Multiple concurrent and convergent stages of genome reduction in bacterial symbionts across a stink bug family

Abstract

Nutritional symbioses between bacteria and insects are prevalent and diverse, allowing insects to expand their feeding strategies and niches. A common consequence of long-term associations is a considerable reduction in symbiont genome size likely influenced by the radical shift in selective pressures as a result of the less variable environment within the host. While several of these cases can be found across distinct insect species, most examples provide a limited view of a single or few stages of the process of genome reduction. Stink bugs (Pentatomidae) contain inherited gamma-proteobacterial symbionts in a modified organ in their midgut and are an example of a long-term nutritional symbiosis, but multiple cases of new symbiont acquisition throughout the history of the family have been described. We sequenced the genomes of 11 symbionts of stink bugs with sizes that ranged from equal to those of their free-living relatives to less than 20%. Comparative genomics of these and previously sequenced symbionts revealed initial stages of genome reduction including an initial pseudogenization before genome reduction, followed by multiple stages of progressive degeneration of existing metabolic pathways likely to impact host interactions such as cell wall component biosynthesis. Amino acid biosynthesis pathways were retained in a similar manner as in other nutritional symbionts. Stink bug symbionts display convergent genome reduction events showing progressive changes from a free-living bacterium to a host-dependent symbiont. This system can therefore be used to study convergent genome evolution of symbiosis at a scale not previously available.

Figure 1

Genomic characteristics of pentatomid symbionts. (a–f) Genomic characteristics as a function of genome size for stink bug symbionts. (a) number of genes annotated, (b) GC % of the entire genome, (c) percentage of conserved genes found according to the BUSCO gammaproteobacterial set (Dotted line in indicates the recommended 95% threshold of BUSCO completeness for new species descriptions). (d) tRNAs annotated, (e) coding density of the genome, (f) number of pseudogenes annotated by Prokka. Blue line indicates the LOWESS fitted curve. (g) Number of conserved genes between each genome and all larger genomes (excluding pseudogenes). (h) COG composition of the shared genes for non-symbiotic and large genome symbionts (> 3.8 Mb, dashed bar) and for all genomes including genome reduced symbionts (< 1.4 Mb, solid bar).

Figure 2

Host and symbiont relationships: (a) Stink bug mitochondrial tree with their respective symbiotic bacterial genome size. Nodes with lower than 60% bootstrap support were collapsed. Numbers above nodes represent bootstrap support while numbers underneath represent posterior probability from Bayesian Inference run. X indicates lower than 0.8 posterior probability. NA-genome size was not available. (b) Consensus cladogram of representative genomes of Erwiniaceae and stink bug symbionts. Individual FastTree reconstructions for the 10 longest protein sequences identified to be common for all taxa using reciprocal best hit Blast. E. coli was used as an outgroup. NCBI accession numbers for genomes are shown in parenthesis. Squares indicate a symbiont of a member of the Pentatominae while circles indicate symbionts of Edessinae. Numbers at nodes indicate Shimodaira-Hasegawa (SH)-like local support values. Taxa that were individually reconstructed are shown as polytomies in blue.

Figure 3

Presence of genes involved in branched chain amino acid biosynthesis pathway. The presence of genes in each genome according to the order of the pathway (a) and gene order and presence in the ilv operon region (b). Numbers in parentheses represent genome sizes in Mb. Colors are used to match genes in between panels for ease of visualization. Unlabeled grey pentagons denote genes not directly involved in the pathway.

Figure 4

Genes involved in the production of Lipid A and attachment of O-antigen. (a) Intermediate metabolites are represented in bold and the presence or absence of a gene is represented in the table, with a crossed box indicating not present in all members. (b) Progressive elimination of lpxA, lpxB, and lpxD in the SoE. (c) Loss of lpxK kdsB, and adjacent genes. A double grey line indicates a region of approximately 15 kb containing several genes not shown here. Genes labeled ‘hypoth.’ or unlabeled indicate hypothetical proteins or interrupted CDS. Asterisk indicates a fragmented CDS. Numbers in parentheses correspond to genome sizes in Mb.