Content uploaded by Meredith Blackwell
Author content
All content in this area was uploaded by Meredith Blackwell on May 16, 2014
Content may be subject to copyright.
995
Mol. Biol. Evol. 18(6):995–1000. 2001
q
2001 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
Insect Symbiosis: Derivation of Yeast-like Endosymbionts Within an
Entomopathogenic Filamentous Lineage
Sung-Oui Suh,* Hiroaki Noda,†and Meredith Blackwell*
*Department of Biological Sciences, Louisiana State University at Baton Rouge; and †National Institute of Sericultural and
Entomological Sciences, Tsukuba, Ibaraki, Japan
Yeast-like endosymbionts (YLSs) of insects often are restricted to specific hosts and are essential to the host’s
survival. For example, in planthoppers (Homoptera: Delphacidae), endosymbionts function in sterol utilization and
nitrogen recycling for the hosts. Our study, designed to investigate evolutionary changes in the YLS lineage involved
in the planthopper association, strongly suggests an origin of the YLSs from within the filamentous ascomycetes
(Euascomycetes), not the true yeasts (Saccharomycetes), as their morphology might indicate. During divergence of
the planthopper YLSs, dramatic changes would have occurred in the insect-fungus interaction and the fungal mor-
phology that have previously been undescribed in filamentous ascomycetes. Phylogenetic trees were based on
individual and combined data sets of 2.6 kb of the nuclear small- and large-subunit ribosomal RNA genes for YLSs
from three rice planthoppers (Laodelphax striatellus, Nilaparvata lugens, and Sogatella furcifera) compared with
56 other fungi. Parsimony analysis placed the planthopper YLSs within Cordyceps (Euascomycetes: Hypocreales:
Clavicipitaceae), a genus of filamentous insects and a few fungal pathogenic ascomycetes. Another YLS species
restricted to the aphid Hamiltonaphis styraci (Homoptera: Aphididae) was a sister taxon to the planthopper YLSs.
Filamentous insect pathogens (Metarhizium and Beauveria) specific to the same species of insect hosts as the YLSs
also formed lineages within the Clavicipitaceae, but these were distinct from the clade comprising YLS species.
Trees constrained to include the YLSs in families of the Hypocreales other than the Clavicipitaceae were rejected
by the Kishino-Hasegawa test. In addition, the results of this study support a hypothesis of two independent origins
of insect-associated YLSs from among filamentous ascomycetes: the planthopper YLSs in the Clavicipitaceae and
the YLSs associated with anobiid beetles (Symbiotaphrina species). Several lineages of true yeasts (Saccharomy-
cetes) also formed endosymbiotic associations with beetles, but they were not closely related to either group derived
from the filamentous ascomycetes.
Introduction
Over the last century, the discovery of microbial
endosymbionts in a wide variety of arthropods has been
a significant finding in arthropod biology. For example,
the recognition that prokaryotic rickettsial endosymbi-
onts were widespread among arthropods and may induce
sterility in their hosts brought a new perspective to stud-
ies of arthropod speciation (Shoemaker, Katju, and Jae-
nike 1999). In contrast, although a number of fungal
endosymbionts of insects were previously reported, rel-
atively few were substantiated (e.g., Buchner 1965, pp.
24–25, 236). Obligate fungal gut endosymbionts are
known, however, in planthoppers and aphids (Homop-
tera) and three families of beetles (Coleoptera: Anobi-
idae, Cerambycidae, and Scolytidae; Nardon and Gren-
ier 1989). The fungal endosymbionts all appear to play
important roles in insect nutrition, broadening the range
of available resources by supplying enzymes for deg-
radation or detoxification of plant material. For example,
Symbiotaphrina kochii occurs in the gut of the anobiid
tobacco beetle Lasioderma serricorne, a pest in stored
tobacco. The single-celled fungus detoxifies plant ma-
terials ingested by the beetles (Dowd 1989, 1991).
Most known fungal endosymbionts of insects are
true yeasts (Saccharomycetes), but phylogenetic analy-
ses based on partial sequences of the nuclear small-sub-
Key words: coevolution, parasitism, host switching, molecular
phylogeny.
Address for correspondence and reprints: Sung-Oui Suh, Depart-
ment of Biological Sciences, Louisiana State University, BatonRouge,
Louisiana 70803. E-mail: ssuh@unix1.sncc.lsu.edu.
unit ribosomal RNA gene (rDNA) have suggested that
two groups of endosymbionts were derived from within
the filamentous ascomycetes (Euascomycetes): the an-
obiid beetle yeast-like endosymbionts (YLSs) (S. kochii
and Symbiotaphrina buchneri) and several unnamed
YLSs of planthoppers (Homoptera, Delphacidae: Lao-
delphax striatellus, Nilaparvata lugens, and Sogatella
furcifera) and an aphid (Homoptera, Aphididae: Ham-
iltonaphis styraci). Studies of the anobiid YLSs indi-
cated that they were derived from a filamentous asco-
mycete lineage outside the pyrenomycetes. Because of
problems in acquiring and sampling vast numbers of
taxa, the close filamentous relatives of the Symbiota-
phrina species could not be determined to provide more
information on changes occurring during evolution of
endosymbiosis (Jones and Blackwell 1996; Noda and
Kodama 1996; Jones, Dowd, and Blackwell 1999).
The planthopper YLSs, also suggested to be more
closely related to euascomycetes than to true yeasts,
were tentatively placed among taxa in the Hypocreales
on the basis of partial sequences of the nuclear small-
subunit rDNA (Noda, Nakashima, and Koizumi 1995).
More recently, Fukatsu and Ishikawa (1996) found that
the aphid YLS was closely related to those of the plan-
thoppers. Although these YLSs were placed phyloge-
netically among taxa in Hypocreales, a more exact phy-
logenetic position was not determined because of in-
complete sequence data and taxon sampling. A number
of workers have suggested a need for additional se-
quences such as large-subunit rDNA to obtain better res-
olution and statistical support within the Hypocreales
(Spatafora and Blackwell 1993; Noda, Nakashima, and
Koizumi 1995; Rehner and Samuels 1995; Fukatsu and
996 Suh et al.
Table 1
Taxa Sequenced in this Study and GenBank Accession Numbers of Sequences
F
UNGUS
S
OURCE
a
H
OST
G
EN
B
ANK
A
CCESSION
N
O
.
SSU rDNA LSU rDNA
Ls YLS .............................
Nl YLS .............................
Sf YLS .............................
Beauveria bassiana ...................
Metarhizium flavoviride var. minus ......
Metarhizium anisopliae ................
H. Noda
H. Noda
H. Noda
ARSEF 2427
ARSEF 1764
ARSEF 2037
ARSEF 3822
Delphacidae: Laodelphax striatellus
Delphacidae: Nilaparvata lugens
Delphacidae: Sogatella furcifera
Delphacidae: Nilaparvata lugens
Delphacidae: N. lugens
Delphacidae: N. lugens
Aphididae: Diuraphis noxia
AF267232
AF267233
AF267234
AF280633
AF280632
—
AF280631
AF267235
AF267236
AF267237
AF280637
AF280635
AF280636
AF280634
a
H. Noda is with the National Institute of Sericultural and Entomological Sciences, Japan; ARSEF
5
United States Department of Agriculture, Agricultural
Research Service (USDA-ARS) Collection of Entomopathogenic Fungal Cultures.
Ishikawa 1996; Suh et al. 1998; Suh and Blackwell
1999). In contrast to the situation encountered in the
study of Symbiotaphrina, taxon sampling was much less
problematic among the better-understood pyrenomyce-
tes, including the Hypocreales. The dramatic changes
required for the transition in morphology (filamentous
growth to budding cells) and host relations (pathogen-
esis to obligate intracellular symbiosis) in the Hypocre-
ales drew us to the planthopper YLSs in an attempt to
pinpoint the lineage from which the endosymbionts
were derived.
The planthopper YLSs have never been found to
be free-living in nature; they occur in the host fat body
and are transmitted to the offspring through the ovary
(Nasu 1963; Noda 1977). The YLSs apparently are in-
volved in an obligate association in which they utilize
sterol and help to recycle nitrogen within the fat bodies
of the insect hosts (Wetzel et al. 1992; Sasaki, Kawa-
mura, and Ishikawa 1996; Hongoh and Ishikawa 1997).
Planthopper YLSs have not been cultured; their cells,
however, can be separated by Percoll buoyant density
gradient centrifugation to allow for DNA extraction
(Noda and Omura 1992). In this study, we determined
about 2.6 kb of partial sequences of nuclear small-sub-
unit (SSU) and large-subunit (LSU) rDNA from each
planthopper YLS and compared these with other major
groups of ascomycetes. We also report sequences of
rDNA for species of the filamentous entomopathogenic
fungi Metarhizium and Beauveria that have been isolat-
ed from planthopper and aphid hosts.
Materials and Methods
Strains Used in the Study
YLSs from L. striatellus (Ls YLS), N. lugens (Nl
YLS), and S. furcifera (Sf YLS) were isolated and pu-
rified by the methods of Noda and Omura (1992), and
the freeze-dried cells were used directly for extracting
DNA. Isolates of Metarhizium and Beauveria were
grown for 1 week in 2% malt extract broth in 1.5-ml
tubes. Information on the sources and GenBank acces-
sion numbers for the sequences determined in this study
is given in table 1.
DNA Extraction, PCR, and Sequencing
Nucleic acids were extracted and purified following
the procedure of Lee and Taylor (1990). The primer sets
NS1–NS8 (White et al. 1990) and LS1–LSD (Hausner,
Reid, and Klassen 1993) were used for amplifying SSU
and LSU rDNA, respectively, by the polymerase chain
reaction (PCR). PCR products were purified using a
DNA purification kit (Bio-Rad Laboratories, Hercules,
Calif.), and the purified double-stranded PCR products
were used directly as templates for sequencing with an
ABI PRISM BigDye Terminator Cycle sequencing kit
(PE Applied Biosystems, Foster City, Calif.). Primers
used in sequencing were NS1, NS2, NS3, 18H, and NS8
for SSU rDNA and LS1, LR3, and LR5 for LSU rDNA
(White et al. 1990; Hausner, Reid, and Klassen 1993;
Rehner and Samuels 1995). DNA sequences were de-
termined by an ABI PRISM 377 automated DNA se-
quencer (PE Applied Biosystems).
Data Analyses
Sequences were aligned with other data obtained
from nucleotide sequence libraries by using the multi-
alignment program Clustal X (Thompson et al. 1997).
Species included in this study and their GenBank ac-
cession numbers of SSU and LSU rDNA sequences, re-
spectively, were as follows: Aphysiostroma stercorar-
ium (U32398; U47820), Aspergillus fumigatus
(M60300; —), Atkinsonella hypoxylon (U44034;
U57087), Balansia sclerotica (U32399; U47821), Beau-
veria bassiana IFO 4848 (AB027336; AB027382),
Beauveria brongniartii (AB027335; AB027381), Blas-
tomyces dermatitidis (M55624; —), Candida albicans
(M60302; —), Candida tropicalis (M55527; —), Cer-
atocystis fimbriata (U32418; —), Cercophora septen-
trionalis (U32400; —), Chromocleista malachitea
(D88323; —), Claviceps paspali (U32401; U47826),
Claviceps purpurea (U44040; U57085), Cordycepioi-
deus bisporus (AH006986; AF009654), Cordyceps cap-
itata (U44041; U57086), Cordyceps ophioglossoides
(U46881; U47827), Cryphonectria parasitica (L42441;
—), Daldinia concentrica (U32402; —), Diaporthe
phaseolorum (L36985; —), Diatrype disciformis
(U32403; U47829), Echinodothis tuberiformis (U44042;
Yeast-like Endosymbionts 997
F
IG
. 1.—Consensus of 272 most-parsimonious trees obtained
from SSU rDNA sequence data. The planthopper yeast-like endosym-
biont clade appears in boldface type, and orders of ascomycetes and
families of Hypocreales are indicated. Tree length
5
561; consistency
index
5
0.5455; homoplasy index
5
0.4545; retention index
5
0.8175; rescaled consistency index
5
0.4459. Numbers on tree branch-
es indicate the percentages of bootstrap samplings derived from 1,000
samples supporting the internal branches by
$
50%.
U57083), Epichloe¨ amarillians (U35034; U57680), Ep-
ichloe¨ typhina (U32405; U17396), Eurotium rubrum
(U00970; —), Exophiala jeanselmei (L36996; —), Fon-
secaea pedrosoi (L36997; —), Hirsutella thompsonii
(U32406; U47831), Hypocrea lutea (D14407; —), Hy-
pocrea schweinitzii (L36986; U47833), Hypomyces
chrysospermus (M89993; —), Hypomyces polyporinus
(U32410; —), Hypoxylon atroroseum (U32411; —),
Leucostoma persoonii (M83259; —), Metarhizium ani-
sopliae IFO 5940 (AB027337; AB027383), Microascus
trigonosporus (L36987;—), Myriogenospora atramen-
tosa (U44114; U57084), Nectria haematococca
(U32413; —), Neocosmospora vasinfecta (U44117 ;
U47836), Neotyphodium coenophialum (U45942;
U57681), Neurospora crassa (X04971; —).Ophiostoma
stenoceras (M85054; —), Ophiostoma ulmi (M83261;
—), Paecilomyces tenuipes (D85136; U47838), Penicil-
lium chrysogenum (M55628; —), Phialophora verru-
cosa (L36999; —), Protomyces inouyei (D11377; —),
Saccharomyces cerevisiae (Z75578; —), Sordaria fimi-
cola (X69851; —), Sporothrix schenckii (M85053; —),
Taphrina deformans (U00971; —), Trichoderma kon-
ingii (AF218790; —), Trichoderma viride (AF218788;
—), Xylaria hypoxylon (U20378; U47841), yeast-like
symbiont of Hamiltonaphis styraci (D55719; —).
Alignments were optimized visually, and ambigu-
ous regions were excluded from the analyses. Maxi-
mum-parsimony analyses were performed using
PAUP*, version 4.0b4a (Swofford 2000). Heuristic tree
searches were executed using the tree bisection-recon-
nection branch-swapping algorithm with random se-
quence analysis. The bootstrap values in most-parsi-
monious trees were obtained from 1,000 replications.
Maximum-likelihood analyses (Kishino and Hasegawa
1989) for tree scoring were performed using PAUP*,
version 4.0b4a, or the DNAML program in the PHYLIP
package, version 3.572c (Felsenstein 1995), with em-
pirical frequencies and a transition/transversion ratio of
2.0. The partition homogeneity test option in PAUP*
with 1,000 replicates was used to determine whether the
SSU and LSU rDNA data sets were in conflict. Mac-
Clade software was used to trace host preferences of the
clavicipitaceous fungi (Maddison and Maddison 1999).
Results and Discussion
Approximately 1,730 bp of SSU rDNA represent-
ing most of the gene were obtained from PCR products
of the YLSs of the planthoppers and Metarhizium and
Beauveria isolates. The SSU rDNA of B. bassiana (AR-
SEF 2427) and M. flavoviridae var. minus (ARSEF
1764) had insertions of several hundred bases. The in-
sertions occurred at positions 943 and 1199 of the SSU
rDNA of Escherichia coli and were found to be major
groups of group I introns (IC or IE) based on secondary
structure analysis (Michel and Westhof 1990; Suh,
Jones, and Blackwell 1999). Intron sequences were ex-
cluded from the analyses, and details of the introns will
not be discussed here. For the LSU rDNA, about 900
bp of the gene including the variable D1/D2 region were
sequenced from the PCR products. Two isolates of M.
flavoviridae var. minus had identical sequences in LSU
rDNA.
Partial sequences of SSU rDNA from 56 taxa,
which were selected from most major groups of asco-
mycetes, were compared with those of the planthopper
YLSs using parsimony criteria; two archiascomycetes
were used as outgroup taxa (fig. 1). Of the 771 char-
acters remaining after the ambiguous regions were ex-
cluded, 522 were constant, 56 were parsimony-uninfor-
mative, and 193 were parsimony-informative. The spe-
cies of ascomycetes compared were well defined at the
level of order, with comparatively high bootstrap values
in the consensus of 272 most-parsimonious trees pro-
duced by the analysis. The phylogenetic relationships of
the pyrenomycetes depicted here agree well with pre-
vious reports (Berbee and Taylor 1992; Spatafora and
Blackwell 1993; Blackwell 1994), although some deeper
branches were neither well resolved nor supported sta-
tistically. Although a Clavicipitaceae clade was not well
supported, the three traditional families within the order
Hypocreales (Hypocreaceae, Nectriaceae, and Clavicip-
itaceae) represented independent lineages (fig. 1). The
planthopper YLSs grouped together on a long branch
with the aphid (H. styraci) YLS with a 100% bootstrap
998 Suh et al.
F
IG
. 2.—The best of five most-parsimonious trees obtained by
analysis of combined SSU and LSU rDNA data sets places the yeast-
like endosymbiont (YLS) clade in Clavicipitaceae with somewhat bet-
ter resolution and support than with analyses of the individual gene
sequences (see also fig. 1). Tree length
5
684; consistency index
5
0.5760; homoplasy index
5
0.4240; retention index
5
0.6827; rescaled
consistency index
5
0.3933;
2
ln likelihood
5
6,366.76796. Trees con-
strained to place the YLS lineage within Hypocreaceae or Nectriaceae
had log likelihoods significantly worse than the most-parsimonious tree
shown here.
F
IG
. 3.—Tree diagram based on the combined data of figure 2
(branches with
,
80% bootstrap values collapsed) indicates that host
preferences of clavicipitaceous taxa, including the insect endosymbi-
otic habit, have a phylogenetic distribution. The yeast-like endosym-
bionts are derived within an insect-pathogenic Cordyceps clade (Cor-
dycepioideus bispous and Hirsutella thompsonii).
value within the Clavicipitaceae. The comparison of
SSU rDNA sequences could not fully resolve the phy-
logenetic relationships among species of the Clavicipi-
taceae, but two insect parasites that arose in an insect
parasitic Cordyceps lineage (C. bisporus and H. thomp-
sonii) were the closest sister taxa of the YLS clade.
In the partition homogeneity test for the SSU and
LSU rDNA sequence data, the observed summed tree
lengths of 675 steps fell within the distribution of ran-
domized data sets (P
5
0.501), indicating that there was
not significant conflict between them, and the data were
combined. The combined data set from 28 taxa using
two species of the Xylariales as outgroup taxa was an-
alyzed using parsimony criteria (fig. 2). After excluding
ambiguous regions of the remaining 1,577 characters in
the combined data set, 1,255 were constant and 228
were parsimony-informative. Of five most-parsimonious
trees, the tree with the best score in the Kishino-Hase-
gawa test provided somewhat better resolution and sup-
port for the branches within the Clavicipitaceae (fig. 2).
As in the case of the tree derived from the independent
SSU data set, the YLSs and the insect parasites, C. bis-
porus and H. thompsonii, formed a clade, but it was
supported by a higher (90%) bootstrap value. Once
again, the YLS lineage was separated from C. bisporus
and H. thompsonii by a comparatively long branch
length. Placement of the planthopper YLSs within the
Clavicipitaceae was supported by the Kishino-Hasegawa
test. Two constraint trees placing the YLSs inother fam-
ilies of the Hypocreales (Hypocreaceae and Nectriaceae)
were significantly worse than the best tree (fig. 2). The
differences of ln Lvalues (
2
ln L), the standard deviation
of the difference (SD), and the Pvalue for significance
between the best and other constraint trees were as fol-
lows: (1) for YLSs in the Hypocreaceae,
2
ln L
5
2
119.39787, SD
5
28.55769, P
,
0.0001; (2) for YLSs
in the Nectriaceae,
2
ln L
52
116.92290, SD
5
27.85217, P
,
0.0001 (fig. 3).
The insect pathogen C. bisporus is characterized by
perithecia produced in a Cordyceps-like stroma, dark as-
cospores, and a Hirsutella anamorph. It has been placed
among species of Cordyceps based on its morphological
and molecular characteristics (Suh et al. 1998). Since
culturing of the YLSs from planthoppers has not been
successful, the morphological characteristics could not
be fully compared with related taxa in the Clavicipita-
ceae, but they appeared very dissimilar. Under the com-
pound microscope, the purified cells were typical yeast-
like cells with budding; they lacked pseudohyphae or
true hyphae. The YLSs appear to have adapted to the
intracellular habit in planthoppers, perhaps even having
lost some genes essential for hyphal growth. Because of
their unique habit as endosymbionts and their restricted
yeast-like growth form, the YLSs not only stand out
among the Clavicipitaceae, but they are also distinct
among all of the Hypocreales.
The host ranges of a set of taxa of Clavicipitaceae
are shown mapped on the tree (fig. 3, based on the com-
bined data of fig. 2). Although some lineages were not
strongly supported by bootstrap analysis and the number
of taxa was limited, host ranges of the taxa were related
to their phylogenetic positions within the family. Mem-
bers of the Clavicipitaceae were divided into several
groups. Plant parasites, endophytes, fungal parasites,
and some insect pathogens were separated from a Cor-
dyceps clade of insect pathogens (C. bisporus and H.
Yeast-like Endosymbionts 999
thompsonii) and the planthopper YLSs. In addition, a
few species of Cordyceps are parasites of false truffles
(Basidiomycetes), and Nikoh and Fukatsu (2000) esti-
mated that a member of the genus Cordyceps jumped
from an arthropod to a fungal host about 43 MYA. If
their estimate is correct, the YLS lineage would be
younger than 43 Myr old because it diverged after the
fungal (C. ophioglossioides and C. capitata)–insect
pathogen split in Cordyceps (fig. 3).
Species of Hypocreales are known for their wide
variety of associations with other organisms; in partic-
ular, host shifts appear to have occurred repeatedly in
the Clavicipitaceae, a family that contains many species
that are leaf endophytes and parasites of arthropods, fun-
gi, and plants (Alexopoulos, Mims, and Blackwell 1996,
p. 330). The work reported here is unique because it
describes yet another host association, obligate endo-
symbiosis with Homoptera, derived from within a Cor-
dyceps lineage of obligate insect pathogens with fila-
mentous growth. The planthopper and aphid YLS line-
age represents a relatively recent independent diver-
gence of endosymbionts within Euascomycetes, and
these fungi are related only distantly to the Symbiota-
phrina YLSs of anobiid beetles. The planthopper YLSs
are even more distantly related to lineages of the true
yeasts that are associates of several groups of beetles.
Acknowledgments
We thank Dr. Joseph W. Spatafora for helpful discus-
sions on taxon sampling. Dr. R. A. Humber provided
cultures of Metarhizium and Beauveria from the ARSEF
collection. Ms. Debra Waters and Ms. Ning Zhang
helped to improve the manuscript. This study was sup-
ported by the National Science Foundation (DEB-
0072741 to M.B.).
LITERATURE CITED
A
LEXOPOULOS
, C. J., C. W. M
IMS
, and M. B
LACKWELL
. 1996.
Introductory mycology. John Wiley and Sons, New York.
B
ERBEE
, M. L., and J. W. T
AYLOR
. 1992. Two ascomycete
classes based on fruiting-body characters and ribosomal
DNA sequence. Mol. Biol. Evol. 9:278–284.
B
LACKWELL
, M. 1994. Minute mycological mysteries: the in-
fluence of arthropods on the lives of fungi. Mycologia 86:
1–17.
B
UCHNER
, P. 1965. Endosymbiosis of animals with plant mi-
croorganisms. John Wiley and Sons, New York.
D
OWD
, P. F. 1989. In situ production of hydrolytic detoxifying
enzymes by symbiotic yeasts of cigarette beetle (Coleop-
tera: Anobiidae). J. Econ. Entomol. 82:396–400.
———. 1991. Symbiont-mediated detoxification in insect her-
bivores. Pp. 411–440 in P. B
ARBOSA
,V.A.K
RISCHIK
, and
C. G. J
ONES
, eds. Microbial mediation of plant-herbivore
interactions. John Wiley and Sons, New York.
F
ELSENSTEIN
, J. 1995. PHYLIP (phylogeny inference package).
Version 3.572c. Distributed by the author, Department of
Genetics, University of Washington, Seattle.
F
UKATSU
, T., and H. I
SHIKAWA
. 1996. Phylogenetic position of
yeast-like symbiont of Hamiltonaphis styraci (Homoptera,
Aphididae) based on 18S rDNA sequence. Insect Biochem.
Mol. Biol. 26:383–388.
H
AUSNER
, G., J. R
EID
, and G. R. K
LASSEN
. 1993. On the sub-
division of Cerarocystis s. l., based on partial ribosomal
DNA sequences. Can. J. Bot. 71:52–63.
H
ONGOH
, Y., and H. I
SHIKAWA
. 1997. Uric acid as a nitrogen
resource for the brown planthopper, Nilaparvata lugens:
studies with synthetic diets and aposymbiotic insects. Zool.
Sci. 14:581–586.
J
ONES
, K. G., and M. B
LACKWELL
. 1996. Ribosomal DNA se-
quence analysis places the yeast-like genus Symbiotaphrina
within filamentous ascomycetes. Mycologia 88:212–218.
J
ONES
, K. G., P. F. D
OWD
, and M. B
LACKWELL
. 1999. Poly-
phyletic origins of yeast-like endocytobionts from anobiid
and cerambycid beetles. Mycol. Res. 103:542–546.
K
ISHINO
, H., and M. H
ASEGAWA
. 1989. Evaluation of the max-
imum likelihood estimate of the evolutionary tree topolo-
gies from DNA sequence data, and the branching order in
hominoid. J. Mol. Evol. 29:170–179.
L
EE
, S. B., and J. W. T
AYLOR
. 1990. Isolation of DNA from
fungal mycelia and single spores. Pp. 282–287 in M. A.
I
NNIS
,D.H.G
ELFAND
,J.J.S
NINSKY
, and T. J. W
HITE
, eds.
PCR protocols—a guide to methods and applications. Ac-
ademic Press, San Diego.
M
ADDISON
, W. P., and D. R. M
ADDISON
. 1999. MacClade. Ver-
sion 4. Sinauer, Sunderland, Mass.
M
ICHEL
, F., and F. W
ESTHOF
. 1990. Modelling of the three-
dimensional architecture of group I catalytic introns based
on comparative sequence analysis. J. Mol. Biol. 216:585–
610.
N
ARDON
, P., and A. M. G
RENIER
. 1989. Endosymbiosis in Co-
leoptera: biological, biochemical, and genetic aspects. Pp.
175–216 in W. S
CHWEMMLER
and G. G
ASSNER
, eds. Insect
endocytobiosis: morphology, physiology, genetics, evolu-
tion. CRC Press, Boca Raton, Fla.
N
ASU
, S. 1963. Studies on some leafhoppers and planthoppers
which transmit virus disease of rice plant in Japan. Bull.
Kyushu Agric. Exp. Stn. 8:153–349.
N
IKOH
, N., and T. F
UKATSU
. 2000. Interkingdom host jumping
underground: phylogenetic analysis of entomoparasitic fun-
gi of the genus Cordyceps. Mol. Biol. Evol. 17:629–638.
N
ODA
, H. 1977. Histological and histochemical observation of
intracellular yeastlike symbiotes in the fat body of the
smaller brown planthopper,Laodelphax striatellus (Homop-
tera: Delphacidae). Appl. Entomol. Zool. 12:134–141.
N
ODA
, H., and K. K
ODAMA
. 1996. Phylogenetic position of
yeastlike endosymbionts of anobiid beetles. Appl. Environ.
Microbiol. 62:162–167.
N
ODA
, H., N. N
AKASHIMA
, and M. K
OIZUMI
. 1995. Phyloge-
netic position of yeast-like symbiotes of rice planthoppers
based on partial 18S rDNA sequences. Insect Biochem.
Mol. Biol. 25:639–646.
N
ODA
, H., and T. O
MURA
. 1992. Purification of yeast-like sym-
biotes of planthoppers. J. Invertebr. Pathol. 59:104–105.
R
EHNER
, S. A., and G. J. S
AMUELS
. 1995. Molecular system-
atics of the Hypocreales: a teleomorph gene phylogeny and
the status of their anamorphs. Can. J. Bot. 73:S816–S823.
S
ASAKI
, T., M. K
AWAMURA
, and H. I
SHIKAWA
. 1996. Nitrogen
recycling in the brown planthopper, Nilaparvata lugens: in-
volvement of yeast-like endosymbionts in uric acid metab-
olism. J. Insect Physiol. 42:125–129.
S
HOEMAKER
, D. D., V. K
ATJU
, and J. J
AENIKE
. 1999. Wolba-
chia and the evolution of reproductive isolation between
Drosophilla recens and Drosophila subquinaria. Evolution
53:1157–1164.
S
PATAFORA
, J. W., and M. B
LACKWELL
. 1993. Molecular sys-
tematics of unitunicate perithecial ascomycetes: the Clavi-
cipitales-Hypocreales connection. Mycologia 85:912–922.
1000 Suh et al.
S
UH
, S.-O., and M. B
LACKWELL
. 1999. Molecular phylogeny
of the cleistothecial fungi placed in Cephalothecaceae and
Pseudeurotiaceae. Mycologia 91:836–848.
S
UH
, S.-O., K. G. J
ONES
, and M. B
LACKWELL
1999. A Group
I intron in nuclear small subunit rRNA gene of Crypten-
doxyla hypophloia: evidence for a new major class of Group
I introns. J. Mol. Evol. 48:493–500.
S
UH
, S.-O., J. W. S
PATAFORA
,G.R.S.O
CHIEL
,H.C.E
VANS
,
and M. B
LACKWELL
. 1998. Molecular phylogenetic study
of a termite pathogen Cordycepioideus bisporus. Mycologia
90:611–617.
S
WOFFORD
, D. L. 2000. PAUP*. Phylogenetic analysis using
parsimony (*and other methods). Version 4. Sinauer, Sun-
derland, Mass.
T
HOMPSON
, J. D., T. J. G
IBSON
,F.P
LEWNIAK
,F.J
EANMOUGIN
,
and D. G. H
IGGINS
. 1997. The CLUSTAL X windows in-
terface: flexible strategies for multiple sequence alignment
aided by quality analysis tool. Nucleic Acids Res. 25:4876–
4882.
W
ETZEL
, J. M., M. O
HNISHI
,T.F
UJITA
,K.N
AKANISHI
,Y.
N
AYA
,H.N
ODA
, and M. S
UGIURA
. 1992. Diversity in ste-
roidogenesis of symbiotic microorganisms from planthop-
pers. J. Chem. Ecol. 18:2083–2094.
W
HITE
, T. J., T. B
RUNS
,S.L
EE
, and J. T
AYLOR
. 1990. Ampli-
fication and direct sequencing of fungal ribosomal RNA
genes for phylogenetics. Pp. 315–322 in M. A. I
NNIS
,D.
H. G
ELFAND
,J.J.S
NINSKY
, and T. J. W
HITE
, eds. PCR
protocols—a guide to methods and applications. Academic
Press, San Diego.
B. F
RITZ
L
ANG
, reviewing editor
Accepted February 12, 2001
