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The roles of balancing selection and recombination in the evolution of rattlesnake venom

Abstract

The origin of snake venom involved duplication and recruitment of non-venom genes into venom systems. Several studies have predicted that directional positive selection has governed this process. Venom composition varies substantially across snake species and venom phenotypes are locally adapted to prey, leading to coevolutionary interactions between predator and prey. Venom origins and contemporary snake venom evolution may therefore be driven by fundamentally different selection regimes, yet investigations of population-level patterns of selection have been limited. Here, we use whole-genome data from 68 rattlesnakes to test hypotheses about the factors that drive genomic diversity and differentiation in major venom gene regions. We show that selection has resulted in long-term maintenance of genetic diversity within and between species in multiple venom gene families. Our findings are inconsistent with a dominant role of directional positive selection and instead support a role of long-term balancing selection in shaping venom evolution. We also detect rapid decay of linkage disequilibrium due to high recombination rates in venom regions, suggesting that venom genes have reduced selective interference with nearby loci, including other venom paralogues. Our results provide an example of long-term balancing selection that drives trans-species polymorphism and help to explain how snake venom keeps pace with prey resistance.

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Fig. 1: Overview of the study system and estimates of population structure and historical demography.
Fig. 2: Genome scans of genetic diversity within populations and differentiation between populations across chromosome housing major venom gene families (SVMPs, SVSPs and PLA2s).
Fig. 3: Signatures of selection in venom gene regions, with comparisons to chromosomal backgrounds and non-venom homologues.
Fig. 4: Trans-species amino acid polymorphisms among SVMP, SVSP and PLA2 genes.
Fig. 5: Predicted probabilities of alternative evolutionary mechanisms (neutrality, negative frequency-dependent selection and heterozygote advantage) across SVMP, SVSP and PLA2 venom gene regions.
Fig. 6: Recombination rate variation in SVMP, SVSP and PLA2 venom gene regions.

Data availability

The genomic data that support the findings of this study are available at NCBI SRA under accession PRJNA593834.

Code availability

Analysis scripts are available on GitHub (https://github.com/drewschield/venom_population_genomics).

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Acknowledgements

We thank R. Orton and N. Balchan for assistance in the field. We thank J. Vindum and the California Academy of Sciences for tissue loans. A. Ludington kindly provided advice on demographic analysis. We thank S. Flaxman and R. Safran for helpful discussion on the study and feedback on the manuscript. This work was supported by National Science Foundation (NSF) postdoctoral research fellowship grant DBI-1906188 to D.R.S., NSF grant DEB-1501886 to D.R.S. and T.A.C. and NSF grant DEB-1655571 to T.A.C., S.P.M. and J.M.M., NSF grants DEB-1949268, BCS-2001063 and DBI-2130666 to M.D. and National Institutes of Health grant R35GM128590 to M.D.

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Contributions

D.R.S. and T.A.C. designed the study. D.R.S., B.W.P., Z.L.N., S.S.G., C.F.S., J.M.P., J.M.M., S.P.M. and T.A.C. collected samples and generated data. D.R.S., B.W.P., R.H.A. and M.D. performed analyses. D.R.S. and T.A.C. wrote the manuscript with contributions from B.W.P., R.H.A., M.L.H. and M.D. All authors provided edits to the manuscript.

Corresponding authors

Correspondence to Drew R. Schield or Todd A. Castoe.

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Nature Ecology & Evolution thanks Giulia Zancolli and Wieslaw Babik for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Overview of filtering strategy to remove potential bioinformatic artifacts of copy-number variation (CNV) in major venom gene regions.

a Schematic representation of the workflow used to filter departures from Hardy–Weinberg equilibrium (HWE) due to excess heterozygosity at SNP positions. Input variant calls were analysed using a one-tailed HWE test (adapted from Wigginton et al. 2005) and SNPs with significant HWE P-values after FDR correction were filtered. Individual CNVs were detected, scored, and regional variation in CNV presence, quantified as the proportion of individuals in a population with a detected CNV (% CNV), was measured across each major venom gene region. CNV calls were used to mask genotypes per individual prior to population genetic analysis. b Regional variation in CNV presence per population across the major venom gene regions. Top panels for each venom gene region show log2 read depths in sliding windows relative to the autosomal median depth for the CV1 reference population. Here values of zero indicate equal coverage to the autosomal median, values of −1 equal half coverage, and values of 1 equal twice the autosomal median coverage. Dark blue segments indicate the locations of venom genes in each region. Lower panels for each region show variation in the proportion of individuals with masked genotypes in a detected CNV (% CNV) per population. c Minor allele frequency spectra across the dataset for each gene in the major venom gene families after using the described HWE and CNV filtering strategy.

Extended Data Fig. 2 Genome scans of genetic diversity within populations and differentiation between populations across chromosome housing major venom gene families (SVMPs, SVSPs, and PLA2s) in CV2 and CO2 populations.

a Sliding windows of nucleotide diversity (π) in C. viridis (CV2) and C. oreganus (CO2) populations and sequence divergence (dxy) and relative differentiation (Fst) between CV1 and CV2 and between CO1 and CO2 across Chromosome 9 (top panels), and in the SVMP region (bottom panels). b π, dxy, and Fst across Chromosome 10 (top), and in the SVSP region (bottom). c π, dxy, and Fst across Chromosome 15 (top), and in the PLA2 region (bottom). Shaded points in top panels show estimates in 10 kb windows and lines show estimates in 100 kb windows. In bottom panels in a and b, shaded points show estimates in 1 kb windows, and lines show estimates in 10 kb windows. In bottom panels in c, shaded points show estimates in 250 bp windows and lines are estimates in 1 kb windows. The regions housing venom genes are shaded in grey in all panels. Chromosome-specific and genome-wide mean values for each statistic are represented by blue and red dashed horizontal lines. The locations of individual venom genes are shown as blue boxes (bottom panels). The non-venom homologue PLA2gIIE is shown in light purple. Gaps in measurements are locations that were masked due to significant evidence of copy-number variation between C. viridis and C. oreganus.

Extended Data Fig. 3 Genetic diversity point estimates for major venom paralogues.

Point estimates (dark blue circles) of π (a), dxy (b), and Fst (c) for paralogues in the three major venom gene families, compared to chromosome-specific background distributions outside of venom gene regions. Boxplots show the median (horizontal lines), interquartile (box limits), and range (whiskers) based on n = 2,253, 1,998, and 1,239 10 kb sliding windows for chromosomes 9, 10, and 15, respectively.

Extended Data Fig. 4 Tajima’s D across major venom regions in CV2 and CO2 populations.

Tajima’s D across SVMP (a), SVSP (b), and PLA2 (c) regions in CV2 and CO2 populations. Top panels show sliding window Tajima’s D estimates in 100 kb (lines) and 10 kb (points points) windows. Venom gene regions are shaded in grey. Middle panels in a and b show zoomed in venom gene regions with sliding window estimates in 10 kb (lines) and 1 kb (points) windows. Middle panels in c show estimates in 1 kb (lines) and 250 bp (points) windows. Gaps in lines represent windows where there was insufficient data to calculate a mean estimate. Dark blue segments show the locations of venom genes in each region, and the non-venom homologue PLA2gIIE is shown in light purple in c. Regional variation in the presence of CNVs (% CNV) in CV2 (orange dashed line) and CO2 (dark blue line) is shown below venom region scans. Individual genotypes in detected CNVs were masked. Bottom panels show distributions of chromosome-specific and non-venom homologue (NV) backgrounds compared to values in each venom gene region, with boxplots showing the median (horizontal lines), interquartile (box limits), and range (whiskers). Asterisks indicate significant differences between venom gene regions and chromosome backgrounds and non-venom homologues based on two-tailed Welch’s two-sample t-tests and n = 2,293, 2,068, and 1,272 10 kb sliding windows for SVMP, SVSP, and PLA2 comparisons, respectively (*P < 0.05; **P < 0.001). Exact P-values for comparisons can be found in Supplementary Table 5.

Extended Data Fig. 5 Genomic scans of iHS and ß selection statistics in CV1 and CO1 populations.

Lines show mean estimates in 1 Mb sliding windows. Shaded points show mean estimates in 100 kb sliding windows.

Extended Data Fig. 6 Scans of iHS and ß selection statistics across major venom regions.

Scans of a|iHS|and b ß selection statistics in CV1 and CO1 populations across SVMP, SVSP, and PLA2 venom gene regions. Lines in SVMP and SVSP panels show mean estimates in 10 kb sliding windows. Shaded points in SVMP and SVSP panels show mean estimates in 1 kb sliding windows. Lines in PLA2 panels show mean estimates in 1 kb sliding windows. Shaded points in PLA2 panels show mean estimates in 250 bp sliding windows. Gaps in lines represent windows where there was insufficient data to calculate a mean estimate. Venom gene locations are shown with dark blue segments. The non-venom PLA2gIIE homologue is shown as a light purple segment.

Extended Data Fig. 7 Selection statistic point estimates for major venom paralogues.

Point estimates (dark blue circles) of Tajima’s D (a), df (b), |iHS|(c), and ß (d) for paralogues in the three major venom gene families, compared to chromosome-specific background distributions outside of venom gene regions. Boxplots show the median (horizontal lines), interquartile (box limits), and range (whiskers) based on n = 2,253, 1,998, and 1,239 10 kb sliding windows for chromosomes 9, 10, and 15, respectively.

Extended Data Fig. 8 Diagnostic parameters in composite-likelihood ratio tests of selection in major venom regions.

Results of composite-likelihood ratio tests of balancing selection and diagnostic parameters in the SVMP (a-b), SVSP (c-d), and PLA2 (e-f) venom gene regions. Top and middle panels show B0,MAF scores, log\(\widehat {(A)}\), and \(\hat x\) parameters as shown in Fig. 3 and Extended Data Fig. 9. Dark blue arrows show locations of venom genes and dashed lines show the genome-wide 95th quantile. Lower panels show the dispersion parameter, log\(\widehat {(a)}\), where positive values indicate balancing selection in regions with high B0,MAF scores and negative values are indicative of positive selection. The dashed horizontal line indicates 0 on the y-axis.

Extended Data Fig. 9 Signatures of selection in the PLA2 venom gene region.

Signatures of selection in the PLA2 venom gene region, with comparisons to chromosomal backgrounds and non-venom homologues (NV). a Tajima’s D across chromosome 15 in C. viridis (CV1) and C. oreganus (CO1) populations (top). Middle panels show variation zoomed into the PLA2 region, shaded in grey. Boxplots show distributions of Tajima’s D for chromosome 15, non-venom homologues of the PLA2 family, and PLA2s. b Proportion of fixed differences (df) between CV1 and CO1. c Integrated haplotype statistics (|iHS|) in CV1 and CO1. d ß statistic measuring allele frequency correlation in CV1 and CO1. Points in chromosome scan panels represent mean estimates in 10 kb sliding windows, and lines represent 100 kb windowed estimates. Points in zoomed PLA2 region scans represent mean estimates in 250 bp windows and lines show 1 kb windowed estimates. Locations of individual PLA2 genes are shown as dark blue segments (bottom panels). The PLA2gIIE non-venom homologue is shown in light purple. Gaps in lines represent windows where there was insufficient data to calculate a mean estimate. Boxplots in a-d show the median (horizontal lines), interquartile (box limits), and range (whiskers). Asterisks indicate significant differences between venom gene regions and chromosome backgrounds and non-venom homologues based on two-tailed Welch’s two-sample t-tests (Tajima’s D and |iHS|) and Mann–Whitney tests (df and ß) and n = 1,272 10 kb sliding windows for comparisons (*P < 0.05; **P < 0.001; ***P < 2.2 × 10−16). Exact P-values for comparisons can be found in Supplementary Table 5. e-f Results of composite-likelihood ratio tests of balancing selection versus neutrality using B0,MAF scores in the PLA2 region in CV1 and CO1. Upper panels show B0,MAF scores, with higher values indicating greater evidence for balancing selection. Arrows show locations of PLA2 genes. Lower panels show inferred footprint size, log\(\widehat {(A)}\), as solid grey lines and equilibrium allele frequency, \(\hat x\), as dashed lines. Dashed lines in top panels of e-f show the genome-wide 95th quantile.

Extended Data Fig. 10 Population-scaled recombination rates in major venom regions.

Population-scaled recombination rate (ρ = 4Ner) across SVMP (a), SVSP (b), and PLA2 (c) venom gene regions in C. viridis and C. oreganus. Upper panels show chromosome-wide variation and lower panels show variation within the venom regions, specifically, highlighted by the grey shading in all panels. Dark blue segments in lower panels show the locations of venom paralogues. The light purple segment in c is the non-venom homologue PLA2gIIE. Shaded points in the upper panels of a-c represent mean ρ in 10 kb windows and black lines represent 100 kb windowed means. In lower panels of a and b, points and lines represent 1 kb and 10 kb windowed ρ. In lower panels of c, lines represent 1 kb windowed ρ and points are estimates from all SNP intervals. Vertical dashed lines show the locations of inferred recombination hotspots from Schield et al. (2020). Panels at the bottom show regional variation in the proportion of C. viridis (orange dashed line) and C. oreganus (dark blue line) individuals per population with evidence of copy-number variation (% CNV) across the venom gene regions.

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Schield, D.R., Perry, B.W., Adams, R.H. et al. The roles of balancing selection and recombination in the evolution of rattlesnake venom. Nat Ecol Evol 6, 1367–1380 (2022). https://doi.org/10.1038/s41559-022-01829-5

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