Department of Biological Sciences, Louisiana State University at Baton Rouge; and
National Institute of Sericultural and Entomological Sciences, Tsukuba, Ibaraki, Japan
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Abstract |
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Introduction |
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Most known fungal endosymbionts of insects are true yeasts (Saccharomycetes), but phylogenetic analyses based on partial sequences of the nuclear small-subunit ribosomal RNA gene (rDNA) have suggested that two groups of endosymbionts were derived from within the filamentous ascomycetes (Euascomycetes): the anobiid beetle yeast-like endosymbionts (YLSs) (S. kochii and Symbiotaphrina buchneri) and several unnamed YLSs of planthoppers (Homoptera, Delphacidae: Laodelphax striatellus, Nilaparvata lugens, and Sogatella furcifera) and an aphid (Homoptera, Aphididae: Hamiltonaphis styraci). Studies of the anobiid YLSs indicated that they were derived from a filamentous ascomycete lineage outside the pyrenomycetes. Because of problems in acquiring and sampling vast numbers of taxa, the close filamentous relatives of the Symbiotaphrina 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 planthoppers. Although these YLSs were placed phylogenetically among taxa in Hypocreales, a more exact phylogenetic position was not determined because of incomplete sequence data and taxon sampling. A number of workers have suggested a need for additional sequences such as large-subunit rDNA to obtain better resolution and statistical support within the Hypocreales (Spatafora and Blackwell 1993
; Noda, Nakashima, and Koizumi 1995
; Rehner and Samuels 1995
; Fukatsu and 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 pyrenomycetes, including the Hypocreales. The dramatic changes required for the transition in morphology (filamentous growth to budding cells) and host relations (pathogenesis to obligate intracellular symbiosis) in the Hypocreales 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 involved 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, Kawamura, 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-subunit (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 isolated from planthopper and aphid hosts.
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Materials and Methods |
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Data Analyses
Sequences were aligned with other data obtained from nucleotide sequence libraries by using the multialignment program Clustal X (Thompson et al. 1997
). Species included in this study and their GenBank accession numbers of SSU and LSU rDNA sequences, respectively, were as follows: Aphysiostroma stercorarium (U32398; U47820), Aspergillus fumigatus (M60300; ), Atkinsonella hypoxylon (U44034; U57087), Balansia sclerotica (U32399; U47821), Beauveria bassiana IFO 4848 (AB027336; AB027382), Beauveria brongniartii (AB027335; AB027381), Blastomyces dermatitidis (M55624; ), Candida albicans (M60302; ), Candida tropicalis (M55527; ), Ceratocystis fimbriata (U32418; ), Cercophora septentrionalis (U32400; ), Chromocleista malachitea (D88323; ), Claviceps paspali (U32401; U47826), Claviceps purpurea (U44040; U57085), Cordycepioideus bisporus (AH006986; AF009654), Cordyceps capitata (U44041; U57086), Cordyceps ophioglossoides (U46881; U47827), Cryphonectria parasitica (L42441; ), Daldinia concentrica (U32402; ), Diaporthe phaseolorum (L36985; ), Diatrype disciformis (U32403; U47829), Echinodothis tuberiformis (U44042; U57083), Epichloë amarillians (U35034; U57680), Epichloë typhina (U32405; U17396), Eurotium rubrum (U00970; ), Exophiala jeanselmei (L36996; ), Fonsecaea pedrosoi (L36997; ), Hirsutella thompsonii (U32406; U47831), Hypocrea lutea (D14407; ), Hypocrea schweinitzii (L36986; U47833), Hypomyces chrysospermus (M89993; ), Hypomyces polyporinus (U32410; ), Hypoxylon atroroseum (U32411; ), Leucostoma persoonii (M83259; ), Metarhizium anisopliae IFO 5940 (AB027337; AB027383), Microascus trigonosporus (L36987;), Myriogenospora atramentosa (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), Penicillium chrysogenum (M55628; ), Phialophora verrucosa (L36999; ), Protomyces inouyei (D11377; ), Saccharomyces cerevisiae (Z75578; ), Sordaria fimicola (X69851; ), Sporothrix schenckii (M85053; ), Taphrina deformans (U00971; ), Trichoderma koningii (AF218790; ), Trichoderma viride (AF218788; ), Xylaria hypoxylon (U20378; U47841), yeast-like symbiont of Hamiltonaphis styraci (D55719; ).
Alignments were optimized visually, and ambiguous regions were excluded from the analyses. Maximum-parsimony analyses were performed using PAUP*, version 4.0b4a (Swofford 2000
). Heuristic tree searches were executed using the tree bisection-reconnection branch-swapping algorithm with random sequence analysis. The bootstrap values in most-parsimonious 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 empirical 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. MacClade software was used to trace host preferences of the clavicipitaceous fungi (Maddison and Maddison 1999
).
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Results and Discussion |
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Partial sequences of SSU rDNA from 56 taxa, which were selected from most major groups of ascomycetes, were compared with those of the planthopper YLSs using parsimony criteria; two archiascomycetes were used as outgroup taxa (fig. 1
). Of the 771 characters remaining after the ambiguous regions were excluded, 522 were constant, 56 were parsimony-uninformative, and 193 were parsimony-informative. The species of ascomycetes compared were well defined at the level of order, with comparatively high bootstrap values in the consensus of 272 most-parsimonious trees produced by the analysis. The phylogenetic relationships of the pyrenomycetes depicted here agree well with previous reports (Berbee and Taylor 1992
; Spatafora and Blackwell 1993
; Blackwell 1994
), although some deeper branches were neither well resolved nor supported statistically. Although a Clavicipitaceae clade was not well supported, the three traditional families within the order Hypocreales (Hypocreaceae, Nectriaceae, and Clavicipitaceae) represented independent lineages (fig. 1
). The planthopper YLSs grouped together on a long branch with the aphid (H. styraci) YLS with a 100% bootstrap value within the Clavicipitaceae. The comparison of SSU rDNA sequences could not fully resolve the phylogenetic relationships among species of the Clavicipitaceae, but two insect parasites that arose in an insect parasitic Cordyceps lineage (C. bisporus and H. thompsonii) were the closest sister taxa of the YLS clade.
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The host ranges of a set of taxa of Clavicipitaceae are shown mapped on the tree (fig. 3 , based on the combined 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. Members of the Clavicipitaceae were divided into several groups. Plant parasites, endophytes, fungal parasites, and some insect pathogens were separated from a Cordyceps clade of insect pathogens (C. bisporus and H. thompsonii) and the planthopper YLSs. In addition, a few species of Cordyceps are parasites of false truffles (Basidiomycetes), and Nikoh and Fukatsu (2000) estimated 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 particular, host shifts appear to have occurred repeatedly in the Clavicipitaceae, a family that contains many species that are leaf endophytes and parasites of arthropods, fungi, and plants (Alexopoulos, Mims, and Blackwell 1996
, p. 330). The work reported here is unique because it describes yet another host association, obligate endosymbiosis with Homoptera, derived from within a Cordyceps lineage of obligate insect pathogens with filamentous growth. The planthopper and aphid YLS lineage represents a relatively recent independent divergence of endosymbionts within Euascomycetes, and these fungi are related only distantly to the Symbiotaphrina 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.
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Acknowledgements |
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Footnotes |
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1 Keywords: coevolution
parasitism
host switching
molecular phylogeny
2 Address for correspondence and reprints: Sung-Oui Suh, Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803. ssuh{at}unix1.sncc.lsu.edu
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