Symbiosis and insect diversification: an ancient symbiont of sap-feeding insects from the bacterial phylum Bacteroidetes

Abstract

Several insect groups have obligate, vertically transmitted bacterial symbionts that provision hosts with nutrients that are limiting in the diet. Some of these bacteria have been shown to descend from ancient infections. Here we show that the large group of related insects including cicadas, leafhoppers, treehoppers, spittlebugs, and planthoppers host a distinct clade of bacterial symbionts. This newly described symbiont lineage belongs to the phylum Bacteroidetes. Analyses of 16S rRNA genes indicate that the symbiont phylogeny is completely congruent with the phylogeny of insect hosts as currently known. These results support the ancient acquisition of a symbiont by a shared ancestor of these insects, dating the original infection to at least 260 million years ago. As visualized in a species of spittlebug (Cercopoidea) and in a species of sharpshooter (Cicadellinae), the symbionts have extraordinarily large cells with an elongate shape, often more than 30 mum in length; in situ hybridizations verify that these correspond to the phylum Bacteroidetes. "Candidatus Sulcia muelleri" is proposed as the name of the new symbiont.

FIG. 1.

Phylogenetic relationships of auchenorrhynchan lineages represented in the current study plus other major lineages of Hemiptera. The tree represents relationships with substantial support, as compiled from recent studies. Nodes for which no firm support is available or for which conflicting results have been reported are collapsed. Abbreviations in parentheses indicate study species as indicated in Table 1. Letters next to nodes refer to relevant studies giving support to particular clades, as follows (clade names are given for those with available nomenclature): a, Auchenorrhyncha-Sternorrhyncha-Coleorrhyncha-Heteroptera (36, 42, 48, 49); b, Fulgoromorpha (4, 6, 36, 42, 46); c, unnamed clade (4, 36); d, Dictyopharidae-Fulgoridae (reference and references therein); e, Cicadomorpha (36, 46); f, Cicadoidea (16); g, Cercopoidea (and relationships within Cercopoidea) (14); h, Membracoidea (16, 46); i, Cicadellinae-Coelidiinae (and others) (16); j, Deltocephalinae-Membracidae (and others) (16); k, Membracidae (and relationships within Membracidae) (12, 13, 16). Dates are minimum ages for the subsequent node, based on fossils assigned to the descendant clade (4, 20, 21, 27, 38, 39, 40). My, million years.

FIG. 2.

Maximum-likelihood phylogram based on 16S rRNA genes of the symbionts (sym.) isolated from auchenorrhynchous insects and closely related members of the phylum Bacteroidetes. The topology of the likelihood tree is almost identical to that derived from Bayesian and parsimony analyses. Symbiont terminals are labeled with the host taxon, and outgroup terminals are labeled with the bacterial species name (see Table 1 for abbreviations) and GenBank accession number. Support values from analyses in which outgroups were included are indicated below the branch as nonparametric parsimony bootstrap value/nonparametric likelihood bootstrap value/Bayesian posterior probability, for branches on which all three support values are greater than 50. Asterisks indicate cases in which all three of the support values are 100.

FIG. 3.

Comparison of the a-symbiont as depicted by Müller (33) and FISH with symbionts from Auchenorrhyncha bacteriomes. (A) Reproduction of drawing (33) of a-symbionts dissected from the bacteriome of Philaenus spumarius (Cercopoidea). (B and C) FISH of the Bacteroidetes symbiont (“Candidatus Sulcia muelleri” [Sm]) dissected from the bacteriomes of Clastoptera arizonana (Cercopoidea) intermixed with round Buchnera aphidicola (Ba) cells from a pea aphid as controls. (B) FISH results from a Bacteroidetes diagnostic rRNA probe with 6-carboxytetramethylrhodamine (red); (C) FISH results with a Gammaproteobacteria-diagnostic rRNA probe with Alexa488 (green). (D) Superimposed FISH images of bacteriome contents of the sharpshooter Homalodisca lacerta, showing “Candidatus Sulcia muelleri” (Sm) (red) and “Candidatus Baumannia cicadellinicola” (Bc) (green).

FIG. 4.

Proposed history of the association between the Bacteroidetes symbiont and its insect hosts, as reconstructed from the insect phylogeny presented in Fig. 1. Abbreviations in parentheses indicate study species as indicated in Table 1. Heavy lines indicate lineages that were continuously associated with the Bacteroidetes symbiont, assuming a single initial infection in an ancestral lineage. Depending on the as-yet-unknown resolution of basal branches within Hemiptera, heteropteran or coleorrhynchan lineages either have lost ancestral infections, have never been infected, or retain undetected infections. Phylogenetic analyses of the symbionts provide strong support for clades defined by the marked nodes and do not conflict with the remaining nodes in the host tree.