Targeted sequencing of venom genes from cone snail genomes reveals coupling between dietary breadth and conotoxin diversity

Abstract

1Although venomous taxa provide an attractive system to study the genetic basis of adaptation and 2 speciation, the slow pace of toxin gene discovery through traditional laboratory techniques (e.g., cDNA 3 cloning) have limited their utility in the study of ecology and evolution. Here, we applied targeted 4 sequencing techniques to selectively recover venom gene superfamilies and non-toxin loci from the 5 genomes of 32 species of cone snails (family, Conidae), a hyper diverse group of carnivorous marine 6 gastropods that capture their prey using a cocktail of neurotoxic proteins (conotoxins). We were able to 7 successfully recover conotoxin gene superfamilies across all species sequenced in this study with high 8 confidence (> 100X coverage). We found that conotoxin gene superfamilies are composed of 1-6 exons 9and adjacent noncoding regions are not enriched for simple repetitive elements. Additionally, we 10 provided further evidence for several genetic factors shaping venom composition in cone snails, including 11 positive selection, extensive gene turnover, expression regulation, and potentially, presence-absence 12 variation. Using comparative phylogenetic methods, we found that while diet specificity did not predict 13 patterns of conotoxin gene superfamily size evolution, dietary breadth was positively correlated with total 14 conotoxin gene diversity. These results continue to emphasize the importance of dietary breadth in 15 shaping venom evolution, an underappreciated ecological correlate in venom biology. Finally, the 16 targeted sequencing technique demonstrated here has the potential to radically increase the pace at which 17 venom gene families are sequenced and studied, reshaping our ability to understand the impact of genetic 18 changes on ecologically relevant phenotypes and subsequent diversification.

Lack of signal for the impact of venom gene diversity on speciation rates in cone snails

“…We used a targeted sequencing approach to recover markers for phylogenetic inference and obtain an estimate of conotoxin gene diversity from Conidae species. For the phylogenetic markers, we identified loci using a previous Conidae targeted sequencing dataset (Phuong & Mahardika 2017) and the Conidae transcriptome data from (Phuong et al . 2016).…”

“…2016). In the Conidae targeted sequencing dataset, the authors generated a phylogeny using 5883 loci across 32 species (Phuong & Mahardika 2017). For our sequencing experiment, we only retained loci that were >180bp and were present in at least 26 out of 32 taxa with at least 10X coverage.…”

“…To recover conotoxin loci, we targeted sequences generated from both the previous targeted sequencing dataset (Phuong & Mahardika 2017) and the transcriptome dataset (Phuong et al . 2016).…”

“…2016). For conotoxin sequences discovered from the targeted sequencing dataset (Phuong & Mahardika 2017), we performed the following to generate our target sequences: (1) we trimmed each sequence to only retain the coding region and included 100bp flanking the exon, (2) merged sequences using cd-hit v4.6.4 (Li & Godzik 2006) at 95% sequence similarity to reduce redundancy among conotoxin loci (3) masked repeats using the RepeatMasker v4.0.6 web server (Smit et al . 2015), and (4) retained loci >120bp to ensure that the locus was longer than our desired bait sequence length.…”

“…We also targeted conotoxin loci from the transcriptomes described in (Phuong et al . 2016) to obtain conotoxin loci from gene superfamilies that were not targeted in (Phuong & Mahardika 2017) or performed poorly. We performed the following to generate a set of conotoxin loci from the transcriptome data: (1) we trimmed sequences from (Phuong et al .…”