Physiological and evolutionary contexts of a new symbiotic species from the nitrogen-recycling gut community of turtle ants

Abstract

While genome sequencing has expanded our knowledge of symbiosis, role assignment within multi-species microbiomes remains challenging due to genomic redundancy and the uncertainties of in vivo impacts. We address such questions, here, for a specialized nitrogen (N) recycling microbiome of turtle ants, describing a new genus and species of gut symbiont—Ischyrobacter davidsoniae (Betaproteobacteria: Burkholderiales: Alcaligenaceae)—and its in vivo physiological context. A re-analysis of amplicon sequencing data, with precisely assigned Ischyrobacter reads, revealed a seemingly ubiquitous distribution across the turtle ant genus Cephalotes, suggesting ≥50 million years since domestication. Through new genome sequencing, we also show that divergent I. davidsoniae lineages are conserved in their uricolytic and urea-generating capacities. With phylogenetically refined definitions of Ischyrobacter and separately domesticated Burkholderiales symbionts, our FISH microscopy revealed a distinct niche for I. davidsoniae, with dense populations at the anterior ileum. Being positioned at the site of host N-waste delivery, in vivo metatranscriptomics and metabolomics further implicate I. davidsoniae within a symbiont-autonomous N-recycling pathway. While encoding much of this pathway, I. davidsoniae expressed only a subset of the requisite steps in mature adult workers, including the penultimate step deriving urea from allantoate. The remaining steps were expressed by other specialized gut symbionts. Collectively, this assemblage converts inosine, made from midgut symbionts, into urea and ammonia in the hindgut. With urea supporting host amino acid budgets and cuticle synthesis, and with the ancient nature of other active N-recyclers discovered here, I. davidsoniae emerges as a central player in a conserved and impactful, multipartite symbiosis.

Domestication and evolutionary histories of specialized gut symbionts across cephalotine ants

“…Phylogenetic inferences for turtle ant symbionts have previously relied on partial sequencing of the 16S rRNA gene. While useful as a first step, relationships between symbionts and broader placement on the tree of life remained partially ambiguous due to an occasional lack of strong bootstrap support, and conflicting results between studies (Anderson et al., 2012; Béchade et al., 2023; Hu et al., 2014, 2018). To more finely resolve our understanding of the phylogenetic relationships between host‐specific symbionts of Cephalotes and Procryptocerus , we used concatenated protein, or protein‐coding gene alignments obtained from symbiont genomes, inferring phylogenies for each of the nine core symbiont orders.…”

“…Cephalotine ants (Cephalotes and Procryptocerus spp.) comprise one social insect group that relies on a diverse nutritionally beneficial symbiont community, with evidence for roles in nitrogen recycling and amino acid provisioning (Béchade et al, 2023;Duplais et al, 2021;Hu et al, 2018). Prior studies suggest a strongly conserved community composition within the gut of worker ants, consisting of host-specific bacteria from nine orders (Anderson et al, 2012;Hu et al, 2014Hu et al, , 2018Hu et al, , 2023.…”

“…Despite extensive 16S rRNA-based work suggesting separate domestications of cephalotine symbionts (Hu et al, 2014(Hu et al, , 2018 and recent lineage-specific, small-scale protein phylogenetics towards the same end (Béchade et al, 2023;Hu et al, 2023), a full and rigorous description of symbiont domestication history and subsequent evolution requires a multi-locus approach. Fortunately, a moderate fraction of gut community members are cultivable, enabling the generation of genomic data with as of yet untapped potential for higher resolution phylogenetics.…”

Molecular ecology of microbiomes in the wild: Common pitfalls, methodological advances and future directions

“…While microbial community composition and diversity have been extensively studied in microbiome ecology, there is a significant need to explore the functional attributes of whole communities, localized populations, and individual microorganisms (genes and pangenomes) within a microbiome. Investigating microbial functions, such as metabolic pathways and molecular interactions between members of the microbiome and with the host, can provide important insights into the contributions of specific microbial taxa/consortia and their functional roles in host and ecosystem health (Béchade et al, 2023;e.g. Hicks et al, 2018;Karmacharya et al, 2019).…”

Molecular ecology and microbiomes in the wild: methodological advances, common pitfalls and future directions

“…While microbial community composition and diversity have been extensively studied in microbiome ecology, there is a significant need to explore the functional attributes of whole communities, localized populations, and individual microorganisms (genes and pangenomes) within a microbiome. Investigating microbial functions, such as metabolic pathways and molecular interactions between members of the microbiome and with the host, can provide important insights into the contributions of specific microbial taxa/consortia and their functional roles in host and ecosystem health (Béchade et al, 2023;e.g., Hicks et al, 2018;Karmacharya et al, 2019). Furthermore, isolation and culturing of microbial strains can provide complementary information not otherwise accessible through community -omics alone (e.g., physiological profiling of microbial growth rates and chemical/antibiotic sensitivity), while also paving the way for future experimental work using such host-associated microbial isolates.…”