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 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, protecting the beetle's eggs and larvae from antagonistic fungi. Here, we use metagenomics to assemble 11 additional genomes of lagriamide-producing symbionts from 7 different host species within Lagriinae from 5 countries, to unravel the evolutionary history of this symbiotic relationship. In each host, we detected one dominant genome-eroded Burkholderia symbiont encoding the lagriamide biosynthetic gene cluster. However, we did not find evidence for host-symbiont co-diversification or for monophyly of the lagriamide-producing symbionts. Instead, our analyses support a single ancestral acquisition of the gene cluster followed by at least four independent symbiont acquisitions and subsequent genome erosion in each lineage. By contrast, a clade of plant-associated relatives retained large genomes but secondarily lost the lagriamide gene cluster. Our results, therefore, reveal a dynamic evolutionary history with multiple independent symbiont acquisitions characterized by a high degree of specificity and highlight the importance of the specialized metabolite lagriamide for the establishment and maintenance of this defensive symbiosis.

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

Figure 1

Beetle mitogenome phylogenetic tree using 13 mitochondrial protein coding genes constructed using MrBayes [45]. Branch values represent posterior probabilities. Mitogenomes recovered in this study are highlighted with bold lettering. Pictures depicting a representative species of each subfamily are included (Pimeliinae: Pimelia obsoleta; Lagriinae: Lagria hirta; Diaperinae: Trachyscelis aphodioides; Tenebrioninae: Tenebrio molitor; Blaptinae: Blaps lethifera; Stenochiinae: Strongylium cultellatum; Alleculinae: Cteniopus sulphureus). Photography credits: Udo Schmidt [54] (CC BY-SA 2.0).

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, although we have omitted these numbers for the smallest 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 separate continuous arcs in the outer ring (ring 5). Coding sequences (CDS) which are core genes or pseudogenes are shown in the innermost ring (ring 1) and next innermost ring (ring 2), respectively, whereas the rest are indicated the next two rings (rings 3 and 4). (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 shaded block in the matrix indicates a shared HOG. HOGs have been hierarchically clustered on the x-axis. Bootstrap values are indicated on nodes. 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). The branches of other Burkholderia and outgroups have been collapsed for the sake of clarity.

Figure 5

Schematic representation of proposed evolutionary scenario. The lga BGC was acquired by the common ancestor but lost in the free-living relatives. Burkholderia carrying the lga BGC were independently acquired multiple times by the beetle hosts.

Figure 6

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 (center) and the lga BGC, as inferred via two models GTRCAT (left) and GTRGAMMAI (right). In all panels, the four conserved clades are highlighted in purple, green, blue, and orange. Gray dots on nodes indicate congruence between the compared phylogenies, whereas red dots indicate incongruence.