopsis and are
not definitive taxonomic characters.
Our findings contribute to the understanding of the
evolution of morphological characters. Versicolorous
median cells appear to have evolved from concolorous
median cells and spathulate apical appendages from nonspathulated apical appendages. Pigmentation and apical
appendage tip morphology appear to be synapomorphies
for Pestalotiopsis species. While not intended as a comprehensive survey of the genus Pestalotiopsis, our study is
significant in evaluating the relative importance and
utility of morphological characters in segregating Pestalotiopsis species. We propose that Pestalotiopsis species
be delineated, in order of taxonomic weighting, on the
basis of: (1) the degree of pigmentation of median cells
(either brown concolorous or versicolorous), (2) apical
appendage tip morphology (presence or absence of
spathulation), (3) apical appendage length, and (4) spore
length.
Acknowledgments
We are grateful to the following mycologists who
provided cultures and specimens: Dr. A. Aptroot, Dr.
R. Fogel, Dr. E.H.C. Mckenzie, T. Nikulin, Dr. R.G.
Shivas, Dr. J.E. Taylor, and Prof. M.J. Wingfield. This
research was funded by the Hong Kong Research
Grants Council. Dr. R. Dulymamode and G.J.D. Smith
are thanked for thoughtful reviews and support. Rajeeta
Jeewon is thanked for her continuous support during
the course of the study. Heidi Kong and Helen Leung
are thanked for laboratory assistance. This paper represents part of a dissertation submitted by the first author in partial fulfillment of a doctoral degree.
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