SummaryVirtually all aphids maintain an obligate mutualistic symbiosis with bacteria from theBuchneragenus, which produce essential nutrients for their aphid hosts. Most aphids from the Lachninae subfamily have been consistently found to house additional endosymbionts, mainlySerratia symbiotica. This apparent dependence on secondary endosymbionts was proposed to have been triggered by the loss of the riboflavin biosynthetic capability byBuchnerain the Lachninae last common ancestor. However, an integral large-scale analysis of secondary endosymbionts in the Lachninae is still missing, hampering the interpretation of the evolutionary and genomic analyses of these endosymbionts. Here, we analysed the endosymbionts of selected representatives from seven different Lachninae genera and nineteen species, spanning four tribes, both by FISH (exploring the symbionts’ morphology and tissue tropism) and 16S rRNA gene sequencing. We demonstrate that all analysed aphids possess dual symbiotic systems, and while most harbourS. symbiotica, some have undergone symbiont replacement by other phylogenetically-distinct bacterial taxa. We found that these secondary associates display contrasting cell shapes and tissue tropism, and some appear to be lineage-specific. a scenario for symbiont establishment in the Lachninae, followed by changes in the symbiont’s tissue tropism and symbiont replacement events, thereby highlighting the extraordinary versatility of host-symbiont interactions.Originality-Significance StatementA key question in evolutionary biology is that of how mutualism evolves. One way to approach this problem is to investigate recently-established mutualistic associations, particularly by comparing various symbiotic systems in closely related hosts. Here, we present a most comprehensive study to investigate co-obligate symbioses in aphids, focusing in the Lachninae subfamily. While most aphids keep an obligate vertically-transmitted association with intracellularBuchnerabacteria, some, such as members of the Lachninae subfamily, host an additional putative co-obligate symbiont. Thus, the Lachninae dual symbiotic systems offer a unique opportunity to understand the evolutionary dynamics of host-symbiont associations, in particularly how secondary symbionts become obligate and eventually may be replaced. Through genome sequencing of three aphid species belonging to distantly related tribes within the subfamily, we have previously corroborated that they have indeed established co-obligate mutualistic associations with theS. symbioticasecondary endosymbiotic bacterium. This was putatively facilitated by an ancient pseudogenisation of the riboflavin biosynthetic pathway inBuchnera, rendering it unable to provide the essential vitamin to the host. However, not all Lachninae members harbourS. symbiotica, some species being associated to at least four different bacterial taxa. To correctly interpret the genomic data and to understand the evolutionary dynamics of these symbiotic associations, a wide-range analysis of both the phylogenetic relations as well as of the secondary symbionts’ localisation within the bacteriome is needed. To tackle this, we have combined phylogenetic analyses of the symbionts’ 16S rRNA gene sequences and FISH microscopy, to understand the symbiont’s identity as well as the morphological characteristics and tissue tropism. The phylogenetic affinities and patterns of co-divergence of the symbionts, in combination with previously published genomic data, have enabled us to build an evolutionary scenario for the establishment, changes in tissue tropism such as “stable” internalisation into bacteriocytes, and replacements of the putative “ancient” secondary endosymbiont from the Lachninae last common ancestor. Also, we were able to determine through phylogenetic analyses that some putative co-obligate endosymbionts may have evolved from once facultative ones. The evolutionary framework presented here reveals a dynamic pattern for the more recent evolutionary history of these symbioses, including replacement and novel acquisition of phylogenetically different co-obligate symbionts. This study opens new research avenues on this symbiont-diverse subfamily, providing insight into how mutualism in endosymbiotic associations can evolve, and the role these bacteria have played in the species’ adaptation and even in the speciation process.
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