Arsenophonus and Sodalis replacements shape evolution of symbiosis in louse flies

Abstract

Symbiotic interactions between insects and bacteria are ubiquitous and form a continuum from loose facultative symbiosis to greatly intimate and stable obligate symbiosis. In blood-sucking insects living exclusively on vertebrate blood, obligate endosymbionts are essential for hosts and hypothesized to supplement B-vitamins and cofactors missing from their blood diet. The role and distribution of facultative endosymbionts and their evolutionary significance as seeds of obligate symbioses are much less understood. Here, using phylogenetic approaches, we focus on the Hippoboscidae phylogeny as well as the stability and dynamics of obligate symbioses within this bloodsucking group. In particular, we demonstrate a new potentially obligate lineage of Sodalis co-evolving with the Olfersini subclade of Hippoboscidae. We also show several likely facultative Sodalis lineages closely related to Sodalis praecaptivus (HS strain) and suggest repeated acquisition of novel symbionts from the environment. Similar to Sodalis, Arsenophonus endosymbionts also form both obligate endosymbiotic lineages co-evolving with their hosts (Ornithomyini and Ornithoica groups) as well as possibly facultative infections incongruent with the Hippoboscidae phylogeny. Finally, we reveal substantial diversity of Wolbachia strains detected in Hippoboscidae samples falling into three supergroups: A, B, and the most common F. Altogether, our results prove the associations between Hippoboscoidea and their symbiotic bacteria to undergo surprisingly dynamic, yet selective, evolutionary processes strongly shaped by repeated endosymbiont replacements. Interestingly, obligate symbionts only originate from two endosymbiont genera, Arsenophonus and Sodalis, suggesting that the host is either highly selective about its future obligate symbionts or that these two lineages are the most competitive when establishing symbioses in louse flies.

Keywords: Arsenophonus; Louse flies; Phylogeny; Replacements; Sodalis; Wolbachia.

Figure 1. Host phylogeny.

(A) Host phylogeny derived from concatenation of three genes: 16S rRNA, EF, and COI. The phylogeny was reconstructed by BI analysis. Posterior probabilities and bootstrap support are printed upon branches, respectively (asterisk was used for very low or missing bootstrap branch support). Taxa labelled with voucher are newly sequenced in this study. Genomic COI sequences are labelled with rRNA. Three smaller trees on the top of the figure represent outlines of three separate phylogenetic trees based on BI analyses of 16S rRNA (B), EF (C), and COI (D) genes. Full versions of these phylogenies are included in Figs. S6–S8. Three main families of Hippoboscidae are colour coded: yellow for Lipopteninae (one group), brown for Hippoboscinae (one group), and orange for Ornithomiinae (three groups). Colour squares label branches where are placed main Hippoboscidae groups. This labelling corresponds with labelling of branches at smaller outlines, which are in addition to this highlighted with the same colour. All host trees are included in Figs. S1–S8.

Figure 2. 16S rRNA phylogeny of Arsenophonus in Hippoboscidae inferred by BI analysis.

Posterior probabilities and bootstrap support are printed upon branches, respectively (asterisk was used for very low or missing bootstrap branch support). Taxa labelled with voucher are newly sequenced in this study. Genomic sequences are labelled with rRNA. Taxa in dark purple represent Arsenophonus bacteria which genome was sequenced. Numbers behind these taxa correspond to their GC content of 16S rRNA, GC content of genome, and genome size, respectively. Numbers behind other taxa correspond to GC content of their 16S rRNA. The smaller picture on the right side represents host phylogeny to which symbiont phylogeny was compared. Red lineages correspond to obligate symbionts while orange lineage is symbiont of recent origin. The blue A represent likely facultative Arsenophonus infection. To achieve this, we also used the information available on groEL gene by Morse et al. (2013) and Duron et al. (2014). Phylogenetic reconstructions of Arsenophonus of entire Hippoboscoidea and all Arsenophonus bacteria are included in Figs. S9 and S10.

Figure 3. 16S rRNA phylogeny of Sodalis in Hippoboscidae inferred by BI analysis.

Posterior probabilities and bootstrap support are printed upon branches, respectively (asterisk was used for very low or missing bootstrap branch support). Taxa labelled with voucher are newly sequenced in this study. Taxa in dark purple represent Sodalis-like bacteria which genome was sequenced. Numbers behind these taxa correspond to their GC content of 16S rRNA, GC content of genome, and genome size, respectively. Numbers behind other taxa correspond to GC content of their 16S rRNA. Red lineages correspond to obligate symbionts while orange lineage is symbiont of recent origin. The red dashed line shows that co-evolution between Icosta spp. and their obligate endosymbiont imperfect.

Figure 4. Wolbachia phylogeny.

(A) Wolbachia phylogeny inferred from 16S rRNA by BI analysis. (B) Wolbachia phylogeny inferred from MLST genes by BI analysis. Posterior probabilities and bootstrap support are printed upon branches, respectively (asterisk was used for very low or missing bootstrap branch support). Colour letters upon branches correspond to Wolbachia supergroups. Taxa in red represent Wolbachia bacteria from Hippoboscidae and Nycteribidae which are newly sequenced in this study. Taxa labelled with # in the 16S tree represent taxa which were used for the MLST analysis. Wolbachia from O. biloba, which was obtained from genomic data, is labelled with rRNAob. Supergroup E was used for rooting both trees.