Comparative phylogenetic analysis of bacterial associates in Pyrrhocoroidea and evidence for ancient and persistent environmental symbiont reacquisition in Largidae (Hemiptera: Heteroptera)
Authors: Gordon Eric Robert Lucien , McFrederick Quinn , Weirauch Christiane 
Published: July 2016

AbstractThe ancient insect order Hemiptera, one of the most well-studied insect lineages with respect to bacterial symbioses, still contains major branches which lack robust phylogenies and comprehensive characterization of associated bacterial symbionts. The Pyrrhocoroidea (Largidae [220 species]; Pyrrhocoridae [~300 species]) is a superfamily of the primarily-herbivorous hemipteran infraorder Pentatomomorpha, though relationships to related superfamilies are controversial. Studies on bacterial symbionts of this group have focused on members of Pyrrhocoridae, but recent examination of species of two genera of Largidae demonstrated divergent symbiotic complexes between these putative sister families. We surveyed bacterial diversity of this group using paired-end Illumina and targeted Sanger sequencing of bacterial 16S amplicons of 30 pyrrhocoroid taxa, including 17 species of Largidae, in order to determine the identity of bacterial associates and similarity of associated microbial communities among species. We also constructed the first comprehensive phylogeny of this superfamily (4,800 bp; 5 loci; 57 ingroup + 12 outgroup taxa) in order accurately trace the evolution of symbiotic complexes among Pentatomomorpha. We undertook multiple lines of investigation (i.e., experimental rearing, FISH microscopy, phylogenetic and co-evolutionary analyses) to understand potential transmission routes of largid symbionts. We found a prevalent, specific association of Largidae with plant-beneficial-environmental cladeBurkholderiahoused in midgut tubules. As in other distantly-related Heteroptera, symbiotic bacteria seem to be acquired from the environment every generation. We review current understanding of symbiotic complexes within the Pentatomomorpha and discuss means to further investigations of the evolution and function of these symbioses.ImportanceObligate symbioses with bacteria are common in insects, particularly for Hemiptera wherein varied forms of symbiosis occur, though knowledge of symbionts remains incomplete for major lineages. Thus, an accurate understanding of how these partnerships evolved and changed over millions of years is not yet achievable. We contribute to our understanding of the evolution of symbiotic complexes in Hemiptera by characterizing bacterial associates of Pyrrhocoroidea focusing on the family Largidae and by constructing a phylogeny to establish evolutionary relationships of and within this group. Members of Largidae are associated with specific symbioticBurkholderiafrom a different clade thanBurkholderiasymbionts in other Hemiptera and are members of the earliest-diverging superfamily ofBurkholderia-associated Hemiptera. Evidence suggests that species of Largidae reacquire specific symbiotic bacteria every generation environmentally, a rare strategy for insects with potentially volatile evolutionary ramifications, but one that has persisted in Largidae and other related lineages since the Cretaceous.