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Nova Hedwigia
72 3-4
311-328 Stuttgart, Mai 2001
A revision of
Verticillium
sect.
Prostrata. II.
Phylogenetic analyses of SSU and LSU nuclear rDNA sequences
from anamorphs and teleomorphs of the ClavicipitaceaeO)
by
Gi-Ho Sung!, Joseph W. Spatafora
1
*, Rasoul Zare
2,
Kathie T. Hodge
3
and Walter Gams4
I
Department of Botany and Plant Pathology, Oregon State University
Corvallis, OR 97331, USA
2CABI Bioscience, Bakeham Lane, Egham, Surrey TW20 9TY, UK. - Present address: Depart-
ment of Botany, Plant Pests and Diseases Research Institute, P.O. Box 1454, Tehran 19395, Iran
3
Dept. of Plant Pathology and L.H. Bailey Hortorium, Cornell University
Ithaca, N.Y. 14850-4203, USA
4
Centraalbureau vom Schimmelcultures, P.O. Box 85167, 3508 AD Utrecht, The Netherlands
With 2 figures and 1 table
Sung, G.-H.,
J.w.
Spatafora, R. Zare, K.T. Hodge
&
w.
Gams (2001): A revision of Verticillium
sect. Prostrata. II. Phylogenetic analyses of SSU and LSU nuclear rDNA sequences from anamorphs
and teleomorphs of the Clavicipitaceae. - Nova Hedwigia 72: 311-328.
Abstract: Parsimony analyses were conducted on partial nucleotide sequences from the small and
large subunits of the nuclear ribosomal DNA from representatives of Verticillium sect. Prostrata and
related ascomycetes. The majority of species from V. sect. Prostrata were supported as members of
the Clavicipitaceae, but they did not form a monophyletic group within the family. Three to six
groups of fungi in V.sect. Prostrata were inferred in these analyses and were designated groups B 1-
D3 following the convention of Zare et al. (2000). These groups integrated with other anamorph and
teleomorph genera including Cordyceps, which was also not supported as being monophyletic.
Group B 1 included the anamorph of C. militaris, V.lecanii, V.psalliotae, V.fusisporum, V.aranearum,
and V. antillanum. It was part of a larger clade designated Cordyceps s. stricto, which included
entomopathogenic species of Cordyceps and anamorphic species of Beauveria, Engyodontium,
Microhilum, and Paecilomyces. Group B2 included V.lamellicola, 'Cephalosporium' lanosoniveum,
and 'Acremonium' obclavatum and was the most closely related clade to Cordyceps s. stricto. Group
C represented a monophyletic clade of nematophagous species that included V. balanoides, V.
campanulatum, and V. sinense. It was part of a weakly supported clade designated the C. ophio-
glossoides clade, which included fungicolous and entomopathogenic species of Cordyceps and
*
corresponding author
0)
Part I: Zare et aI., Nova Hedwigia 71: 465-480. 2000.
0029-5035/01/0072-0311
$
4.50
© 2001 J. Cramer in der Gebrtider Borntraeger
Verlagsbuchhandlung, D-14129 Berlin· D-70176 Stuttgart
311
anamorphic species of Hirsutella, Harposporium, and Paecilomyces. Within the C. ophioglossoides
clade, V. sect. Prostrata group C was well supported as closely related to C. gunnii, a parasite of
lepidopteran larvae. Group D was not monophyletic and consisted of three lineages including the
rust parasite V. epiphytum (Dl), the mainly nematophagous species V. chlamydosporium, V.
suchlasporium, V. cf. bactrosporum, and V. gonioides (D2), and the homopteran pathogen V.
pseudohemipterigenum (D3). These data did not confidently address the relationships of the three
lineages of group D to one another or to other groups within the Clavicipitaceae.
Key words: Anamorph, Cordyceps, host-jumping, parsimony, systematics, teleomorph.
Introduction
Verticillium Nees is a large and heterogeneous anamorph genus that has been linked
to several families of the Ascomycota including Clavicipitaceae, Hypocreaceae,
Nectriaceae, and Phyllachoraceae (Gams
&
van Zaayen 1982, Gams 1988, Samuels
1988, Messner et a1. 1996, Zare et a1. 2000). The genus is characterized by phialidic
conidiophores produced directly from the mycelium as either erect or prostrate
structures. The verticillate phialides are generally aculeate and are typically produced
in whorls of three to five. Conidia are hyaline and range in morphology from
cylindrical to ellipsoidal to falcate according to species. In addition, some species
produce varying forms of chlamydospores that are thought to serve as resistant
propagules. Classification of Verticillium currently consists of four different sections
and a residual group, based on morphological and cultural characters (Gams
&
van
Zaayen 1982). This classification is recognized as polyphyletic and a more natural
classification is necessary. Current attempts to produce such a classification, however,
are hampered by the fact that several of the important morphological traits can vary
with culture conditions and age of isolate (Gams 1971, Gams
&
van Zaayen 1982,
Gams 1988).
Verticillium sect. Prostrata W. Gams was introduced by Gams (1971) for species
producing prostrate conidiophores that are often poorly differentiated from a fine,
white or yellowish mycelium. Phialides either possess or lack inflated bases according
to species. A number of species produce dictyochlamydospores, but production varies
amongst and within species and across a range of culture conditions. Conidia often
aggregate on the tips of phialides in heads, but in a few species they adhere in chains.
In addition to variation in morphological traits, delineation of the section is
complicated by the fact that some species are known to produce differentiated erect
conidiophores (e.g., Verticillium suchlasporium). Recognition of monophyletic
lineages within V. sect. Prostrata is also complicated by the fact that several other
anamorph genera, including Tolypocladium, Engyodontium, Aphanocladium, and
Acremonium, are morphologically similar to V. sect. Prostrata and are hypothesized
to be closely related, but the exact nature of their relationships remains disputed
(reviewed in Zare et a1.2000).
Several species of V. sect. Prostrata have been linked to entomopathogenic (e.g.,
Torrubiella confragosa Mains) and fungicolous (e.g., Cordyceps ophioglossoides (Fr.)
Link) teleomorphs of the Clavicipitaceae, including the anamorph of C. militaris
(L. : Fr.) Link which is the type of V.sect. Prostrata. The majority of species within
312
the section, howeve
teleomorphs of the
great promise in.~
integration of anam(J
1995). Broad taxo~
only result in more
1994), but theywi
morphologies and e
Verticillium
has a b~
mogenous, and
nell
sect.
Prostrata
inch
and some fungicol<l
with teleomorphs
0
include species of A
Hosts of the fungi
including fleshy fu
Zaayen
&
Gams 19l
of
V.
sect.
Prostratt
adults or cysts (Bal'l
soil samples and pIa
host affiliation are
diverse and the pal
unknown. Like oth
the subject of num
(Mankau 1980, Kel
et a1.1995), and fu]
more accurate unc
desirable for the dl
introduction of suc
In a phylogenetic
distinct groups of ~
and phialide morpl
dictyochlamydospl
Clavicipitaceae, bl
provides the most '
the foundation for
series of systematic
a molecular phylol
V.
sect.
Prostrata .•
Prostrata
with da
Ascomycota, ii) to
for its relationship
and iv) to develop
the section, however, are only known from anamorphs and their relationship to
teleomorphs of the Clavicipitaceae is speculative. Molecular phylogenetics holds
great promise in developing more robust phylogenetic hypotheses through the
integration of anamorphic and teleomorphic fungi in systematic studies (Taylor 1993,
1995). Broad taxon sampling of representatives with diverse life histories will not
only result in more accurate hypotheses of relationships (Blackwell
&
Spatafora
1994), but they will also result in a better understanding of the evolution of
morphologies and ecologies, such as host affiliation and nutritional mode.
Verticillium has a broad host range with mainly plant-pathogenic, fungicolous, ento-
mogenous, and nematophagous groups (van Zaayen & Gams 1982). Verticillium
sect. Prostrata includes the majority of entomogenous and nematophagous species
and some fungicolous taxa within the genus and overlaps in part of its host range
with teleomorphs of the Clavicipitaceae. Hosts of the entomogenous (s.l.) species
include species of Arachnida, Coleoptera, Homoptera, and Lepidoptera (Gams 1971).
Hosts of the fungicolous include a variety of Ascomycota and Basidiomycota,
including fleshy fungi of Hymenomycetes and rusts of the Urediniomycetes (van
Zaayen
&
Gams 1982, Lim
&
Wan 1983, Gams et al. 2001). Nematophagous species
of Vsect. Prostrata have a fairly broad host range, but species specialize on either
adults or cysts (Barron 1977, Gams 1988). Many species, however, are isolated from
soil samples and plant litter and the exact nature of their nutritional mode and potential
host affiliation are unknown. The host range of Vsect. Prostrata is therefore quite
diverse and the patterns and processes by which this host range arose are largely
unknown. Like other fungi of the Clavicipitaceae, species of Vsect. Prostrata are
the subject of numerous biological control studies, including those of nematodes
(Mankau 1980, Kerry & Crump 1977), arthropods (Schuler et al. 1991, Vestergaard
et al. 1995), and fungal pathogens (Saksirirat & Hoppe 1991, Verhaar et al. 1996). A
more accurate understanding of their systematics and phylogenetic affinities is
desirable for the design of biological control experiments and the application and
introduction of such agents in nature.
In a phylogenetic study of ITS rDNA, Zare et al. (2000) revealed at least three
distinct groups of Vsect. Prostrata that were consistent with differences in conidium
and phialide morphology, host affiliation, and - to a lesser extent - production of
dictyochlamydospores. Their data also supported a phylogenetic affinity with the
Clavicipitaceae, but few teleomorphs were included in their analyses. Their study
provides the most thorough insight into the polyphyly of Vsect. Prostrata and laid
the foundation for an eventual revision of its nomenclature. As part of a continuing
series of systematic investigations into Verticillium and the Clavicipitaceae, we initiated
a molecular phylogenetic study of nuclear ribosomal DNA (rDNA) sequences from
Vsect. Prostrata. The major goals of the study were i) to integrate data from Vsect.
Prostrata with data from the Clavicipitaceae and other related members of the
Ascomycota, ii) to test the monophyly of Vsect. Prostrata, iii) to develop hypotheses
for its relationship to the major teleomorphs and anamorphs of the Clavicipitaceae,
and iv) to develop hypotheses for character evolution and host association.
313
Material and methods
Taxon sampling. - Taxa were sampled to include a broad representation of V. sect. Prostrata,
representative teleomorphs of the Clavicipitaceae (e.g., Cordyceps, Torrubiella, Claviceps, Epichloe),
and other possible anamorphs ofthe Clavicipitaceae (e.g., Metarhizium, Beauveria, Paecilomyces).
Both host affiliation and morphology were used in taxon selection. Several sequences were included
to represent a diversity of outgroup taxa from other families and orders of perithecial ascomycetes.
A total of 93 isolates, representing 80 species and varieties, were included in this study. Sources of
isolates and host affiliation of specimens used in this study are listed in Table I.
DNA extraction. - DNA was extracted from both dried herbarium specimens and live cultures.
Crude DNA extracts were prepared by a modified CTAB method (Gardes
&
Bruns 1993, Spatafora
et al. 1998). Samples were ground in microcentrifuge tubes with disposable pestles attached to an
electric hand drill. Six-hundred III 65°C 2x CTAB buffer was then added to the ground tissue and
incubated at 65°C for 30 min. Two chloroform: isoamylalcohol (24: I) extractions were conducted
after the incubation. DNA was purified from the aqueous phase ofthe second chloroform extraction
using the GeneClean III kit (Bio 101 Inc., Vista, CA).
Polymerase chain reaction and DNA sequencing. - To integrate V.sect. Prostrata into a database of
clavicipitalean fungi, 1150 bp of the small subunit (SSU) and 950 bp of the large subunit (LSU)
nuclear ribosomal DNA (nrDNA) were amplified in the conventional polymerase chain reactions
(PCR) (Mullis
&
Faloona 1987). The SSU rDNA was amplified with primers NS I and NS4 (White
et al. 1990). The LSU rDNA was amplified with primers LROR and LR5 (Vilgalys
&
Sun 1994).
PCRs were performed in 50-Ill reactions as follows: [94°C (1 min), 50-52°C (30 sec), 72-73°C
(1 min)] x 35-40 cycles. Success ofPCRs was confirmed by agarose gel electrophoresis of 5 IIIof
the reaction mix. PCR products of SSU and LSU rDNA were purified using QIAquick PCR
purification kits (Qiagen Inc., Valencia, CA). Purified PCR products were visualized on a 1%
agarose gel stained with ethidium bromide and quantified using Gibco-BRL low DNA Mass Ladder.
The purified product was sequenced usingABI Prism BigDye Terminator Cycle Sequencing chemistry
with AmpliTaq DNA polymerase, FS on an ABI Prism Model 377 (version 2.1.1) automated DNA
sequencer (Perkin-Elmer) at the Central Services Laboratory ofthe Center for Gene Research and
Biotechnology at Oregon State University. The template strands of purified PCR-products were
directly sequenced utilizing the primers NS I, SR7, NS3, NS4 for SSU rDNA and LROR, LR5 for
LSU rDNA (White et al. 1990; Vilgalys
&
Sun 1994).
Phylogenetic analyses. - DNA sequences were edited in SeqEd Ver. 1.0.3, manually aligned using a
color font, and appended to apreexisting data set of the Clavicipitaceae and other perithecial fungi
(Spatafora et al. 1998). The phylogenetic analyses were performed with PAUP* 4.0 (Swofford
1998). Parsimony analyses were performed on the combined data set of SSU and LSU nrDNA
using the following heuristic search options: 100 replicates of random sequence addition, TBR (Tree
bisection-reconnection) branch swapping, and MULTRE in effect. Insertions and deletions (indels)
were minimized in alignments and gaps were treated as missing data in the analyses. Ambiguously
aligned sequence regions were excluded from the data matrix before analysis. Relative support for
the resulting trees was determined by 2500 bootstrap replications (Felsenstein 1985) on informative
characters only with the previously mentioned search options except that only one tree was retained
during each replication (Moncalvo et al. 2000). The phylogenetic trees generated from the combined
data set with SSU and LSU nrDNA data sets were rooted with Xylaria curta and X. hypoxylon.
To test alternative phylogenetic hypotheses for V.sect. Prostrata, i.e., monophyly of V. sect. Prostrata,
constraint topologies were constructed in MacClade 3.0 (Maddison
&
Maddison 1992). Constraint
topologies forced the monophyly of V.sect. Prostrata, but left all other nodes of the tree as unresolved.
These topologies were used as starting trees in maximum parsimony analyses; search options were
as described above except that MAXTREES was set to 1000. The most parsimonious trees recovered
from the constraint analyses were statistically compared to the trees recovered from the maximum
parsimony analyses using the Templeton WSR test implemented in PAUP* 4.0b3 (Swofford 1998).
Host affiliation was mapped onto one of the most parsimonious trees using MacClade 3.0 (Maddison
&
Maddison 1992) with equal weights for all character state transformations.
314
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Results
The combined SSU and LSU nrDNA dataset included 2040 aligned nucleotide
positions, with SSU rDNA comprising 1100 and the LSU rDNA comprising 940
positions. One-hundred and ten SSU rDNA positions and 128 LSU rDNA positions
were excluded due to either ambiguously aligned regions or an excess of missing
data near the 5' and 3' ends. The final data set included] 802 nucleotide positions of
which 347 positions - 146 from the SSU rDNA and 201 from the LSU rDNA -
were identified as parsimony-informative. The alignment is available from Treebase
as study accession number S573, matrix accession number M 866. Maximum
parsimony analysis of the 93 taxa dataset yielded 329 equally most parsimonious
trees of 1710 steps. For each of these trees, the consistency index (CI) was 0.322 and
the retention index (RI) was 0.690. Although a large number of trees were recovered
in these analyses, many of the nodes amongst the major genera and groups of the
Clavicipitaceae were resolved in the strict consensus tree (Fig. 1). One of the most
parsimonious trees from the maximum parsimony analyses was chosen at random
and is shown in Fig. 2 for the purpose of displaying branch lengths and mapping
host affiliation.
Maximum parsimony analyses of the combined SSU and LSU rDNA data support
the inclusion of all isolates sampled from Vsect. Prostrata in the Clavicipitaceae
except for V incurvum. However, the monophyly of the Clavicipitaceae is weakly
supported by bootstrap values and is characterized by a relatively short branch. The
clavicipitalean isolates of Vsect. Prostrata did not form a monophyletic group and
were placed in at least three separate parts of the Clavicipitaceae clade. To test the
monophyly of Vsect. Prostrata, two topological constraint analyses were performed.
One topological constraint that forced the monophyly of Vsect. Prostrata including
V incurvum (constraint]), while the second constraint forced the monophyly of V
sect. Prostrata without V incurvum (constraint 2). The ITS-5.8S sequences of V
incurvum could not be aligned with the remaining taxa of Verticillium (Zare & Gams,
unpublished). Maximum parsimony analyses using constraint 1 and constraint 2
topologies as starting trees resulted in trees that were 75 and 55 steps longer,
respectively. The most parsimonious trees from both constraint 1 and constraint 2
were both rejected as being significantly worse explanations of the data than the
trees from maximum parsimony analyses (P
=
<
0.0001). This finding is consistent
with the observation that the ITS-5.8S sequences of V incurvum could not be aligned
with the remaining taxa of Verticillium.
Discussion
Familial relationships of Verticillium sect. Prostrata. - Verticillium is known to be
phylogenetic ally related to many families of perithecial ascomycetes (Gams ]971,
Gams & van Zaayen 1982, Samuels 1988, Zare et al. 2000). In these analyses, the
specimens sampled from Vsect. Prostrata grouped within the Clavicipitaceae with
the exception of V incurvum, which grouped more closely with members of the
Hypocreaceae. The clavicipitaceous affinity of Vsect. Prostrata is consistent with
318
-
-
&
H
-
Fig. 1. Strict consensl
of the combined SS
differentiated by acce
are listed in Table 1.
major groups of
Vertic
the convention of ZaJ
nodes representing th
V. sect. Pros/rata
group
Bl
93
100
lOO
95
H
c
7J
88
80
100
100
~:t:~;~;
%~:';:(~~"(}I!
I
Xylariales
AJj~:i~iti:.~~rIJ~f(7n()sfJoru:y
I
Microascales
tJ~.;;;~~:~'~:'h~~·:~~~:.:;a
rlum
Sphaerostilhe/laaureonitens
Hypomrces p(ll-"pllrinu~'
V. il1l;urmm CBS 460.88
Nec/riacinn(/hari'J(l
Haemafonec/rra huemalo('occa
Neocos/nos!'of{/vasinjecta
Aphml/lchldimN
alhum
CBS 401 .70
~~~2~~1~~11
Paeci/omyces telwipes
C
hifusisporo
V.
sect
Prostrata
~~~~l:gp
I
v. lecani;
I
group
B 1
~~R}J:020
IB. ha.uiana
B.hrfJI1M/liartii
Belluveriacaledl!llica
C.
smrabaeicola
P.javtllJicus
Croseosfrmrww
Cordrcepsmilitarl.\'
Verticil/illm
sp.
CBS 402.78
'CephalosfJrlrlum'fa/lIFsporum CBS 126.27
CBS ]001721
t~~
l~jn~
v.
psa/lio/af
CBS 363.86
Verticil/;um
sp.
CBS 101284
Microhilum 0I1coperae ARSEf 4358
V.
p.wlliowe
CBS 639.85
V.
arallearum
CBS
726.73a
V
'~~:~:/II%I;;I::~
fg§
j~..;~
X~~J~9i~~91
Engrodollfillm aranearum
~~'S3i~~~
I'Cepllalosporillm'lanoSOlliverllll
I
V.
sect.
Prostrata
~cl:';~~II:~;~I;'~(~(t{j'61fBS
311.74 group
B2
E.amari!!all,\'
Epichloef)phina
NeOfrphodium coenophialum
V.
sect.
Prostrata
§:~
~~:~ll
v.
eplflhyrum
I
Dl
ARSEF 2037
1
group
1~§~~
j1~~
Me/(lrhizium
IF05940
V.
,l'Uch/1I.\porirll/l
CBS 464_88
I
V.
XOllioides
CBS. 891.72
V.
sect.
Prostrata
~:%;;/~7/~~~::;~~'~';;~
1~~_~cioI433
group
D2
V.
ch!wnydllsprlUllm
CBS 504.66
Atk;/l.wmel/a hypoxrloll
Baltlllsi(/arisfidae
B. hennillKsilllw
M.,-r/oxeno.\pOfl/ (rtr(rlnenlosa
C/avicep.lpUrpllrea
~'d~~~(~~871 - -
V.
sect.
Prostrata
196-1013
V. {lJeudohemlpterrgenllln
I
ROIifemphtho/"{l allKusrispum
group D3
Cordlupioidells hisporus
Hin-lItel!al/lOmpJOnii
C.
cochlidiicofa
C.
coccidiico/(,
Ha/]los{!oriwll helicoides
Atricllrdw:epJ harpl!S{lorifem
§:~
~j~:~61
v.
halanoides
I
v.
campollulatlllll
V.
sect.
Prostrata
~~§~~~:~~
I
V.
,~illeIlJe
group C
V. haltl/wides
CBS
522,80
C. Ii
11Il~1
ii
Ccaplt(//(I
C.
01'/lIoXlo.H/Jides
P./ifacinl/s
Co/fe."."'.'ii:hllmgl.oeosporioides
I
Glomerella clIlgu/ala
Phyllachorales
Vertlnllmmdahlwe
Cerwphoraseplenrrirmllli.\
I
Chaefomilllll gfoho,mm
Sordariales
NeUfO,I'I'0I"/lC/"{lJ.\'(/
Ophiosrmna l,iMman -
Ophiostomatales
Draporrhe phaseolawm -
Dlaporthales
g;:/~?;;;~
~;;~:~::r'ri~::1I
Xylariales
Fig. I. Strict consensus tree of 329 most parsimonious trees from the maximum parsimony analyses
of the combined SSU and LSU rDNA dataset. Species represented by multiple isolates are
differentiated by accession number; acceession numbers for species represented by a single isolate
are listed in Table I. Bootstrap values of >50% are noted above their respective nodes. The three
major groups of Verticillium sect. Prostrata are designated to the right of the species names following
the convention of Zare et al. (2000); subgroups are designated numerically, i.e., BI, DI-D3. The
nodes representing the Hypocreales and Clavicipitaceae are noted by Hand C, respectively.
319
1{JQJ--
Aphyslostroma stercorarium
'L....-
Hypocrea schweinitzii
Sphaerostilhella aureonitens
Hypomyces polyporinus
V. incurvum CBS 460.88
Nectria cinnabarina
~ Haematonectria haematococca
-~ Neocosmospora vasinfecta
Aphanocladium album CBS 40 1.70
~
3
Paecilomyces tenuipes OSC 764031
100
P. tenuipes AB027334 Lepidoptera
L
C.
bifusispora
·j88V. lec~~~r~,;~~un~~S 726.73a - Arachnida
V. lecanii IMI 304807 - Homoptera
63~
B. bassiana NRRL 28020
Pi. ;
B. bassiana IFO 4848
56 6~
B. brongniartii
. Beauveria caledonica - Saprobe
C. scarabaeicola - Coleoptera
Verticillium sp. CBS 101284
. Microhilum oncoperae - Lepidoptera
~ C.
roseostromata
I
Le 'd
t
a
~ C. militaris
pi
op er
P. javanicus - Coleoptera
V. psalliotae IMI 163640 - Soil
.~' V. psalliotae CBS 100172 - Arachnida
• V.fusisporum CBS 164.70 - Aphyttophorales
• V, antillunum CBS 350.85 - Agaricales
731
U'.
98
Engyodontium urunearum CBS 309.85
I
Arachnida
Engyodon/lum uruneurum ARSEF 2929
•• V. psulliotae CBS 639.85 - Rhizosphere
V. psulliotue CBS 532.8 I - Soil
V. psulliotue CBS 363.86 - Agaricales
Verticillium sp. CBS 402.78 - Leaf litter
.~ 'Cephulosporium'longisporumCBS 126,27 - Homoptera
~:6
'Cephulosporium' lunosoniveum IMI317442/ U d' I
100 :Cephulospor!um' lunosoniveumCBS 704.86 re ma es
'Acremonlum
ohclavatum - Air
V. lumellicolu CBS I
J
6.25 - Agaricales
V. chlumydosporium - Soil
HXl
rf.8
E. amariLlans
I
8Epichloe typhinu Poaceae
Neotyphodium coellophialum
V. epiphytum CBS 154.61
I
Uredinales
V. eplphytum CBS 384.8 I
- M. flavoviride var. minus - Homoptera
M. amsopliue var. frigidum
____ "_"•..•[" M. anisopliae var. mujus - Coleoptera
Metarhizium anisopliae
V. suchlusporium - Nematoda
V. gonioides - Aphyttophorales
V. bulbillosum - Pinaceae
V. cf. buctrosporum
S8.---
Atkinsonellu hypoxylon
- Balansia aristidae
B. henningsiana
I
Poaceae
Myriogenospora atramentosa
Cluviceps purpureu
C.
puspali
~ V. pseudohemipterigellum ARSEF 5687
V. pseudohemlpterigenum I 96- to 13 - Homoptera
Rotiferophthora angustispora - Rotifera
~ Cordycepioideus bisporus - Isoptera
R7.---o....- Hirsutella thompsonii - Arachnida
99 __
C. cochlidiicolu - Lepidoptera
- C. coccidiicola - Homoptera
Harposporiunt helicoides - Nematoda
Atricorayceps harpospuriferu - Diplopoda
V. balulloides CBS 250.82
I
Nematoda
- V. balanOldes CBS 335.80
V. campanulatum - dung of nematode
!JJ!!j ~
~;~=:::;=~~~
UU~
=
~~~atoda
V. bulunoides CBS 522.80 - Nematoda
C. gum!ii - Lepidoptera
C.
capi tatu
I .
C. ophioglossoides Elaphomyces
P. lilucinus - Nematoda
_ 5 changes Fig. 2. A phylogram
0
tree is shown to emphl
as in Figure 1 with the
asterisks. Host or subs
names.
These data are alsc
Prostrata and those
the SSU rDNA, the
to the Phyllachoral
supported by the d
lengths. Here we ir
larger sampling of]
attraction (Felsenst
for a close relationsl
Penz.
&
Sacco and il
and the inclusion
I
strengthened the h)
and
V.
sect.
Nigres£
hypotheses from
ffiI
phylogenetic anal
Clavicipitaceae wel
supported by the da
by relatively short
monophyly of the
f
ceae are united by t
thickened apex and
(Diehl 1950, Main!
1993) .
Polyphyly of
Vertici
as being included i
rejected. The most
which forced the
Il
were rejected as sig
=
<
0.0001). These
represent a natural
possibly six, sepa
Clavicipitaceae.
Wl
several of the group
analyses (Figs 1, :
morphological and
follow the convenl
Prostrata as B-D
c
320
hypotheses from morphological studies (Gams 1971, Gams & van Zaayen 1982) and
phylogenetic analyses using the ITS rDNA (Zare et al. 2000). Although the
Clavicipitaceae were inferred to be monophyletic, their monophyly was not strongly
supported by the data. The clavicipitaceous region of the rDNA tree is characterized
by relatively short basal branches that received low bootstrap support (Fig. 2). The
monophyly of the family is, however, consistent with morphology. The Clavicipita-
ceae are united by the synapomorphies of long cylindrical asci with a conspicuously
thickened apex and filiform ascospores that typically disarticulate into part-spores
(Diehl 1950, Mains 1958, Rogerson 1970, Kobayasi 1982, Spatafora
&
Blackwell
1993).
These data are also consistent with a distant relationship between fungi of
V.
sect.
Prostrata and those of V.sect. Nigrescentia (Zare et al. 2000).
In
a previous study of
the SSU rDNA, the plant pathogen V.dahliae Kleb. was shown to be closely related
to the Phyllachoraceae (Messner et al. 1996). This relationship was not strongly
supported by the data and the node in question was characterized by long branch
lengths. Here we included sequence data from both the SSU and LSU rDNA and a
larger sampling of perithecial ascomycetes, which may serve to disrupt long branch
attraction (Felsenstein 1978, Graybeal 1998). These analyses increased the support
for a close relationship between V.dahliae and Colletotrichum gloeosporioides (Penz.)
Penz.
&
Saccoand its teleomorph Glomerella cingulata (Stonem.) Spauld.
&
Schrenk
and the inclusion of V. dahliae in the Phyllachoraceae (Fig. 1). These data also
strengthened the hypothesis of the disparate relationship between V. sect. Prostrata
and V.sect. Nigrescentia (Zare et al. 2000).
Polyphyly of Verticillium sect. Prostrata. - Although V.sect. Prostrata was confirmed
as being included in the Clavicipitaceae, the monophyly of V. sect. Prostrata was
rejected. The most parsimonious trees from the analyses of constraint topologies,
which forced the monophyly of V. sect. Prostrata with and without V. incurvum,
were rejected as significantly worse explanations of the data (Templeton WSR test P
=
<
0.0001). These results suggest that V.sect. Prostrata is polyphyletic and does not
represent a natural group of fungi within the Clavicipitaceae. At least three, and
possibly six, separate groups of V. sect. Prostrata were resolved within the
Clavicipitaceae. We emphasize groups - rather than clades - out of convenience as
several of the groups were not supported as monophyletic in the maximum parsimony
analyses (Figs 1, 2), but represent closely related sets of species. Also, certain
morphological and ecological traits are discussed in the context of these groups. We
follow the convention of Zare et al. (2000) and designate the groups of
V.
sect.
Prostrata as B-D with group A representing V. dahliae of V. sect. Nigrescentia.
Fig. 2. A phylogram of one of the 329 most parsimonious trees. Only the Hypocreales section of the
tree is shown to emphasize the Clavicipitaceae and Verticillium sect. Prostrata. Tree descriptors are
as in Figure 1 with the exception that nodes which collapse in the strict consensus are designated by
asterisks. Host or substratum, where known for the specific isolate, is provided to the right of species
names.
321
~
Groups Band D are not supported as monophyletic. Group B comprises two closely
related groups that are designated Bland B2. Group D includes three separate lineages
of V.sect. Prostrata (DI-D3), a finding consistent with Zare et al. (2000), that were
part of a poorly resolved region of the tree (Figs 1, 2).
Group B 1 includes the type of V. sect. Prostrata, the anamorph of C. militaris, and
morphologically similar species of V. lecanii, V. psalliotae, V. fusisporum, V.
aranearum, and V.antillanum; group B2 includes V.lamellicola, 'Cephalosporium'
lanosoniveum, and 'Acremonium' obclavatum. Species in group B are characterized
by phialides produced on prostrate conidiophores, nonadhesive conidia that vary in
morphology from oval to cylindrical to falcate, and the lack of dictyochlamydospore
production. Group C includes the nematophagous species V.balanoides, V.campa-
nulatum, and V.sinense. It is distinguished from the other groups of V.sect. Prostrata
by the basal inflation of the phialides, which are similar to Tolypocladium, and
adhesive conidia that are balanoid and rarely elongated.
Group D is grossly paraphyletic in these analyses and includes three lineages that are
part of an unresolved region of the tree (Fig. 1). Group Dl consists of the rust
parasite V.epiphytum, which produces falcate conidia that are morphologically similar
to V.psalliotae, but it differs from the latter species in its sparse production of thick-
walled, cyanophilic chlamydospores. Group D2 comprises the species V.chlamydo-
sporium, V.suchlasporium, V. cf. bactrosporum, and V.gonioides. It is not resolved
as monophyletic, largely due to the different placements of V. chlamydosporium
among the most parsimonious trees (Figs 1, 2). These species occur primarily on
nematode cysts and eggs and are characterized by the often prominent production of
dictyochlamydospores and nonadhesive conidia that are oval to subglobose to truncate.
Verticillium suchlasporium is unusual among V.sect. Prostrata in that it forms erect
conidiophores. Group D3 consists of the homopteran pathogen V.pseudohemipteri-
genum, which produces a compact whorl of phialides on erect conidiophores that
distinguishes it from other species of the group (Hywel-Jones et al. 1997). The
considerable morphological differences that exist among the fungi of group D may
serve to divide them into more natural groups in future analyses (Zare et al. 2000).
However, the data presented here do not robustly address this issue.
Discrepancies exist whether some species of V.sect. Prostrata (e.g., V.balanoides)
are capable of dictyochlamydospore production and considerable debate exists over
the taxonomic utility of dictyochlamydospore production among species of Verticil-
lium. Because of its variable formation in culture, Gams (1971, 1988) regarded the
dictyochlamydospore as a poor taxonomic character, unsuitable for a subdivision of
V.sect. Prostrata. In contrast, Kamyschko (1962) erected Diheterospora and Batista
&
Fonseca (1965) introduced Pochonia, largely on the basis of dictyochlamydospores,
both genera with type species that turned out to be identical with V.chlamydosporium.
This taxonomy was followed by Barron
&
Onions (1966) and Barron (1985, 1991)
who considered the dictyochlamydospore as a valuable character in the taxonomy of
nematophagous fungi and parasites of rotifers (e.g. Rotiferophthora). These analyses
suggest that while production of dictyochlamydospores may distinguish some taxa,
i.e., group D2, their production may be polymorphic for some verticillate fungi of
322
the Clavicipitaceae
gained several tiilll
not possible until
i
taceae.
Relationships of
V.
pitaceae. - Isolate~
of the Clavicipitac
only was Verticillil
not to be monoph~
militaris and relatil
included the parasi
variety of arthrop
Furthermore, the gr
group, although tht
WSR
P
=
0.85).
Ve
Cordyceps s. strict(
dontium, Microhill
militaris, the type
related Cordyceps
and Coleoptera (e
Verticillium that at
hypothesized as '
entomopathogen a
1982). Verticillium
Torrubiella confra
aranearum has als(
the culture sample
link to Torrubiella
clade to Cordyceps
a teleomorph in v
unpubl.). These res
affinity among the
stricto.
In the strict conser
related to teleomor
gunnii) and the clo
2). Teleomorphs of
that show consider
two isolates, Cord;
currently classified
not strongly supp
underrepresents th
However, C. gunm
1982), is well supp
(Fig. 2). An anamo
the Clavicipitaceae and the ability to produce such structures may have been lost and
gained several times. A more accurate character state reconstruction of this trait is
not possible until its distribution is confirmed for more anamorphs of the Clavicipi-
taceae.
Relationships of V.sect. Prostrata with anamorphs and teleomorphs of the Clavici-
pitaceae. - Isolates of V. sect. Prostrata integrate with anamorphs and teleomorphs
of the Clavicipitaceae at several points in the rDNA phylogeny. Importantly, not
only was Verticillium revealed to be polyphyletic, but Cordyceps was also inferred
not to be monophyletic. It consisted of two separate clades; one that included C.
militaris and relatives and will be referred to as Cordyceps sensu stricto, and one that
included the parasites of Elaphomyces (e.g., C. ophioglossoides) and pathogens of a
variety of arthropods and will be referred to as the C. ophioglossoides clade.
Furthermore, the grass endophytes of the Clavicipitaceae did not form a monophyletic
group, although their monophyly could not be rejected by these analyses (Templeton
WSR P
=
0.85). Verticillium sect. Prostrata group B is included with teleomorphs of
Cordyceps s. stricto and the closely related anamorphic species of Beauveria, Engyo-
dontium, Microhilum, and Paecilomyces. The teleomorphs of the clade include C.
militaris, the type species of Cordyceps, and morphologically similar and closely
related Cordyceps species that are pathogens of Lepidoptera (e.g., C. bifusispora)
and Coleoptera (e.g. C. scarabaeicola). Of particular interest are the isolates of
Verticillium that are known to be linked with teleomorphs of Torrubiella, a genus
hypothesized as closely related to Cordyceps because of its ecology as an
entomopathogen and its ascus and ascospore morphology (Mains 1949, Kobayasi
1982). Verticillium lecanii (isolate
IMI
304807) was established from an isolate of
Torrubiella confragosa pathogenic on scale insects (Jun et al. 1991). Verticillium
aranearum has also been linked to Torrubiella (T. alba Petch, Petch 1932), although
the culture sampled here was not isolated from a teleomorphic specimen. Another
link to Torrubiella may exist in V. sect. Prostrata group B2, the most closely related
clade to Cordyceps s. stricto. 'Cephalosporium' lanosoniveum CBS 740.86 produced
a teleomorph in vitro that is tentatively identified as Torrubiella sp. (W. Gams,
unpubl.). These results suggest that Torrubiella may display its closest phylogenetic
affinity among the brightly pigmented, fleshy stromatic species of Cordyceps s.
stricto.
In
the strict consensus tree (Fig. 1), V. sect. Prostrata group C is resolved as being
related to teleomorphs of the Cordyceps ophioglossoides clade (e.g., C. capitata, C.
gunnii) and the closely related anamorphs of Hirsutella and Harposporium (Figs 1,
2). Teleomorphs of this clade are represented by darkly pigmented, stromatic species
that show considerable diversity in morphology and host affiliation to the point that
two isolates, Cordycepioideus bisporus and Atricordyceps harposporioides, are not
currently classified in Cordyceps (Suh et al. 1988). The C. ophioglossoides clade is
not strongly supported by the data (Fig. 2) and the current sampling certainly
underrepresents the phylogenetic diversity of teleomorphs of the Clavicipitaceae.
However, C. gunnii, an Australian parasite of lepidopteran larvae (Kobayasi 1941,
1982), is well supported as a teleomorph closely related to V.sect. Prostrata group C
(Fig. 2). An anamorph has not been established by culture methodology for C. gunnii
323
and these data do not establish that C. gunnii is the teleomorph of any member of this
group of V. sect. Prostrata. Rather, these results provide predictive value for future
sampling of teleomorphs that may further illuminate teleomorph-anamorph relationships
and connections within the Clavicipitaceae. Production of Verticillium anamorphs is
known for C. ophioglossoides, a parasite of Elaphomyces, but the phialides lack the
basal inflation as in V.sect. Prostrata group C and these data do not confidently establish
a close relationship between C. ophioglossoides and V.sect. Prostrata group C.
Although V.sect. Prostrata group D is confirmed as a member of the Clavicipitaceae,
its relationships to teleomorphs and other anamorphs of the family are poorly resolved
(Figs 1,2). Group Dl
(v.
epiphytum) is included in the clade containing the teleo-
morphs and anamorphs of Epichloe and group D3 (V.pseudohemipterigenum), along
with the nematophagous species and Rotiferophthora angustispora, grouped most
closely with the teleomorphs of Atkinsonella, Balansia and Claviceps. None of these
relationships were strongly supported by the data (Fig. 2). All of these teleomorphs
are symbionts of the Poaceae (Rogerson 1970, Clay 1988) and some are linked to
anamorphs that are morphologically similar to V.sect. Prostrata (e.g., Acremonium,
Neotyphodium) (Diehl 1950, Morgan-Jones
&
Gams 1982, Glenn etal. 1996, Schardl
et al. 1991, 1997). The similarity in anamorphs, however, exists for many teleomorphs
of the Clavicipitaceae and is by itself not proof of a close relationship. Group D2 of
V.sect. Prostrata and Metarhizium, an entomopathogenic anamorph, are also placed
in this poorly resolved region of the tree, but again their relationship is not strongly
supported by the data (Fig. 2) and remains speculative.
Evolution of host association. - In the classification of V. sect. Prostrata and the
Clavicipitaceae, host affiliation has been regarded as an important taxonomic character
(Diehl 1950, Gams & van Zaayen 1982, Zare et al. 2000). In an attempt to better
understand the evolution of host-jumping, host association was mapped onto one of
the most parsimonious trees (Fig. 2). Because of the lack of support for much of the
basal nodes of the Clavicipitaceae and the presence of multiple most parsimonious
trees, we present these results as working hypotheses and emphasize only the more
strongly supported resolutions (Fig. 2).
Verticillium sect. Prostrata group B includes pathogens of insects, spiders and fungi.
Its placement within Cordyceps s. stricto expands the host range of this clade beyond
that of Lepidoptera and Coleoptera and bolsters arguments that the inclusion of
anamorphic taxa in phylogenetic analyses improves our understanding of the evolution
of host affiliation and life histories. Pathogens and parasites of fungi are located in
four regions of the rDNA tree of the Clavicipitaceae (Fig. 2), a pattern consistent
with multiple origins of fungal pathogens on distantly related groups of fungi, i.e.,
homo basidiomycetes, rusts, and ascomycete truffles. Nikoh & Fukatsu (2000)
proposed that the parasites of Elaphomyces originated from a host jump from
arthropods onto truffle-like fruitbodies. While these data are consistent with their
finding, they do not provide unequivocal support for the polarity of a single arthropod-
to-Elaphomyces host-jump (Fig. 2).
At least two separate groups of nematophagous fungi exist among the species of
Verticillium, suggesting that two independent origins of parasitism of nematodes
324
may have occurred
basal nodes of the
do not allow us to (
parasitism of nem:
jumps. Furthermon
from plant or funga
in nature is unknm
Conclusion. - The
morphs andteleomc
testable phylogene
evolutionary histo
supported the inclt
but rejected the n
morphology is dist
in phial ide and coni
production are con
the basal nodes in t
short branch 1engtl
increased samplin
anamorphic fungi
define monophylet
This research was SUI
Most cultures were pr
Entomopathogenic Fu
Mei and R.C. Summel
BARRON, G.L. (197'
KENDRICK (eds): Bi
BARRON, G.L. (1985)
BARRON, G.L. (199
endoparasites of rotife
BARRON, G.L. &A.
Diheterospora, Stemp
BATISTA, A.c. & 0
entidade fungica dos s
BLACKWELL, M. &
detecting morphologic
Problems and Perspec
CLAY, K. (1988): Clay
to mutualism. - In:
Hi
Plants and Animals:
7'
may have occurred within the Clavicipitaceae (Fig. 2). However, the poorly resolved
basal nodes of the Clavicipitaceae and the paraphyly of Vsect. Prostrata group D3
do not allow us to distinguish between the hypotheses of two independent origins of
parasitism of nematodes and a single origin followed by multiple losses or host-
jumps. Furthermore, many isolates of nematophagous Verticillium species are isolated
from plant or fungal material and the extent to which they may exist saprotrophically
in nature is unknown.
Conclusion. - The data presented here illustrate the need for the inclusion of ana-
morphs and teleomorphs in common phylogenetic analyses. Gene phylogenies provide
testable phylogenetic hypotheses of anamorph-teleomorph relationships and their
evolutionary history, despite disparate morphologies and ecologies. These data
supported the inclusion of most isolates of Vsect. Prostrata in the Clavicipitaceae,
but rejected the monophyly of these fungi within the family. The Verticillium
morphology is distributed throughout much of the Clavicipitaceae, but differences
in phialide and conidium morphology, and - to a lesser extent - dictyochlamydospore
production are consistent with certain groups of Vsect. Prostrata. Finally, many of
the basal nodes in the rDNA phylogeny of the Clavicipitaceae were characterized by
short branch lengths and poor statistical support. Molecular analyses that include
increased sampling of additional loci and taxa of the Clavicipitaceae and their
anamorphic fungi are needed to more confidently resolve these nodes and better
define monophyletic, infrafamilial clades.
Acknowledgements
This research was supported by a grant from the National Science Foundation (DEB-9806936).
Most cultures were provided by the Centraalbureau voor Schimmelcultures, some by USDA ARS
Entomopathogenic Fungal Collection, and CABI Bioscience. We thank Drs K.A. Seifert, D. van der
Mei and R.c. Summerbell for critical comments on the paper.
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