Vertical Inheritance Facilitates Interspecies Diversification in Biosynthetic Gene Clusters and Specialized Metabolites

Abstract

While specialized metabolites are thought to mediate ecological interactions, the evolutionary processes driving chemical diversification, particularly among closely related lineages, remain poorly understood. Here, we examine the evolutionary dynamics governing the distribution of natural product biosynthetic gene clusters (BGCs) among 118 strains representing all nine currently named species of the marine actinobacterial genus Salinispora. While much attention has been given to the role of horizontal gene transfer (HGT) in structuring BGC distributions, we find that vertical descent facilitates interspecies BGC diversification over evolutionary timescales. Moreover, we identified a distinct phylogenetic signal among Salinispora species at both the BGC and metabolite level, indicating that specialized metabolism represents a conserved phylogenetic trait. Using a combination of genomic analyses and liquid chromatography-high-resolution tandem mass spectrometry (LC-MS/MS) targeting nine experimentally characterized BGCs and their small molecule products, we identified gene gain/loss events, constrained interspecies recombination, and other evolutionary processes associated with vertical inheritance as major contributors to BGC diversification. These evolutionary dynamics had direct consequences for the compounds produced, as exemplified by species-level differences in salinosporamide production. Together, our results support the concept that specialized metabolites, and their cognate BGCs, can represent phylogenetically conserved functional traits with chemical diversification proceeding in species-specific patterns over evolutionary time frames. IMPORTANCE Microbial natural products are traditionally exploited for their pharmaceutical potential, yet our understanding of the evolutionary processes driving BGC evolution and compound diversification remain poorly developed. While HGT is recognized as an integral driver of BGC distributions, we find that the effects of vertical inheritance on BGC diversification had direct implications for species-level specialized metabolite production. As such, understanding the degree of genetic variation that corresponds to species delineations can enhance natural product discovery efforts. Resolving the evolutionary relationships between closely related strains and specialized metabolism can also facilitate our understanding of the ecological roles of small molecules in structuring the environmental distribution of microbes.

Keywords: Salinispora; evolution; evolutionary biology; homologous recombination; microbial ecology; salinosporamide.

FIG 1

Genetic relatedness of Salinispora strains (n = 118). (A) Core genome phylogeny. Colors denote species. Bar, 0.1 nucleotide substitutions per position. Red triangles indicate nodes with bootstrap support >90%. (B) Heatmap depicting pairwise similarities in flexible genome content among strains (generated from a Jaccard distance matrix). (C) Total number of biosynthetic gene cluster (BGCs; both whole and fragments) identified across Salinispora species. Black bars represent medians, red diamonds represent means, and dashed green line denotes average across the genus. “N” indicates number of genomes per species. (D) Nonmetric multidimensional scaling (NMDS) plot depicting biosynthetic gene cluster family (GCF) composition for each Salinispora strain. Circles represent strains colored by species.

FIG 2

Gene cluster family (GCF) genetic similarity since divergence from a common ancestor (inferred from core genome divergence). Each point represents a pairwise comparison calculated from whole-BGC alignments as a function of core genome divergence for the same strain pairs. Linear regression lines are denoted for each GCF, with the regression line for expected neutral divergence (i.e., diverging at the same rate as the core genome) in gray. Predicted interspecies transfer events are circled in gray for sal, slc, and spt (see Fig. S4A at https://osf.io/entqc/).

FIG 3

Salinispora metabolomics and GCF distributions across nine species (n = 30 strains). (A) Principal-components analysis (PCA) showing differences in metabolomes. Each point represents a strain colored by species. Standard deviations are shown as black lines for each strain, and 95% confidence intervals are denoted as colored ellipses around each species with ≥3 strains. (B) Targeted analysis for the products of nine GCFs. Hierarchical clustering generated from a Euclidean distance matrix. Green/white columns show the BGC presence/absence within a GCF. Light green indicates the detection of a BGC fragment. Gray/black columns show the relative production of each identified analog, NP, no production. x axis lists GCF name followed by product names. y axis lists strains colored by species with abbreviations for geographic origin (see Table S1 in the supplemental material for more information on geographic locations).

FIG 4

Variations in the salinosporamide GCF (sal) and its products. (A) Phylogeny of the sal BGC across species. Colors denote species. Bar, 0.01 nucleotide substitutions per position. Bootstrap values indicate >99% support. Brackets indicate number of genomes in each species encoding a version of the BGC. (B) Representative BGC for each species. Genes are colored by predicted biosynthetic function with gene blocks colored by their role in compound production. (C) Salinosporamide production across species. Analogs are listed in descending order based on R group size (inset). (D) Sequence alignments of the salA AT₁ domain showing three signature motifs associated with substrate specificity. Conserved amino acid regions in gray.