Repeated horizontal acquisition of lagriamide-producing symbionts in Lagriinae beetles

Abstract

Microbial symbionts associate with multicellular organisms on a continuum from facultative associations to mutual codependency. In some of the oldest intracellular symbioses there is exclusive vertical symbiont transmission, and co-diversification of symbiotic partners over millions of years. Such symbionts often undergo genome reduction due to low effective population sizes, frequent population bottlenecks, and reduced purifying selection. Here, we describe multiple independent acquisition events of closely related defensive symbionts followed by genome erosion in a group of Lagriinae beetles. Previous work in Lagria villosa revealed the dominant genome-eroded symbiont of the genus Burkholderia produces the antifungal compound lagriamide and protects the beetle's eggs and larvae from antagonistic fungi. Here, we use metagenomics to assemble 11 additional genomes of lagriamide-producing symbionts from seven different host species within Lagriinae from five countries, to unravel the evolutionary history of this symbiotic relationship. In each host species, we detected one dominant genome-eroded Burkholderia symbiont encoding the lagriamide biosynthetic gene cluster (BGC). Surprisingly, however, we did not find evidence for host-symbiont co-diversification, or for a monophyly of the lagriamide-producing symbionts. Instead, our analyses support at least four independent acquisition events of lagriamide-encoding symbionts and subsequent genome erosion in each of these lineages. By contrast, a clade of plant-associated relatives retained large genomes but secondarily lost the lagriamide BGC. In conclusion, our results reveal a dynamic evolutionary history with multiple independent symbiont acquisitions characterized by high degree of specificity. They highlight the importance of the specialized metabolite lagriamide for the establishment and maintenance of this defensive symbiosis.

Keywords: Burkholderia; Lagriinae; biosynthetic gene cluster; chemical defence; lagriamide; metagenomics; secondary metabolism; symbiont replacement; symbiosis.

Figure 1.

Beetle mitogenome phylogenetic tree using 13 mitochondrial protein coding genes constructed using MrBayes [33]. Branch values represent posterior probabilities. Mitogenomes recovered in this study are highlighted with bold lettering.

Figure 2.

Analysis of representative lga BGCs extracted from eleven Lagriinae beetle metagenomes. A) Comparison of representative lga BGC gene organization. Individual genes in the lga BGCs are represented by arrows oriented in the predicted direction of transcription and colored according to identity. Pairwise amino acid similarity between BGCs is indicated in the shaded areas between genes. A scale bar is provided for gene size. Dashed lines indicate fragments missing from the respective assemblies. B) Comparison of predicted enzyme domain organization in the representative lga BGCs, where genes are ordered according to biosynthetic order. Boxes around the domains indicate differences between the BGCs.

Figure 3.

A) Circular representation of LvStB_2023 genome from the L. villosa 2023 sample. Individual chromosomes are indicated by shades of purple. Coding sequences (CDS) which are core genes or pseudogenes, as indicated by shades of green, while the rest are indicated in shades of blue. B) Raw count of COG categories present on different contigs of the LvStB_2023 genome (with and without pseudogenes) from the L. villosa 2023 sample.

Figure 4.

RAxML phylogenetic tree (left) and shared hierarchical orthogroups (HOGs) (non-pseudogenes) between different Burkholderia genomes (matrix on the right). Each blue line indicates a shared HOG. HOGs have been hierarchically clustered on the x-axis to improve visualization. Values on nodes indicate bootstrap values. Genome size and coverage is represented in brackets next to MAG ID. Outgroups include - Paraburkholderia acidiphila (GCF_009789655.1), Cupriavidus necator (GCF_000219215.1), Herbaspirillum seropedicae (GCF_001040945.1). Tree has been truncated for visualisation purposes, full tree can be found in Fig. SI 4 (RAxML phylogeny) and Fig. SI 5 (bayesian phylogeny).

Figure 5.

Congruence between phylogenies of beetle host, Burkholdria symbionts and lga BGCs in all samples. A) Tanglegram between lga-carrying symbionts and beetle host phylogeny. B) Tanglegram between lga-carrying symbionts (centre) and the lga BGC, as inferred via two models GTRCAT (left) and GTRGAMMAI (right). Clades within the beetle host mitogenome phylogenetic tree are highlighted in shades of grey. In all panels, the three conserved clades are highlighted in purple, green and orange. Dashed brown lines represent nodes that are unique between the respective phylogenies, except when the node is absent from the other tree. Blue circles indicate clade nodes that are poorly supported.