Coevolution by Common Descent of Fungal Symbionts (EpichZoZ spp.) and
Grass Hosts
Christopher L. Schardl,* Adrian LeuchtmannJ
Malcolm R. Siegel*
Kuang-Ren
Chung,* David Penny,+ and
*Department of Plant Pathology, University of Kentucky; ?_Geobotanisches Institut, Eidgenijssische Technische Hochschule,
Ztirich; and $School of Biological Sciences, Massey University
Epichlog species are ascomycetous fungi (family Clavicipitaceae) that are ecologically obligate symbionts of grasses.
Because they can enhance host fitness by protection from biotic and abiotic stresses, but can also reduce host seed
production, these symbionts span a continuum from antagonistic (highly pathogenic) to mutualistic. Their mutualistic
or antagonistic effects are directly related to the relative importance of their sexual and asexual life cycles. The
sexual cycle of the fungus occurs only on “choked” tillers on which no seeds are produced, so the more antagonistic
Epichlod species permit almost no host seed production and only transmit horizontally (contagiously). Other Epichlok’ species are called “pleiotropic symbionts” because they have both mutualistic and antagonistic effects, being
transmitted both vertically (in host seeds) and horizontally. The possibility of coevolution by common descent of
Epichlol?’and grass species was addressed by surveying grasses for pleiotropic or antagonistic symbionts, identifying
the biological species of EpichZo2 associated with each host, conducting molecular phylogenetic analysis of the
symbionts based on noncoding portions of nuclear genes for l3-tubulin and rRNA, and comparing gene trees with
each other and with the established molecular phylogeny of the host tribes. A total of nine confirmed or likely
biological species of Epichlod were identified, eight of which were specialized to groups of related host species or
genera within a single tribe. Five of the Epichlok’ species were pleiotropic whereas the other four species were
antagonistic. Phylogenetic analysis indicated that the five pleiotropic species and two of the four antagonistic species
coevolved by common descent with their hosts. Of the two species for which common descent was not evident,
one had a broad host range and was paraphyletic to a pleiotropic species, and the other had discordant gene
phylogenies suggestive of a hybrid origin. These results suggested that common descent is more likely in pleotropic
than in antagonistic symbioses of grasses with these fungi.
Introduction
Coevolution
by common descent of fungal parasites (particularly
biotrophs) and plant hosts has often
been asserted, but seldom rigorously
tested (Barrett
1983; Thompson 1989). In other systems, specialization
apparently
without common descent has been documented for monotropoid
plants parasitizing ectomycorrhizal fungi (Cullings, Szaro, and Bruns 1996), and for
algal mutualists of corals (Rowan and Powers 1991),
whereas common descent has been indicated in some
vertically transmissible,
highly interdependent
symbioses (Chapela et al. 1994; Hinkle et al. 1994; Baumann et
al. 1995). In contrasting
evolutionary
trends of mutualistic and antagonistic symbioses, these cases should be
compared with caution because they involve unrelated
hosts, unrelated symbionts, and very different currencies
mediating costs and benefits to the partners. A more
direct comparison
is afforded by Epichloi; species and
cool-season
grasses (subfamily Pooideae), whose symbioses span a continuum of mutualism and antagonism.
Relative mutualism or antagonism of an EpichZo&
grass symbiosis is largely related to the path or paths of
Abbreviations:
ML, maximum likelihood; MP maximum parsimony, MP-I through MP-IX, biological species (mating populations) I
through IX; NJ, neighbor joining; rDNA-ITS, internal transcribed spacers of the nuclear rRNA genes; ti/tv, transition-to-transversion
ratio;
tub2, P-tubulin gene.
Key words: cospeciation,
es, mutualism, symbiosis.
clavicipitaceous
fungi, Epichlok!, grass-
Address for correspondence
and reprints: Christopher
Department of Plant Pathology, University of Kentucky,
Kentucky 40546-0091. E-mail: [email protected].
Mol. Biol. Evol. 14(2):133-143.
1997
0 1997 by the Society for Molecular Biology
and Evolution.
L. Schardl,
Lexington,
ISSN: 0737-4038
symbiont transmission
(Clay 1988) (fig. 1). Many of
these fungi can propagate clonally in the floral meristems and, ultimately, in the seed progeny of infected
mother plants (vertical transmission).
Alternatively,
strains can transmit horizontally (contagiously)
via sexual spores (Chung and Schardl 1996) in a life cycle that
also requires a third symbiont-the
dipteran fly Phorbia
phrenione-to
mediate fungal mating. Grass tillers bearing the fungal sexual structures (stromata) fail to set
seeds, so that the greater the sexual expression by the
fungus the less seed is produced by the infected host
plant. In the most antagonistic symbiota seed production
is abolished and the fungi only transmit horizontally. At
the other extreme, asexual EpichZoL species (classified
by convention
in form genus Neotyphodiurn
or Acremonium sect. Albo-Zanosa
[Morgan-Jones
and Gams
1982; Glenn et al. 19961) are capable only of vertical
transmission.
Some EpichZoL; strains develop stromata
on only some tillers while the remaining tillers of the
plant produce viable, symbiotic seeds. Such EpichZo2
strains are pleiotropic symbionts (Michalakis et al. 1992;
Schardl 1996), meaning they have both mutualistic and
antagonistic effects. Epichloe characteristics
that mediate mutualism are their protective, antiherbivore metabolites (Siegel et al. 1990) and their tendency to enhance
host growth and stress tolerance (Clay 1990; Kimmons,
Gwinn, and Bernard 1990; Gwinn and Gavin 1992; West
1994). Vertically transmitted
strains (pleiotropic
and
asexual) provide benefits directly to the seeds they infect
and to each successive generation of the host’s maternal
lineage. The strongly mutualistic character of pleiotropic
symbionts is indicated by their high frequency of sym133
134
Schardl
et al.
FIG. l.-An
example of pleiotropic symbiosis involving coordinated life cycles of EpichloE festucae (MP-II) and its host grass, Festuca
rubru. The alternative life cycles (clockwise and counterclockwise
loops) occur on different tillers of a symbiont-infected
plant. In the symbiont’s
asexual life cycle (clockwise loop) it systemically
infects the seeds produced on the mother plant and is thus transmitted vertically through
successive host generations. The symbiont’s sexual cycle (counterclockwise
loop) is initiated by a fungal growth (stroma) that envelops a young
flowering tiller and prevents the inflorescence from maturing. Following fertilization and maturation, meiotic spores are ejected and mediate
horizontal (contagious) transmission.
Antagonistic
EpichloP species sterilize most or all flowering tillers of their host plants and are never (or
perhaps rarely) transmitted vertically.
biosis with certain grass species in many or all natural
habitats (Funk, Belanger, and Murphy 1994; Bucheli and
Leuchtmann
1996).
Coevolution of hosts with Epichlog species or asexual relatives is possible if the fungal symbionts are host
specific and symbioses persist over evolutionary
time
scales. With rare exceptions
(Leuchtmann
and Clay
1993) symbiont isolates are highly specialized to their
hosts of origin and closely related species (Leuchtmann
1992; Koga, Christensen, and Bennett 1993; Christensen
1995). However, the condition of long-term persistence
is not always met. In particular, the asexual symbionts
often hybridize with diverse sexual EpichZo2 genotypes,
probably by cellular and nuclear fusions in doubly infected plants (Schardl et al. 1994; Tsai et al. 1994). Because such hybridizations
can involve genotypes spanning the diversity of the genus, the hybrid symbionts
have not strictly cospeciated
with their hosts despite
their vertical transmissibility.
Interestingly,
although evidence of past hybridization
is common in asexual lineages, there has been no report of interspecific hybrids
among the sexual genotypes. Therefore, it is possible
that symbiotic systems involving
sexual EpichZoL; species and host grass lineages may be specific and of long
duration.
Pleiotropic symbioses are finely balanced such that
unknown variables (perhaps epigenetic or microenvironmental) determine whether each flowering tiller of the
host plant will manifest sex of the fungus or plant. We
hypothesize
that such a balance requires fungus and
plant to be more coadapted than symbioses where either
host sex or symbiont sex is lacking. Interspecific
hybridization (Schardl et al. 1994; Tsai et al. 1994) or symbiont transfers to new host taxa (An et al. 1992; Leuchtmann and Clay 1993) may disrupt this balance. Conversely, long-term cospeciation without hybridization
or
host transfers may help maintain pleiotropic symbiosis.
Based on this hypothesis, we predict that phylogenetic
evidence of cospeciation
between Epichloti species and
their hosts will be strongest for pleiotropic symbioses.
In this study we conducted a survey of EpichZo&-grass
associations and molecular phylogenetic
analysis of the
symbionts to test whether pleiotropic,
antagonistic,
or
both types of symbiosis may have coevolved by common descent with hosts.
Material and Methods
Biological Materials
Epichlot species isolates (table 1) were cultured
from stromata or systemically
infected plant tissues as
previously
described (Schardl and An 1993; Leuchtmann, Schardl, and Siegel 1994). To help ensure diversity among isolates from each Epichlol?’species, two or
more isolates were obtained from different locales located at least 200 km from each other. The exceptions
were the two MP-VI isolates used for sequence analysis,
although several populations were sampled for isozyme
analysis. Isolates were sampled from each host species
so far identified.
Mating Tests
To identify biological
conducted
as previously
species, mating tests were
described
(Leuchtmann,
EpichloeXrass
Cospeciation
135
Table 1
EpichZoB Species Isolates Subjected to DNA Sequence Analysis
Biological
Species
Morpho-species
Isolate
MP-I. .......
MP-I ........
MP-I ........
MP-I ........
MP-I ........
MP-I ........
MP-Ib .......
MP-Ib .......
MP-I ........
MP-I ........
MP-I ........
MP-I ........
MP-I ........
MP-I ........
MP-I ........
MP-I ........
MP-II .......
MP-II .......
MP-II .......
MP-II .......
MP-II .......
MP-III ......
MP-III ......
MP-IVb. .....
MP-IVb. .....
MP-IV ......
MP-Vb ......
MP-Vb ......
MP-V .......
MP-V .......
MP-VI ......
MP-VI ......
MP-VII. .....
MP-VII. .....
MP-VIII .....
MP-VIII .....
MP-IXb. .....
MP-IXb. .....
MP-IXb. .....
E. typhina
E. typhina
E. typhina
E. typhina
E. typhina
E. typhina
E. typhina
E. clarkii
E. clarkii
E. clarkii
E. typhina
E. typhina
E. typhina
E. typhina
E. typhina
E. typhina
E. festucae
E. festucae
E. festucae
E. festucae
E. festucae
Epichloe sp.
Epichloe sp.
E. amarillans
E. amarillans
E. amarillans
E. baconii
E. baconii
E. baconii
E. baconii
Epichlod sp.
Epichloe sp.
Epichlod sp.
Epichloe sp.
Epichloe sp.
Epichloe sp.
Epichloe sp.
Epichlod sp.
Epichloe sp.
E358
E470
E469
E2466
E246 1
E8
E432
90168”
E426
E427
E348
E425
E428
E430
E43 1
E505
E28
E32
90660”
90661”
E434
E56
El84
E57
El14
E52
90167c
E248
E424
El031
E501
E502
E354
E503
E277
E2772
El040
El045
El046
Host Tribe
Host Species
Anthoxanthum odoratum
A. odoratum
Dactylis glomerata
D. glomerata
D. glomerata
Lolium perenne
L. perenne
Holcus lanatus
H. lanatus
H. lanatus
Phleum pratense
P. pratense
Poa trivialis
Poa pratensis
Poa silvicola
Brachypodium pinnatum
Festuca longifolia
Festuca rubra subsp. commutata
F.r. subsp. commutata
F.r. subsp. rubra
F. gigantea
Elymus canadensis
Elymus virginicus
Agrostis hiemalis
A. hiemalis
Sphenopholis obtusata
Agrostis capillaris
Agrostis stolonifera
Agrostis tenuis
Calamagrostis villosa
Bromus erectus
B. erectus
Brachypodium sylvaticum
B. sylvaticum
Glyceria striata
G. striata
Brachyelytrum erectum
B. erectum
B. erectum
Aveneae
Aveneae
Poeae
Poeae
Poeae
Poeae
Poeae
Poeae
Poeae
Poeae
Aveneae
Aveneae
Poeae
Poeae
Poeae
Brachypodieae
Poeae
Poeae
Poeae
Poeae
Poeae
Triticeae
Triticeae
Aveneae
Aveneae
Aveneae
Aveneae
Aveneae
Aveneae
Aveneae
Bromeae
Bromeae
Brachypodieae
Brachypodieae
Meliceae
Meliceae
Brachyelytreae
Brachyelytreae
Brachyelytreae
Geographical
Origin
Switzerland
Switzerland
Switzerland
Switzerland
Britain
Europea
France
Britain
Switzerland
Switzerland
Japan
Switzerland
Switzerland
Switzerland
Switzerland
Switzerland
Europea
Europea
Europea
Europea
Switzerland
Texas, USA
Missouri, USA
Texas, USA
Alabama, USA
Georgia, USA
Britain
Britain
Switzerland
Switzerland
Switzerland
Switzerland
Japan
Switzerland
New York, USA
Indiana, USA
Kentucky, USA
Indiana, USA
Indiana, USA
a From cultivated grass native to Europe.
b Tentative assignment.
c Accession number for American Type Culture Collection.
Schardl, and Siegel 1994). All matings were conducted
on the female fungal structures (stromata) produced in
association with host inflorescences.
If matings between
two strains resulted in viable meiotic spores (ascospores), or if the strains were both interfertile
with a
third strain, they were classified in the same biological
species, designated as mating populations MP-I through
MP-IX. Because matings cannot be conducted in culture, these tests were sometimes limited by the low numbers of stromata produced on some infected plants.
Therefore, some biological species assignments
remain
tentative.
Isozyme
Electrophoretic
Analysis
Allozyme profiles of twelve polymorphic loci were
analyzed by horizontal starch gel electrophoresis
as previously described (Leuchtmann
and Clay 1990; Leuchtmann, Schardl, and Siegel 1994; Bucheli and Leuchtmann 1996).
Phylogenetic
Analysis
DNA isolation, DNA fragment amplification
by
PCR, and sequence determinations
were as described
previously (Schardl et al. 1994), with modifications. Oligodeoxynucleotide
primers used for amplification of the
introns
1 to 3, were 5’tub2 5 ’ region, including
GTTTCGTCCGAGTTCTCGAC-3’
(antisense
extending from codons
199 to 193), and 5’-ACCGAGAAAATGCGTGAGAT-3’
(sense, to codon 4). Amplification with these primers was best achieved when the
PCR buffer contained 2.5 mM MgCl*. Amplified fragments were chromatographed
on silica gel using the
Wizard DNA Cleanup kit (Promega Corp., Madison,
Wis.), then sequenced by linear amplification
with random chain termination and direct incorporation
of label
(from cx-[35S]-dATP) using the Promega fmol kit. Parameters for sequencing reactions were as previously described (Schardl et al. 1994). Sequencing primers were
5’-GAGAAAATGCGTGAGATTGT-3’
(sense, through
136
Schardl
et al.
codon 4 and the 5’-GT of intron l), 5’-CATCTTCAAACCGGTCAGTG-3’
(sense, codons 6 to 12), 5’CAAATTGGTGCTGCTTTCTGG-3’
(sense, codons 15
to 21), 5’-TGGTCAACCAGCTCAGCACC-3’
(antisense, codons 115 to 109), 5’-TCGTTGAAGTAGACACTCAT-3’
(antisense,
codons 53 to 47), and 5’ACGCACTGACCGGTTTGAAG-3’
(antisense, codons
12 to 7). The internal transcribed spacers of the nuclear
rRNA gene repeats (rDNA-ITS) were amplified and sequenced as described earlier (Schardl et al. 1994). Sequences of tub2 introns and rDNA-ITS were aligned by
eye, and were similar to the alignments previously reported (Schardl et al. 1991, 1994; Tsai et al. 1994).
Where alignments were ambiguous, insertion or deletion
(indel) gaps were placed so as not to introduce informative nucleotide polymorphisms.
Maximum parsimony (MP) was by the branch-andbound algorithm for exact solutions, as implemented
in
PAUP (Swofford 1993). Characters were unordered and
unweighted, and unambiguous
indels were treated either
as informative (each regardless of size considered equivalent to a single nucleotide change) or as missing information. Maximum likelihood (ML) and neighbor joining
(NJ) employed PHYLIP 3.51~ (Felsenstein
1993). NJ
trees were inferred from a Jukes-Cantor (one-parameter)
distance matrix and a Kimura (two-parameter)
distance
matrix with a transition-to-transversion
ratio (ti/tv) of 2
(MP trees of tub2 introns indicated ti/tv of 1.9). ML
trees were also calculated for ti/tv = 1 and ti/tv = 2.
Regardless
of parameters
used, there were no differences in topology between MP and ML trees, and these
had only a minor difference with NJ trees. ML and NJ
trees were determined at least three times with random
addition of sequences, using a different random number
seed each time, again with no differences in the inferred
topology. Probability estimates for branches in the ML
tree were determined
by the likelihood ratio test, although this test probably
overestimates
significance
(Felsenstein
1993).
Signals for and against partitions in the taxa (sequences) were determined by spectral analysis using the
hadamard matrix method of Lento et al. (1995). This
approach integrates distance and parsimony
methods,
correcting
for multiple changes by standard distance
methods, then, as in cladistic methods, directly examining signals for and against any aspect of the tree (Penny et al. 1996). Spectral analysis tests the support for
inferred clades as well as for other possible clades that
are not in the inferred tree.
Results
Characterization
of EpichZoF Biological
Species
Recent taxonomic evaluations
of EpichZoF species
have considered interfertile populations,
but several biological species have not yet been formally described.
To fully evaluate the biological relevance of molecular
phylogenetic
relationships,
it was important to also determine biological
species relationships.
Mating tests
unambiguously
assigned EpichZoF isolates to biological
species designated mating populations
MP-I to MP-V,
and MP-VII (table 1). Isolates from Agrostis hiemalis
and Sphenopholis obtusata were tentatively assigned to
the same biological species, MP-IV, because of similarities of allozyme profiles, DNA sequences (this study),
and morphology (White 1994). A tentative identification
of three additional biological species, MP-VI, MP-VIII,
and MP-IX, was based on limited mating tests because
infrequent or untimely expression of stromata precluded
complete tests.
All isolates of MP-I, MP-II, MP-V, MP-VI, and
MP-VII were from wild or cultivated grasses native to
Europe, but not from North American grasses. Two of
the isolates were from Japan, but may have been transported there from Europe by human activity. Isolates of
biological species MP-III, MP-IV, MP-VIII, and MP-IX
were all from native North American grasses. There was
good correspondence
between biological
species and
three of the previously described morphological
species,
EpichloF festucae (MP-II), E. amarillans (MP-IV), and
E. baconii (MP-V) (White 1993, 1994; Leuchtmann,
Schardl, and Siegel 1994). Isolates testing as MP-I included those morphologically
similar to Epichloe typhina and E. clurkii, the latter being restricted to the host,
Holcus lanatus.
Allozyme profiles supported the existence of, and
tentative assignments to, nine biological species of Epichlo?. No polymorphisms
were evident among putative
MP-VI, MP-VIII, and MP-IX isolates from each of the
hosts Bromus erectus, Glyceria striata, and Brachyelytrum erectum, respectively. Pairwise Nei’s genetic identities (Nei 1972) between all distinct genotypes were
calculated, and from these were derived the mean genetic identities within and between known or putative
biological species (table 2). Within those species exhibiting polymorphisms,
mean genetic identities were between 0.56 (MP-V) and 0.89 (MP-III). The low average
identity in MP-V reflected the divergence of the isolate
from Calamagrostis
villosa relative to the other MP-V
isolates. Other biological species showing comparably
low average genetic identities were MP-I (0.67) and
MP-VII (0.64). Between species, pairwise comparisons
yielded mean genetic identities between 0 and 0.50.
These values were all lower than intraspecies
mean
identities, though some ranges of interspecies and intraspecies comparisons overlapped considerably.
Host Ranges
and Specificity
of EpichZoF Species
With the exception of MP-I, each biological species
was associated with a host species, genus, or group of
related genera within a single tribe (table 1). Because
grass tribes often include groups of related genera that
hybridize in nature, it was appropriate to consider specialization at the host tribe level. In all, seven tribes of
grasses in the subfamily Pooideae (C3 grasses) were
identified as hosts. The four tribes that hosted only a
single EpichZo2 species each were Brachyelytreae,
Bromeae, Triticeae,
and Meliceae.
Three other tribesPoeae, Aveneae, and Brachypodieae-included
hosts of
MP-I genotypes as well as other species. Within Poeae
and Aveneae, hosts of MP-I were not closely related to
hosts of MP-II, MP-IV, or MP-V. In only one instance
EpichloSGrass
Cospeciation
137
Table 2
Identity Matrix Based on Allozyme Profile Comparisons Within and Between Epichloi; Species
BIOLOGICALSPECIES
No. OF
BIOLOGICALGENOSPECIES TYPEP
MP-I
.....
MP-II.....
21
4
....
3
MP-IV . . . .
2
MP-V.....
3
MP-VI . . . .
2
...
..
2
2
MP-IX . . . .
2
MP-III
MP-VII
MP-VIII.
MP-I
MP-II
MP-III
MP-IV
MP-V
0.67
(0.35-l .OO)
0.26
(0.17-0.42)
0.25
(0.13-0.58)
0.25
(0.09-0.42)
0.12
(o-0.34)
0.32
(0.26-0.35)
0.82
(0.67-0.92)
0.47
(0.25-0.58)
0.40
(0.33-0.50)
0.31
(0.25-0.42)
0.40
(0.33-0.42)
0.89
(0.83-0.92)
0.35
(0.25-0.42)
0.36
(0.25-0.50)
0.42
(0.42-0.42)
0.83
(0.834.83)
0.28
(0.08-0.50)
0.17
(0.17-o. 17)
0.56
(0.33-0.92)
0.33
(0.17-0.42)
(0.34-0.89)
0.50
0.01
(o-0.13)
0.45
(0.34-0.52)
(0.254.38)
0.30
0.15
(0.08-0.17)
0.40
(0.33-0.42)
(0.3::.55)
0.17
(0.17-0.17)
0.31
(0.25-0.33)
(O.lqz.38)
0.08
(O.OSJJOS)
0.29
(0.25-0.33)
(O.ltz.33)
0.28
(0.17-0.33)
0.14
(0.08-0.17)
MP-VI
MP-VII
MP-VII
MP-IX
1.00
(0.20;:.30)
0.33
(0.33-0.33)
0.33
(0.33-0.33)
(0.6tz.64)
0.11
(0.08-0.13)
0.46
(0.42-0.5 1)
1.00
(0:)
1.00
Note.-Values shown are average Nei’s genetic identity, with ranges in parentheses.
a Genotypes distinguishable by sequences, allozyme profiles, or mating types.
were sympatric, congeneric grasses identified as hosts
of distinct Epichlog species. This exceptional
case involved MP-I from Brachypodium pinnatum and MP-VII
from B. sylvaticum. Repeated mating attempts between
fungal stromata of opposite mating types on these two
hosts never resulted in production of viable ascospores.
In contrast, successful mating of stromata on B. pinnaturn with MP-I stromata on Dactylis glomerata was confirmed by segregation of parental isozyme loci (data not
shown).
Symbiotic
Phenotypes
There was wide variation among symbioses for expression of the fungal sexual structures (stromata) arresting development
of host inflorescences.
However,
each biological species consisted only of pleiotropic or
antagonistic
symbionts. Symbioses involving any of the
five biological species, MP-II, MP-III, MP-IV, MP-VII,
and MP-IX, were pleiotropic and heritable at high frequency. In contrast, symbioses involving MP-I, MP-V,
MP-VI, and MP-VIII caused complete or nearly complete abolition of host seed production,
and Epichlog
infection was never observed in the rare seeds that they
produced (Chung and Schardl 1996; unpublished observations).
Sequence
Variation
The first three introns of the P-tubulin genes (tub2)
(Schardl et al. 1994; Tsai et al. 1994) were sequenced
from a minimum of two isolates from each biological
species of Epichloe. Aligned sequences revealed 99 sites
with one or more nucleotide substitutions.
Of these, 69
sites had informative changes (identical sequences were
represented only once for this determination).
In addition, there were 18 indels of which 13 were informative.
Pairwise distances between sequences (table 3) were
largely concordant
with the isozyme results (see table
2). Those interspecies and intraspecies comparisons giving higher mean identity values had lower mean distances of their tub2 intron sequences. Within species the
highest mean distance was in MP-I, with MP-V and
MP-VII also giving high mean distances. As observed
in allozyme profiles, much of the sequence variation
within MP-V was due to the comparisons of the isolate
from C. villosa with those from Agrostis species.
Sequences of the nuclear rDNA internal transcribed
spacers (rDNA-ITS) were determined for representative
isolates of the nine EpichZo@ species. The 12 rDNA-ITS
sequences were aligned, revealing 43 sites with nucleotide substitutions,
of which 24 were informative sites.
Also, there were six indels, of which three were informative.
Gene Phylogenies
The tub2 gene trees derived by maximum parsimony (fig. 2) and distance-based
methods (not shown)
were similar. In almost all instances the tub2 phylogeny
grouped members of each biological species. The exception was the relationship between two European species, MP-I and MP-VII. Most MP-I isolates grouped in
a clade, but isolate E430 grouped separately from other
MP-I isolates, even those from other Poa species. Furthermore, MP-I isolate E505 grouped with MP-VII isolates. Thus, MP-I was exceptionally
diverse in both sequence (table 2) and host range (table I), and appeared
to be paraphyletic with MP-VII (fig. 2).
Another unusual relationship
emerged from comparison of the tub2 gene tree with an rRNA gene tree
based on rDNA-ITS sequences (fig. 3). Due to homoplasy and the smaller number of informative characters
compared to the tub2 sequences,
the rDNA-ITS
tree
lacked strong support of most internal branches. Nevertheless, the rDNA-ITS
tree was nearly concordant
with the tub2 tree except in placement of MP-VIII iso-
138
Schardl
et al.
Table 3
Average Two-Parameter
Distances
of tub2 Intron Sequences Within and Between Epichloi? Species
No. OF
BIOLOGICAL
SPECIES
MP-I
MP-II.
.......
......
BIOLOGICAL SPECIES
GENOTYPE.??
14
5
MP-I
MP-II
(Of41)
MP-III
MP-IV
MP-V
MP-VI
MP-VII
MP-VIII
MP-IX
0
(375-060)
MP-III ......
MP-IV ......
MP-V
......
MP-VI ......
MP-VII .....
4
2
(3::8)
(3z4)
(395-766)
(15%)
(3zL)
(4:-765)
(1 lfl9)
(3::2)
(172-327)
(5Eb5)
(4::8)
(323-437)
(420)
(x?54)
(353-944)
(384-246)
(394-754)
(4izO)
(545-863)
(4245)
(242-832)
(39Y49)
(4zfs3)
(4058)
(424)
(353_535)
(2?32)
(384-244)
(44:46)
(333-538)
3
....
MP-IX ......
(2f21)
10
2
16
2
$36)
MP-VIII
(2:)
3
4
2
2
(4l%7)
42
(28-48)
0
(323-436)
Note.-Values
calculated with a transition-to-transversion ratio of 1.9; shown as distances X lo3 with ranges in parentheses.
a Genotypes distinguishable by sequences, allozyme profiles, or mating types.
late E277. Although the tub2 tree grouped this isolate
with MP-III and MP-VI (fig. 2), its rDNA-ITS sequence
was in a strongly supported clade with MP-I sequences
(fig. 3). These sequence relationships
suggested that
evolution of MP-VIII may have involved interspecific
hybridization.
Comparison
of Epichlol?’ and Grass Phylogenies
Sequence relationships
among seven of the nine
Epichlog species mirror the molecular phylogenetic
relationships of the host tribes (Soreng, Davis, and Doyle
1990; Davis and Soreng 1993) (fig. 4). In the tub2 gene
tree the only taxa that do not fit a mirror phylogeny with
the hosts are MP-I and MP-VIII (fig. 4A). Of these,
MP-I is not specialized to any one host tribe (fig. 4B)
and, as stated above, MP-VIII may have hybrid origin.
A gene tree relating representatives
of the remaining
seven species was supported by spectral analysis (fig.
5). Sister relationships were indicated for MP-II, MP-IV,
and MP-V, which are associated with sister tribes Poeae
and Aveneae (fig. 5A). Likewise, association of MP-III
and MP-VI reflected the relationship
of host tribes Triticeae and Bromeae, respectively.
The remaining
two
EpichZo2 species, MP-VII and MP-IX, are associated
with the most deeply rooted host tribes, Brachypodieae
and Brachyelytreae.
In the spectral analysis (fig. 5B),
there was strong support for grouping symbionts
of
Poeae and Aveneae (values above zero in fig. 5B) and,
similarly, for grouping those of Triticeae and Bromeae.
Also, there was relatively little contradiction
for these
groups (values below zero). Their partitioning
from
symbionts of Brachyelytreae
and Brachypodieae
was not
as strongly supported, but all alternatives had much larger signals against, than for, the respective partitions.
There are, in total, 105 possible bifurcating
trees
relating six taxa, such as six tribes of grass hosts. The
number of strictly bifurcating
trees relating the seven
biological species of EpichZoLi would be 945, but the
sequence data did not resolve the branching orders of
MP-II, MP-IV, and MP-V. Therefore, there are three
equally short MP trees out of the 945 possible trees. Of
these, one was precisely concordant with the grass tribe
phylogeny; the two alternatives differed only in that they
paired one of the two Aveneae-associated
species
(MP-IV or MP-V) with the species (MP-II) symbiotic
with genus Festuca in tribe Poeae.
Discussion
Overall, tub2 and rDNA sequence relationships are
consistent with a tendency of the EpichZoL; species to
coevolve with their hosts, with exceptions involving antagonistic Epichloe species that do not specialize (MP-I)
or that may have hybrid origins (MP-VIII). That the
concordant host and symbiont phylogenies involved all
five species of pleiotropic symbionts suggests that they
are the more highly coadapted with their hosts. Pleiotropic symbioses entail less cost to the hosts in fecundity, and provide the symbionts
with two alternative
means of dissemination:
highly efficient vertical transmission helps ensure dissemination
(Siegel et al. 1984),
whereas horizontal transmission
permits the fungus to
exploit groups of related grass species. Many grasses are
symbiotic with asexual EpichZoL; relatives that are only
clonally propagated and vertically transmitted. Although
vertical transmission
alone could promote host-symbiont cospeciation, the asexual species do not exhibit common descent with their hosts. Instead, many are interspecific hybrids (Schardl et al. 1994; Tsai et al. 1994).
The reason may be that clonal propagation is not sustainable in long-term evolution unless the accumulation
of deleterious
mutations (“Muller’s
ratchet”) (Muller
1964) is counteracted
by a parasexual process such as
interspecific
hybridization.
Thus, Muller’s ratchet may
Epichlo&Grass
MP-I
Lol
Hoi
I%
Poa
I$
Ant
Poa
Dac
Brp
I
-0
E430
&
E32”**
MP-I
Brp
Brp
Poa
MP-II
Fes
Cospeciation
139
MP-I
MP-I
MP-VIII
MP-I
MP-VII
FIG. 3 .-Most
parsimonious
phylogram
(unrooted) by branchand-bound search (Swofford 1993) on rDNA internal transcribed spacer sequences of Epichlok’ species. Isolate numbers are indicated beside
terminal branches, and the biological species are listed as MP-I to
MP-IX at right. Decay indices are as in figure 2, with indels treated
as informative. Percentages below some branches are bootstrap values
(500 replications),
shown only if greater than 60%. There is a high
degree of convergence
of this tree with the tub2 gene tree (fig. 2),
except with regard to E277 (circled). GenBank accession numbers are
L07129,
L07131,
L07132,
LO7137-L07139,
L07141,
L07142,
L20306, L78293, and L78295-L78304.
>+4
0 E1045**
MP-IX Bre
FIG. 2.-Most
parsimonious
(MP) phylogram
by branch-andbound search (Swofford 1993) on tub2 intron sequences of Epichlot?
species. The left edge of the phylogram is the root inferred by midpoint
rooting (sequences from other clavicipitaceous
genera could not be
unambiguously
aligned as outgroups). Epichloti isolates are indicated
at the termini. Filled circles at termini indicate pleiotropic (horizontally
and vertically transmissible)
symbionts, and open circles indicate antagonistic symbionts. The number of asterisks after each isolate designation indicates the number of additional isolates with identical sequence. Biological species are indicated at right as mating populations
MP-I through MP-IX, followed by host genera abbreviated in parentheses as follows: Lolium, Holcus, Dactylis, and Pea (tribe Poeae);
Phleum and Antho~nthum~vene~,
Brachypodium
(Brachypodieae); Festuca (Poeae), Agrostis, Calamagrosti~
and Sphenopholis
7
Glyceria (Mehceae);
Elymus (Triticeae); BGus
(Bro(Aveneae);
meae); and Bxchyelytrum
(BrachyGtreae).
The bar represents a distance of five<uclectide
substitutions or indels (insertions or deletions)
on the horizontal branches (the vertical lines are merely illustrative,
and have no quantitative value). Decay indices relevant to species relationships are indicated above each branch as those calculated with
consideration
of informative indels, followed (if different) by indices
calculated treating indels as missing information. Sequence alignment
was as previously shown for related taxa (Schardl et al. 1994). The
tree involves 88 transitions and 46 transversions,
and the consistency
index, excluding uninformative
characters, is 0.788. The structure of
the MP tree and placement of the midpoint root is similar to that of
those determined by neighbor joining (NJ) on two-parameter distances,
and by maximum likelihood (ML) (Felsenstein 1993), with transitionto-transversion
ratios (ti/tv) of 1 or 2. ML trees (Ln likelihood =
- 1,686 for ti/tv = 2) had significant support (P < 0.05 by likelihod
ratios test) for all internal branches except the branch separating E354
from E503 and E505. NJ trees differed from the MP and ML trees
only in placing MP-VI nearest the root of the clade it shares with
MP-III and MP-VIII. GenBank
accession
numbers are LO6955L06962, L78268-L78292,
and X52616.
select for hybrid asexual species as well as for the maintenance of sex in pleiotropic species. Since there is direct conflict of sexual life cycles of EpichZoL; and grass
hosts, only the pleiotropic symbioses provide benefits of
sex and clonality to both partners. Concordance
of Epichlol; and host phylogenies suggests that such a balance
is most likely achieved and maintained
during coevolution by common descent.
While corresponding
topologies of gene trees suggest common descent of symbiotic partners, further support for such relationships can be obtained by statistical
comparison
of branch lengths in the inferred phylogenies (Hafner and Page 1995). Such analysis should compare trees from homologous genetic information;
however, the available database relating the host tribes is
based on plastid genomes (cpDNA) for which there is
no fungal homolog. Another concern is the placement
of the root of the Epichloe gene trees. Claviceps spp.
and Echinodothis
tuberiformis
were identified as the
most appropriate
outgroups based on comparisons
of
coding sequences
for the 26s large subunit rRNA
(rDNA-LSU)
(Liu 1993; unpublished
data). However,
the rDNA-LSU
sequences are too invariant within the
Epichloe clade for rooting, and the tub2 intron and
rDNA-ITS
sequences of these other species cannot be
aligned unambiguously
with those from Epichlok’ species. Therefore, because tub2 introns represent the most
informative
region thus far investigated
for the genus,
the best available estimate of the root is the midpoint of
the tub2 phylogeny as shown in figure 2.
Phylogenetic
relationships
based on cpDNA restriction endonuclease
sites place the host tribe Brachyelytreae
closest to the root, followed by Meliceae
and Brachypodieae
(Soreng, Davis and Doyle 1990; Davis and Soreng 1993) (fig. 4). The midpoint root of the
EpichZoF tub2 gene tree nearly corresponds to the root
of the host phylogeny, except that the species associated
with Brachypodieae
are the most deeply rooted. Removal of MP-I and MP-VIII to give the seven species
tree (corresponding
to the cladogram in fig. 5A) shifts
the midpoint root to a trifurcation of three clades: one
140
Schardl
et al.
B
le
4
2
1
40412628
77974759
0
FIG. 4.-Comparative
trees from molecular phylogenetic analysis
of pooid grass tribes and Epichlog species. The grass phylogram (at
left in both panels) was inferred by branch-and-bound
search (Swofford 1993) using Wagner parsimony on plastid DNA restriction site
data (Davis and Soreng 1993). The Epichlod species phylogeny was
derived from tub2 sequences as in figure 2, but including only one
representative
each of mating populations
II through IX. Midpoint
roots of the host and fungus phylograms
are at the outside edges. A,
Lines are drawn between fungal mating populations II-IX and their
host tribes. Possible cospeciation events, species duplications, and host
transfers were inferred by comparing grass and fungal cladograms using TREEMAP 1.Ob (Hafner and Page 1995). Possible cospeciation
events are indicated by black circles (0) on the fungal gene tree; a
fungal speciation without corresponding
host speciation is indicated
with an open square (0); and an apparent host species transfer is indicated by an arrow between MP-VIII and its host tribe Meliceae. B,
The broad host range of MP-I is indicated by lines connecting various
isolates with their host tribes. No pattern of cospeciation is expected
for MP-I because this species is not specialized to a grass tribe or
group of related tribes.
FIG. 5.-Molecular
phylogenetic analysis of tub2 intron sequence
relationships
among seven biological species of EpichloP that may
have evolved by common descent with host tribes. A, Sequences of
two isolates from each biological species were compared and one of
the MP trees is presented (the MP trees differed only in branching
orders of sequences 0, 1, and 2). Host tribes are indicated around the
perimeter, and the tree structure mirrors that of the host tribes in that
Aveneae and Poeae are sister tribes, Triticeae and Bromeae are sister
tribes, and Brachypodieae
and Brachyelytreae
are deeply rooted. Sequences are designated by mating population followed by letters as
follows: 0 is the identical sequence from the four MP-II isolates, 1 is
from isolate E421, 2 from El03 1, 3 from E52, 4 from E57, 5 from
E184, 6 from E56, 7 from E354, 8 from E503, 9 from E501, 10 from
E502, and 11 from the three MP-IX isolates. Letters assigned to internal branches are for reference to panel B. B, Hadamard spectrum
(Lento et al. 1995) of partitions of taxa in panel A. The ordinate indicates direct support (corrected for multiple changes) for (positive
values) or against (negative values) each partition. Bars are shaded for
partitions in the optimal tree and labeled with letters as in panel A.
Unshaded bars are for partitions excluded from the optimal tree, each
of which would separate taxa listed above the bar from the other taxa.
Only the most strongly supported and least contradicted partitions are
included. Identical sequences were represented only once when determining the hadamard spectrum.
associated with Brachypodieae
and Brachyelytreae,
one
with Triticeae and Bromeae, and one with Poeae and
Aveneae. Thus, although the rooting of the tree is not
stable to taxon removal, the alternatives
are not very
different from those expected for coevolution by common descent.
Delineations
of biological species were based on
experimental
mating tests, whereas natural mating is almost solely dependent on the activity of a dipteran (fly)
symbiont of the fungus, namely P. phrenione (Bultman
et al. 1995). It is possible that selectivity by fly genotypes or cryptic species keeps some populations isolated, even though they may be highly interfertile in ex-
Epic/do&Grass
perimental matings. Some preliminary observations suggest this. In the past three spring seasons, representatives
of MP-I, MP-II, and MP-III have been induced to form
stromata at the Spindletop farm, north of Lexington, Ky.
The plot included only one mating type (mat-Z) of
MP-III and MP-I, and both mating types of MP-II. In
all 3 years, eggs and larvae of the fly were present on
many of the MP-III stromata andswere associated with
fertile mating, indicating
that females had transferred
mat-2 spermatia from a nearby MP-III population. However, no MP-I or MP-II stromata showed evidence of
feeding or egg laying by the fly. This result suggests
that the fly ignored the native European species and
homed on the North American species in the plot. A
question worthy of investigation
is whether fly genotypes are selective for individual
sympatric species or
even subspecies of Epichloe.
Another
possible
mechanism
isolating
EpichZoL;
species is host specificity. Even in the case of MP-I, with
a broad host range, individual
genotypes can be host
specific. We have been unable to establish stable symbioses of D. glomeratu with isolate E8 from L. perenne,
or of L. perenne with isolates from D. glomeruta (Chung
1996). Interestingly,
stable infections are easily obtained
via seedling inoculations
of L. perenne with isolates
from Holcus Zanatus, with which the L. perenne isolates
have very close sequence relationships.
Allozyme and
sequence profiles indicate close genetic relationships
among MP-I isolates from each of the hosts, A. odoruturn, B. pinnatum, H. Zanatus, and L. perenne, but high
diversity among isolates from D. glomeratu, Poa spp.
and Phleum pratense. There is. also a morphological
character (disarticulation
of the ejected ascospores) that
distinguishes
H. Zanatus-associated
strains with other
MP-I genotypes. This is the taxonomic trait distinguishing morphospecies
E. clarkii from E. typhina (White
1993). The close similarity of allozyme profiles among
E. clarkii isolates suggests that it may be an incipient
biological species even though most E. clarkii isolates
remain interfertile with E. t-yphina in test matings. It is
possible that MP-I as presently delineated includes a
number of populations
isolated by biological processes
such as specificity for the symbiotic fly or host specificity, rather than by genetic barriers to mating. If host
specificity has been a major evolutionary
factor in genus
Epichloe, and if the genus originated early in the evolution of the host subfamily, then a degree of cospeciation would be expected. However, a phylogenetic pattern
of common descent was evident only with Epichloe species separated by genetic barriers to mating.
In most instances,
DNA sequence
comparisons
yielded distinct clades associated with individual
biological species of EpichZoLi. This was the expected pattern in keeping with the documented
tendency for reproductive isolation of fungi to correlate with genetic
divergence
(Vilgalys
1991; Vilgalys and Sun 1994).
However, paraphyly of MP-I with MP-VII and the relationships
among B. sylvaticum- and B. pinnatum-associated genotypes, challenge the concept of cladistic
speciation as a binary event with two new species replacing an ancestral species. The relatively greater di-
Cospeciation
141
versity of MP-I than MP-VII suggests that MP-VII arose
within MP-I. However, another possibility is that a dynamic relationship exists between these two species involving rare interspecific hybridizations.
Fungal species
can be envisaged to hybridize by sexual or parasexual
processes. Sexual hybridization
has not yet been observed, and phylogenetic
evidence indicates that many
asexual relatives of Epichlog are somatic (i.e., parasexual), interspecific hybrids with sexual genotypes (Tsai et
al. 1994). Whether sexual species may hybridize to generate a new sexual species is not known, but MP-VIII
is a possible example, evidenced by its discordant tub2
and rDNA phylogenies.
Such marked discordance
of
gene trees is one hallmark of asexual hybrids (Tsai et
al. 1994), another being their tendency to possess multiple isozyme loci for which there are single loci in sexual isolates (Schardl et al. 1994). Surprisingly, multiple
loci are occasionally observed among MP-I and MP-VII
isolates (Bucheli and Leuchtmann
1996; unpublished
data). Thus, possibilities worth further consideration are
that MP-I occasionally hybridizes with MP-VII, its asexual relatives, or other species, and that hybridization
may generate new sexual species.
When host specialization
and sequence relationships among all Epichloe genotypes are considered, the
pattern indicates diffuse cospeciation
(common descent
with host tribes) with exceptions.
The exceptions include two antagonistic species sampled in this study and
most asexual
species sampled
in previous
studies
(Schardl et al. 1994; Tsai et al. 1994). All exceptions
have indications of interspecific hybridization
(MP-VIII
and at least four of six asexual species) or paraphyly
and broad host specificity (MP-I). In contrast to the cladistic phylogenies of the pleiotropic species, the two extremes of strict horizontal
transmission
(antagonistic
species) and strict vertical transmission (asexual species)
are associated with clear deviations both from cladistic
speciation and from common descent with hosts.
Acknowledgments
We thank A. D. Byrd, M. Crider, W. Hollin, S. Holthaus, R. Jayasekera, and G. Kuldau for technical assistance. We thank K. O’Donnell
(United States Department of Agriculture,
Peoria, Ill.), J. E Wendel (Iowa
State University),
and an anonymous reviewer for critical reading of the manuscript, and R. J. Soreng (Cornell
University and The Smithsonian
Institution) for discussions relating to this study. K. Clay (Indiana University),
C. R. Funk and J. E White, Jr. (Rutgers University), and
D. Schmidt
(Federal
Agricultural
Research
Station,
Nyon, Switzerland)
supplied biological materials. This
research was funded by the National Science Foundation
grant DEB-940801 8. This is publication number 96-12191 of the Kentucky Agricultural
Experiment
Station
published with the approval of the director.
LITERATURE
CITED
AN, Z.-Q., J.-S. LIU, M. R. SIEGEL, G. BUNGE, and C. L.
SCHARDL. 1992. Diversity and origins of endophytic fungal
142
Schardl
et al.
KOGA, H., M. J. CHRISTENSEN,
and R. J. BENNE~. 1993. Celsymbionts of the North American grass Festuca arizonica.
lular interactions of some grass/Acremonium endophyte asTheor. Appl. Genet. 85:366-37 1.
BARRETT,J. A. 1983. Plant-fungus symbioses. Pp. 137-139 in
sociations. Mycol. Res. 97: 1237-l 244.
LENTO, G., R. E. HICKSON,G. K. CHAMBERS,and D. PENNY.
D. J. FUTUYMAand M. SLATKIN,eds. Coevolution. Sinauer,
1995. Use of spectral analysis to test hypotheses on the
Sunderland, Mass.
BAUMANN,P, L. BAUMANN,C. Y. LAI, D. ROUBAKHSH,N. A.
evolutionary origin of the pinnipeds. Mol. Biol. Evol. 12:
MORAN,and M. A. CLARK. 1995. Genetics, physiology, and
28-52.
evolutionary relationships of the genus Buchneru: intracelLEUCHTMANN,A. 1992. Systematics, distribution and host
lular symbionts of aphids. Annu. Rev. Microbial. 4955-94.
specificity of grass endophytes. Nat. Toxins 1:150-162.
BUCHELI,E., and A. LEUCHTMANN.1996. Evidence for genetic
LEUCHTMANN,A., and K. CLAY. 1990. Isozyme variation in
differentiation between choke-inducing and asymptomatic
the AcremoniumlEpichZoe fungal endophyte complex. Phystrains of the Epichloe grass endophyte from Bruchypodium
topathology 80: 1133-l 139.
sylvuticum. Evolution 50: 1879-l 887.
-.
1993. Nonreciprocal compatibility between EpichZo2
BULTMAN,T. L., J. E WHITE JR., T. I. BOWDISH,A. M. WELCH,
and four host grasses. Mycologia 85: 157-163.
and J. JOHNSTON.1995. Mutualistic transfer of Epichlod
LEUCHTMANN,A., C. L. SCHARDL,and M. R. SIEGEL. 1994.
spermatia by Phorbiu flies. Mycologia 87: 182-l 89.
Sexual compatibility and taxonomy of a new species of EpCHAPELA,I. H., S. A. REHNER, T. R. SCHULTZ,and U. G.
ichlol; symbiotic with fine fescue grasses. Mycologia 86:
MUELLER.1994. Evolutionary history of the symbiosis be802-812.
tween fungus-growing ants and their fungi. Science 266:
LIU, J.-S. 1993. Evolution of the fungal tribe Balansieae (Clav1691-1694.
icipitaceae) and related endophytes of grasses. Ph.D. disCHRISTENSEN,
M. J. 1995. Variation in the ability of Acremonsertation, University of Kentucky, Lexington, Ky.
ium endophytes of perennial rye-grass (L.&urn perenne),
MICHALAKIS,Y., I. OLIVIERI,E RENAUD,and M. RAYMOND.
tall fescue (Festucu urundinuceu) and meadow fescue (F.
1992. Pleiotropic action of parasites: how to be good for
prutensis) to form compatible associations in three grasses.
the host. Trends Ecol. Evol. 7:59-62.
Mycol. Res. 99:466470.
MORGAN-JONES,G., and W. GAMS. 1982. Notes on HyphoCHUNG, K.-R. 1996. Genetics of the grass symbiont, Epichlok’
mycetes, XLI. An endophyte of Festucu urundinuceu and
typhinu (Pers.:Fr.) Tul. Ph.D. dissertation, University of
the anamorph of Epichloe typhinu, new taxa in one of two
Kentucky, Lexington, Ky.
new sections of Acremonium. Mycotaxon 15:3 1 l-3 18.
CHUNG, K.-R., and C. L. SCHARDL.1996. Sexual cycle and
MULLER,H. J. 1964. The relation of recombination to mutahorizontal transmission of the grass symbiont, EpichZo2 tytional advance. Mutat. Res. 1:2-9.
phinu. Mycol. Res. (in press).
NEI, M. 1972. Genetic distance between populations. Am. Nat.
CLAY, K. 1988. Clavicipitaceous fungal endophytes of grasses:
106:283-292.
coevolution and the change from parasitism to mutualism.
PENNY,D., M. D. HENDY,l? J. LOCKHART,and M. A. STEEL.
Pp. 79-105 in K. A. PIROZYNSKIand D. HAWKSWORTH,
1996. Corrected parsimony, minimum evolution and Hadeds. Coevolution of fungi with plants and animals. Academamard conjugations. Syst. Biol. 45593-603.
ic Press, London.
ROWAN, R., and D. A. POWERS. 1991. A molecular genetic
1990. Fungal endophytes of grasses. Ann. Rev. Ecol.
classification of Zooxanthellae and the evolution of animalSyst: 21:275-295.
algal symbioses. Science 251: 1348-135 1.
CULLINGS,K. W., T. M. SZARO, and T. D. BRUNS. 1996. EvoSCHARDL,C. L. 1996. EpichZoB species: fungal symbionts of
lution of extreme specialization within a lineage of ectograsses. Ann. Rev. Phytopathol. 34: 109-130.
mycorrhizal epiparasites. Nature 379:63-66.
SCHARDL,
C. L., and Z. AN. 1993. Molecular biology and geDAVIS, J. I., and R. J. SORENG.1993. Phylogenetic structure in
netics of protective fungal endophytes of grasses. Pp. 191the grass family (Poaceae) as inferred from chloroplast
212 in J. K. SETLOW,ed. Genetic engineering, principles
DNA restriction site variation. Am. J. Bot. 80:1444-1454.
and methods. Plenum Press, New York.
FELSENSTEIN,J. 1993. PHYLIP 3.51~. University of WashingSCHARDL,
C. L., A. LEUCHTMANN,
H.-E TSAI, M. A. COLLETT,
ton, Seattle.
D. M. WATT, and D. B. Scorr. 1994. Origin of a fungal
FUNK, C. R., E C. BELANGER,and J. A. MURPHY.1994. Role
symbiont of perennial ryegrass by interspecific hybridizaof endophytes in grasses used for turf and soil conservation.
tion of a mutualist with the ryegrass choke pathogen, EpPp. 201-209 in C. W. BACON and J. E WHITE JR., eds.
ichlod typhinu. Genetics 136: 1307-l 3 17.
Biotechnology of endophytic fungi of grasses. CRC Press,
SCHARDL,C. L., J.-S. LIU, J. E WHITE, R. A. FINKEL,Z. AN,
Boca Raton, Fla.
and M. R. SIEGEL. 1991. Molecular phylogenetic relationGLENN,A. E., C. W. BACON,R. PRICE, and R. T. HANLIN.1996.
ships of nonpathogenic grass mycosymbionts and clavicipMolecular phylogeny of Acremonium and its taxonomic imitaceous plant pathogens. Plant Syst. Evol. 178:27-41.
plications. Mycologia 88:369-383.
SIEGEL,M. R., M. C. JOHNSON,D. R. VARNEY,W. C. NESMITH,
GWINN, K. D., and A. M. GAVIN. 1992. Relationship between
R. C. BUCKNER,L. l? BUSH, l? B. BURRUSII, T. A. JONES,
endophyte infestation level of tall fescue seed lots and Rhiand J. A. BOL~NG.1984. A fungal endophyte in tall fescue:
zoctoniu zeue seedling disease. Plant Dis. 76:911-914.
incidence and dissemination. Phytopathology 74:932-937.
HAFNER,M. S., and R. D. M. PAGE. 1995. Molecular phylogenies and host-parasite cospeciation: Gophers and lice as a SIEGEL,M. R., G. C. M. LATCH, L. l? BUSH, E E FANNIN,D.
D. ROWAN,B. A. TAPPER,C. W. BACON,and M. C. JOHNmodel system. Philos. Trans. R. Sot. Lond. B 349:77-83.
SON. 1990. Fungal endophyte-infected grasses: alkaloid acHINKLE,G., J. K. WETTERER,T. R. SCHULTZ,and M. L. SOGIN.
cumulation and aphid response. J. Chem. Ecol. 16:33011994. Phylogeny of the attine ant fungi based on analysis
3315.
of small subunit ribosomal RNA gene sequences. Science
SORENG,R. J., J. I. DAVIS, and J. J. DOYLE. 1990. A phylo266:1695-1697.
genetic analysis of chloroplast DNA restriction site variaKIMMONS,C. A., K. D. GWINN, and E. C. BERNARD.1990.
tion in Poaceae subfam Pooideae. Plant Syst. Evol. 172:83Nematode reproduction on endophyte-infected and endo97.
phyte-free tall fescue. Plant Dis. 74:757-761.
EpichloSGrassCospeciation 143
SWOFFORD,D. L. 1993. PAUP: phylogenetic analysis using
parsimony. Version 3.1.1. Illinois Natural History Survey,
Champaign, Ill.
THOMPSON,J. N. 1989. Concepts of coevolution. Trends Ecol.
Evol. 4: 179-183.
TSAI, H.-E, J.-S. LIU, C. STABEN,M. J. CHRISTENSEN,G. C.
M. LATCH, M. R. SIEGEL,and C. L. SCHARDL.1994. Evolutionary diversification of fungal endophytes of tall fescue
grass by hybridization with Epichlod species. Proc. Natl.
Acad. Sci. USA 91:2542-2546.
VILGALYS,R. 1991. Speciation and species concepts in the
Collybia dryophila complex. Mycologia 83:758-773.
VILGALYS,R., and B. L. SUN. 1994. Ancient and recent patterns of geographic speciation in the oyster mushroom Pleurotus revealed by phylogenetic analysis of ribosomal DNA
sequences. Proc. Natl. Acad. Sci. USA 91:4599-4603.
WEST, C. I? 1994. Physiology and drought tolerance of endophyte-infected grasses. Pp. 87-99 in C. W. BACONand J. E
WHITE JR., eds. Biotechnology of endophytic fungi of
grasses. CRC Press, Boca Raton, Fla.
WHITE, J. E JR. 1993. Endophyte-host associations in grasses.
XIX. A systematic study of some sympatric species of Epichlo2 in England. Mycologia 85:444-455.
-.
1994. Endophyte-host associations in grasses. XX.
Structural and reproductive studies of Epichlok’ amarillans
sp. nov. and comparisons to E. typhina. Mycologia 86:571580.
BARBARA A. SCHAAL, reviewing
Accepted
October
25, 1996
editor