Phylogenetic significance of morphological characters in the taxonomy of Pestalotiopsis species

Phylogenetic significance of morphological charactersin 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, UKb 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, Australiad 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 China

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 lin-

eages 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.

� 2003 Elsevier Science (USA). All rights reserved.

1. Introduction

Pestalotiopsis Steyaert consists of approximately 205

described species that are easily identified by the pres-

ence of relatively fusiform conidia formed within com-

pact acervuli (CABI Bioscience database, 2001). Theconidia are usually 5-celled, with 3 brown median cells

and hyaline end cells, and with two or more apical ap-

pendages 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€oohlich,1995; Karaca and Erper, 2001; Rivera and Wright, 2000;

Taylor et al., 2001; Tuset et al., 1999). Some of these

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 anamor-

phic 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 con-

ducted in an attempt to devise a suitable classification

scheme for the different species (Guba, 1961; Nag Rag,

1993; Suto and Kobayashi, 1993; Sutton, 1980).

Molecular Phylogenetics and Evolution 27 (2003) 372–383

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MOLECULARPHYLOGENETICSANDEVOLUTION

* Corresponding author. Fax: 852-2517-6082.

E-mail address: [email protected] (R. Jeewon).

1055-7903/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.

doi:10.1016/S1055-7903(03)00010-1

mail to: [email protected]

Steyaert (1949) recognized Pestalotiopsis as a distinctgenus, which is not congeneric to Pestalotia as proposed

by Guba (1929). This was supported by a recent mo-

lecular 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 wasfurther divided into subsections based on differences in

conidial shape, pigmentation of median cells, and pres-

ence 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 me-

dian cells), versicolorous: umber olivaceous (two uppermedian 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 mela-

nin granules within the cell matrix but the origin of such

pigmentation has not been established in all species ex-

cept 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 cul-tural 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 un-

reliable for differentiating certain Pestalotiopsis species.

They observed that Pestalotiopsis glandicola and Pes-

talotiopsis versicolor var. polygoni produced spores ofdifferent 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 Steyaert�ssystem. Another difficulty in the classification of Pes-

talotiopsis species stems from the various degrees of

cultural variation seen within a species, for such char-acters as growth rate, conidial morphology and fruiting

structure charateristics. Dube and Bilgrami (1965) ob-

served morphological variations in the shape, number

and orientation of appendages in cultures of Pestaloti-

opsis darjeelingensis. A similar phenomenon was also

reported by Purohit and Bilgrami (1969) who examined

more than 100 pathogenic isolates of Pestalotiopsis.

Hughes (1953) and Kendrick (1979) pointed out thatdevelopmental 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 classifica-

tion 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 in-terpret (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 es-

tablishing evolutionary relationships in Pestalotiopsis.Recently, the taxonomy of this genus was reviewed

by Morgan et al. (1998) who explored the utility of ar-

tificial 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 com-

bination, and that some species are not sufficiently dif-

ferent to warrant species designation. These studies,however, were restricted in taxonomic sampling and did

not explicitly test phylogenetic hypotheses. The sys-

tematic relationships of Pestalotiopsis species are diffi-

cult to establish as many of them have characters that

overlap in many respects. While all species possess ap-

pendages, pigmented median cells and spores of similar

shape, the major delimiting characters at the species le-

vel have been spore and appendage sizes in a broadrange 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 re-garding the basic criteria used in segregating species.

Molecular studies have shown that Pestalotiopsis is a

monophyletic genus (Jeewon et al., 2002) but relation-

ships at the species level were not addressed. The pur-

pose of the current study is to investigate phylogenetic

relationships among Pestalotiopsis species by analysis of

sequence data derived from the rDNA. The region tar-

geted is the ITS and the 5.8S gene. The specific objec-tives are to elucidate how morphologically different

species are phylogenetically related; to determine whe-

ther morphological-based classification schemes are

congruent with phylogenies derived from molecular

characters; and to test the validity of morphological

characters currently used for differentiating Pestaloti-

opsis species.

R. Jeewon et al. / Molecular Phylogenetics and Evolution 27 (2003) 372–383 373

2. Materials and methods

2.1. Sources of fungal strains

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 listedin Table 1. For each strain, conidia were isolated,

and single spore cultures were grown on PDA at 25 �Cfor 7–10 days. Strain identity was verified by micro-

scopically 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 basedon the keys provided by Guba (1961) and Steyaert

(1949).

2.2. DNA extraction, amplification, and sequencing

For each culture, mycelia were scraped from the

surface of the agar and used for DNA extraction fol-

lowing 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 withthe following thermal cycling profile: initial denatur-

ation of the double stranded DNA for 3min at 94 �C,followed by 30 cycles of 1min denaturation at 94 �C,primer annealing at 54 �C for 50 s, 1.5min extension at

72 �C, and a final extension for 10min at 72 �C. A

small sample of each amplified product was size-veri-

fied 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

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 ICMP 5434 Digitalis purpurea, New Zealand AF409955

Pestalotiopsis aquatica HKUCC 8311 Leucospermum sp., S. Africa AF409956

Pestalotiopsis bilicia BRIP 25718 Xanthorrhoea sp., Australia AF409973

Pestalotiopsis disseminata HKUCC 255 Sonneratia alba, The Philippines AF409976

Pestalotiopsis funerea ICMP 7314 Cupressocyparis leylandii, New Zealand AF405299

Pestalotiopsis gracilis HKUCC 8320 Scaevola hainanensis, Hong Kong, China AF409962

Pestalotiopsis karstenii ICMP 10669 Camellia sp., New Zealand AF405300

Pestalotiopsis leucotho€ees HKUCC 8315 Telopea sp., Hawaii, USA AF409969

Pestalotiopsis maculans CBS 322.76 Camellia sp., France AF405296

Pestalotiopsis microspora HKUCC 8316 Aegiceras cornilatum, Hong Kong AF409958

Pestalotiopsis neglecta HKUM 996 Calamus sp., Australia AF409975

Pestalotiopsis palmarum BRIP 25618 Palm, Australia AF409990

Pestalotiopsis pauciseta ICMP 11874 Ulex europaeus, New Zealand AF409972

Pestalotiopsis rhododendri BRIP 25628 Antidesma ghaesembilla, Australia AF409986

Pestalotiopsis sydowiana HKUCC 8326 Protea mellifera, S. Africa AF409970

Pestalotiopsis theae HKUCC 7982 Protea mellifera, S. Africa AF405297

Pestalotiopsis uvicola BRIP 25613 Verticordia sp., Australia AF409994

Pestalotiopsis versicolor 1 BRIP 25468 Garcia mangostana, Australia AF409993

Pestalotiopsis versicolor 2 BRIP 14534 Psidium guajava, Australia AF405298

Pestalotiopsis vismiae HKUCC 8328 Leucospermum sp., Hawaii, USA AF409977

Pestalotiopsis sp. 1 BRIP 25446 Garcia mangostana, Australia AF409984

Pestalotiopsis sp. 2 HKUCC 8323 Saccharum officinarum, Hong Kong, China AF409968

Pestalotiopsis sp. 3 BRIP 25640 Callistemon sp., Australia AF409985

Pestalotiopsis sp. 4 BRIP 25624 Nepenthes khasiana, Australia AF409989

Pestalotiopsis sp. 5 HKUCC 8322 Unidentified leaf, Hong Kong, China AF409992

Pestalotiopsis sp. 6 BRIP 25619 Nepenthes truncata, The Philippines AF409991

Pestalotiopsis sp. 7 HKUCC 8324 Leucospermum sp., S. Africa AF409961

Pestalotiopsis sp. EN8 HKUCC 7984 Scaevola hainanensis, Hong Kong, China AF405294

Pestalotiopsis sp. 8 STE-U 1755 Leucospermum sp., S. Africa AF409980


Pestalotiopsis sp. 9 HKUCC 8325 Leucospermum sp., S. Africa AF409979


Seiridium cardinale ICMP 7323 Cupressocyparis leylandii, New Zealand AF409995

aBRIP, 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.

374 R. Jeewon et al. / Molecular Phylogenetics and Evolution 27 (2003) 372–383

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=nucleotide&cmd=search&term=AF409955

































sequence both strands of the DNA molecule in anautomated sequencer (ALF Express, Pharmacia-Bio-

tech, Piscataway, NJ, USA) following the manufac-

turer�s 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 consen-

sus sequence for each strain using SeqPup (Gilbert,

1996). All sequences were aligned using Clustal X with

default settings (Thomson et al., 1997). Gaps were in-

troduced to improve alignments, which were finallyoptimized 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 unorderedand branch swapping was performed using the tree bi-

section-reconnection (TBR) swapping algorithm. The

entire dataset was analyzed by treating gaps as missing

data as well as fifth state and with transitions–trans-

versions weighted equally. The dataset consisted of 531

sites of which 79 were parsimony informative. Non-

parametric bootstrapping (Felsenstein, 1985; Sanderson,

1989) with 1000 replications was used to assess theconfidence associated with the various clades. A max-

trees 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 re-

lated genera were attempted, but these created excessive

ambiguous alignment in the highly variable ITS regions.Consistency index (CI) and other tree indices were cal-

culated 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-

sions. Gaps were treated as missing data or fifth state.Insertions/deletions (indels) were included in the anal-

ysis 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, respec-

tively. These two large indel regions were excluded

from the analysis. Support for the inferred trees to-

pologies was evaluated using 1000 bootstrap replica-

tions, as implemented in PAUP* 4.0b8 (Swofford,2001).

2.4.3. Maximum likelihood

Analyses were conducted under the likelihood crite-

rion 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 agamma distribution with no enforcement of a molecu-

lar 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. Boot-

strap values were obtained from 1000 replicates.

To evaluate the statistical significance of all the to-

pologies 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 (CI¼ 0.852, RI¼ 0.970,

)log likelihood¼ 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 abortedafter 20 replicates. Cladistic analysis employing the cri-

terion 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

(TL¼ 214 steps, C¼ 0.813, RI¼ 0.969, )log likeli-hood¼ 1490.6493). The likelihoods of the 3 consensus


trees obtained with gaps treated as newstate and under

different transitions:transversions, however were not

significantly worse (P < 0:05, Table 2). Consensus treesgenerated by both MP and WP analyses (transitionsweighted 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 parame-ter 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, CI¼ 0.852, RI¼ 0.970,

and HI¼ 0.148. Estimated transition:transversion ratio

was 2.0059 and base frequencies were as follows:A¼ 0.2446, C¼ 0.2274, G¼ 0.2, and T¼ 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 bran-

ches (>65%). Tree length was 155, CI¼ 0.742, RI¼0.941, HI¼ 0.258, and )log likelihood¼ 1543.1011.

Table 3 shows the results of the Kishino–Hasegawaand 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 verysimilar 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 re-

lationships 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, thepartition 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 versicolor-

ous median cells. Clade Y and Clade Z group spe-

cies that characterized by brown concolorous median

cells. Characters of conidial morphology are shown inFig. 1.

3.1. Pigmentation of median cells

Two clades are observed for pigmentation of versi-

colorous and concolorous. The versicolorous clade

(Clade X) is monophyletic, while the concolorous clades

(Clade Y and Z) are paraphyletic (Fig. 1). All phyloge-netic analyses support the separation of species

possessing versicolorous median cells from species pos-

sessing 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 um-

ber 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 smallnumber of fuliginous olivaceous median cells. Members

of Subclade b are closely related to those of Subclade a

but are distinguished by having umber olivaceous me-

dian cells with no dark septa separating the two upper

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 )lnL KH/Templeton testa

Gapmode¼missing

1:1 135 0.852 0.970 0.148 1506.4127 P ¼ 0:3178=P ¼ 1:0

1.5:1 159.5 0.862 0.972 0.138 1506.4127 P ¼ 0:3178=P ¼ 1:0

2:1 184 0.870 0.974 0.130 1506.4127 P ¼ 0:3178=P ¼ 1:0

Gapmode¼newstate

1:1 203 0.847 0.975 0.153 1510.5714 P ¼ 0:1025=P ¼ 0:25

1.5:1 222 0.829 0.972 0.171 1496.0495 P ¼ 0:1575=P ¼ 0:5

2:1 241 0.813 0.969 0.187 1492.6901 Best

TT, transition–transversion; TL, tree length; CI, consistency index; RI, retention index; HI, homoplasy index; )lnL¼)log likelihood.a 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.


Fig. 1. Strict consensus of 9 most parsimonious trees based on the ITS and 5.8S dataset of 33 taxa (TL¼ 241 steps, CI¼ 0.813, RI¼ 0.969, and

)log likelihood¼ 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.


Fig. 2. Strict consensus of 2 trees generated from a maximum likelihood analysis of the ITS and 5.8S dataset (TL¼ 135, CI¼ 0.852, RI¼ 0.970, and

)log likelihood¼ 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.


median cells. During the course of our study it was

found that P. pauciseta, P. sydowiana, and Pestalotiopsis

leucotho€ees produced a small number of spores possess-

ing 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.

3.2. Appendage morphology

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 mediancells, long fusiform conidia (greater than 25lm in length),

apical appendages with a length ranging from 25 to 40lmandwhich 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 havingapical appendages of greater than 25lm in length

whereas Subclade b groups species having apical ap-

pendages of 20–25lm in length. There are also other well-

defined Subclades in Clade Z based on the dimension of

apical appendages. Apical appendage length of 15–19lmcan be found in Subclades c, f, and g (except Pestaloti-

opsis adusta which has a length of less than 15lm)whereas species characterized by apical appendages ofless than 15lm 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 ap-

pendages. Species possessing basal appendages less

than or greater than 5lm 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 ap-

pendages did not cluster with others sharing the same

number of apical appendages; they intermingled with

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 pro-

duced an equal number of conidia with 3 apical ap-

pendages.

3.3. Conidial size and shape

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 character-

ized by conidial length greater than 20lm whereasspecies in Subclades e and f possess spore length of less

than 20lm. The only ambiguity is P. adusta, which

possesses a conidial length of less than 20lm but clus-

ters with other species possessing conidial length of

greater than 20lm.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 6lm arepresent in Clades Y and Z only whereas Clade X in-

cludes species characterized by a conidial width of

greater than 6lm. However, conidial form did not

correspond to any particular grouping in the trees.

Species possessing conidia that are fusiform, fusiform-

elliptical, clavate, and reniform intermingle in all the

clades. Different types of ornamentation of the median

cells can be seen in different groups. Members of CladeX 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 spe-

cies in Subclade g that possess spinose and foveate or-

namentation.

4. Discussion

Like most coelomycetes, classification of Pestaloti-

opsis species based on morphological characters has

Table 3

Results of the Kishino–Hasegawa (KH) and Templeton tests for the trees generated by different optimality criteria

MP treea WP treeb (Fig. 1) ML tree (Fig. 2) NJ tree

Consistency index 0.852 0.813 0.852 0.742

)Ln likelihood 1506.4127 1492.6901 1490.6493 1543.1011

KH testc P ¼ 0:3178 P ¼ 1:0 Best P ¼ 0:0001

Templeton Testc P ¼ 0:3178 P ¼ 1:0 Best P ¼ 0:0001aMP tree treating gaps as missing data and equal weighting.bWP 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.


been equivocal because of overlapping variation in sizeand 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 consid-

ered to be of great taxonomic importance at the spe-

cies level. Sequences from the ITS and 5.8S gene

analyzed in this study demonstrated strong bootstrap

support for a close phylogenetic relationship amongspecies 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 (Versi-

colores) and species characterized by concolorous me-

dian cells in another section (Concolorae). However,morphological subgroups of versicolorae as defined by

Guba (1961), who organized species possessing versi-

colorous median cells into 2 sections according differ-

ent 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. ver-

sicolor var. polygoni on the host and in culture, Purohit

and Bilgrami (1969) and Satya and Saksena (1984)

showed that the conidia exhibited different color in-

tensities (concolorous as well as versicolorous) on dif-

ferent 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 Pestaloti-opsis. Molecular data, however, is inconsistent with

their findings as species producing spores with conco-

lorous median cells and versicolorous median cells

form distinct groups supported by high bootstrap val-

ues. Therefore, despite arguments in the morphological

approach over the reliability of pigmentation as a

taxonomic character, results here indicate that pig-

mentation is a sound diagnostic character for speciesdifferentiation.

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 se-

quence of maturation, developing from one spore type

to another, or (ii) produce morphologically distinctspore types. The former conclusion was refuted by

Purohit and Bilgrami (1969). Based on the results ob-

tained 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 knob-

bed 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. Presum-

ably these species have lost the ability to produce vers-

icolorous median cells and have gained the ability to

produce knobbed apical appendages. The presence of aknobbed apical appendage seems to have evolved once

during the evolution of Pestalotiopsis species, and hence

is a phylogenetically reliable taxonomic character.

Steyaert�s and Guba�s 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 todelineate 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). Se-

quence data suggest the possibility that there may be

four phylogenetic groups within the current morpho-

logical concept of Pestalotiopsis. These include species

possessing apical appendage length: (i) less than

15lm, (ii) 15–19lm, (iii) 20–25lm, and (iv) greater than25lm.

Current molecular analyses also resolved long-

standing confusion as to whether species characterized

by different basal appendage dimensions are closely re-

lated. Species with basal appendages greater or less than

5lm 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 ap-pendage as a taxonomic criterion to segregate species

can be misleading. On the other hand, absence of basal

appendages may be quite important for species delimi-

tation. In this study, only 2 species (Pestalotiopsis kar-

stenii and Pestalotiopsis maculans) are characterized by

the absence of basal appendages and they cluster to-

gether with a high bootstrap support.


Based on our morphological observations and mo-lecular 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 possessingmostly 2 appendages (for example, P. adusta, P. mi-

crospora, 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 im-

possible 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; Venkatasubba-iah et al., 1991). The results here, however, indicate that

spore size is homoplasious. Species sharing similar co-

nidial 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 phylogeneti-cally informative and given high taxonomic weighting

since all species in Clade X are characterized by conidial

width of greater than 6lm, whereas those in Clade Y

and Z possess a conidial width of less than 6lm.Pestalotiopsis microspora and Pestalotiopsis sp. EN12

clustered together and are closely related to P. karstenii

and P. maculans. These four species share the same co-

nidium length (less than 20lm) whereas members ofother clades are characterized by spores greater than

20lm 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 se-

quences revealed a high degree of similarity (96%) and a

98% nucleotide similarity among species in Subclade f.

Species of Subclade d, which share 100% sequencesimilarity (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 append-

ages, their lengths ranging from 8–15lm. The principalseparation 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 20lm, whereasthose in Subclade d possess spore lengths of greater than

20lm. 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 phy-

logeny, 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 forspecies 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 poly-

morphic and exhibit different forms under different cul-

tural 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 mediancells. 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 diagnosticmorphological 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 indistin-

guishable using the molecular approach here and its

phylogenetic relationships remain uncertain.

Another point of interest is the clustering of P. mac-

ulans and P. karstenii, which agrees with morphology-based systems. The type species of the genus, P. maculans

is characterized by a spore size of 15–20lm; 1–3 apical

appendages, which are at times branched, and basal ap-

pendages 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-


lationship between these two species on morphologicalgrounds 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 mi-

croscope, the main difference observed was the number of

appendages arising from the apical cell. Our sequence-

based analyses also demonstrate that these two speciesare 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 me-

dian 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 supportedherein by molecular evidence. However, Guba�s sub-

sections 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, orna-

mentation of median cells, and spore form appear to behighly plastic morphologies. A word of caution is nec-

essary 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 ra-ther than individual taxa. Contrarily conidial form and

ornamentation of median cells appear to have under-

gone 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 non-spathulated apical appendages. Pigmentation and apical

appendage tip morphology appear to be synapomorphies

for Pestalotiopsis species. While not intended as a com-

prehensive survey of the genusPestalotiopsis, our study is

significant in evaluating the relative importance and

utility of morphological characters in segregating Pes-

talotiopsis species. We propose that Pestalotiopsis species

be delineated, in order of taxonomic weighting, on thebasis 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 rep-resents part of a dissertation submitted by the first au-

thor in partial fulfillment of a doctoral degree.

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Phylogenetic significance of morphological characters in the taxonomy of Pestalotiopsis species

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