Population Genomic Analysis of a Pitviper Reveals Microevolutionary Forces Underlying Venom Chemistry.

Venoms are among the most biologically active secretions known, and are commonly believed to evolve under extreme positive selection. Many venom gene families, however, have undergone duplication, and are often deployed in doses vastly exceeding the LD50 for most prey species, which should reduce the strength of positive selection. Here, we contrast these selective regimes using snake venoms, which consist of rapidly evolving protein formulations. Though decades of extensive studies have found that snake venom proteins are subject to strong positive selection, the greater action of drift has been hypothesized, but never tested. Using a combination of de novo genome sequencing, population genomics, transcriptomics, and proteomics, we compare the two modes of evolution in the pitviper, Protobothrops mucrosquamatus. By partitioning selective constraints and adaptive evolution in a McDonald-Kreitman-type framework, we find support for both hypotheses: venom proteins indeed experience both stronger positive selection, and lower selective constraint than other genes in the genome. Furthermore, the strength of selection may be modulated by expression level, with more abundant proteins experiencing weaker selective constraint, leading to the accumulation of more deleterious mutations. These findings show that snake venoms evolve by a combination of adaptive and neutral mechanisms, both of which explain their extraordinarily high rates of molecular evolution. In addition to positive selection, which optimizes efficacy of the venom in the short term, relaxed selective constraints for deleterious mutations can lead to more rapid turnover of individual proteins, and potentially to exploration of a larger venom phenotypic space.

Snake venoms are integrated systems, but abundant venom proteins evolve more rapidly.

BACKGROUND: While many studies have shown that extracellular proteins evolve rapidly, how selection acts on them remains poorly understood. We used snake venoms to understand the interaction between ecology, expression level, and evolutionary rate in secreted protein systems. Venomous snakes employ well-integrated systems of proteins and organic constituents to immobilize prey. Venoms are generally optimized to subdue preferred prey more effectively than non-prey, and many venom protein families manifest positive selection and rapid gene family diversification. Although previous studies have illuminated how individual venom protein families evolve, how selection acts on venoms as integrated systems, is unknown. RESULTS: Using next-generation transcriptome sequencing and mass spectrometry, we examined microevolution in two pitvipers, allopatrically separated for at least 1.6 million years, and their hybrids. Transcriptomes of parental species had generally similar compositions in regard to protein families, but for a given protein family, the homologs present and concentrations thereof sometimes differed dramatically. For instance, a phospholipase A2 transcript comprising 73.4 % of the Protobothrops elegans transcriptome, was barely present in the P. flavoviridis transcriptome (<0.05 %). Hybrids produced most proteins found in both parental venoms. Protein evolutionary rates were positively correlated with transcriptomic and proteomic abundances, and the most abundant proteins showed positive selection. This pattern holds with the addition of four other published crotaline transcriptomes, from two more genera, and also for the recently published king cobra genome, suggesting that rapid evolution of abundant proteins may be generally true for snake venoms. Looking more broadly at Protobothrops, we show that rapid evolution of the most abundant components is due to positive selection, suggesting an interplay between abundance and adaptation. CONCLUSIONS: Given log-scale differences in toxin abundance, which are likely correlated with biosynthetic costs, we hypothesize that as a result of natural selection, snakes optimize return on energetic investment by producing more of venom proteins that increase their fitness. Natural selection then acts on the additive genetic variance of these components, in proportion to their contributions to overall fitness. Adaptive evolution of venoms may occur most rapidly through changes in expression levels that alter fitness contributions, and thus the strength of selection acting on specific secretome components.

Quantitative high-throughput profiling of snake venom gland transcriptomes and proteomes (Ovophis okinavensis and Protobothrops flavoviridis).

BACKGROUND: Advances in DNA sequencing and proteomics have facilitated quantitative comparisons of snake venom composition. Most studies have employed one approach or the other. Here, both Illumina cDNA sequencing and LC/MS were used to compare the transcriptomes and proteomes of two pit vipers, Protobothrops flavoviridis and Ovophis okinavensis, which differ greatly in their biology. RESULTS: Sequencing of venom gland cDNA produced 104,830 transcripts. The Protobothrops transcriptome contained transcripts for 103 venom-related proteins, while the Ovophis transcriptome contained 95. In both, transcript abundances spanned six orders of magnitude. Mass spectrometry identified peptides from 100% of transcripts that occurred at higher than contaminant (e.g. human keratin) levels, including a number of proteins never before sequenced from snakes. These transcriptomes reveal fundamentally different envenomation strategies. Adult Protobothrops venom promotes hemorrhage, hypotension, incoagulable blood, and prey digestion, consistent with mammalian predation. Ovophis venom composition is less readily interpreted, owing to insufficient pharmacological data for venom serine and metalloproteases, which comprise more than 97.3% of Ovophis transcripts, but only 38.0% of Protobothrops transcripts. Ovophis venom apparently represents a hybrid strategy optimized for frogs and small mammals. CONCLUSIONS: This study illustrates the power of cDNA sequencing combined with MS profiling. The former quantifies transcript composition, allowing detection of novel proteins, but cannot indicate which proteins are actually secreted, as does MS. We show, for the first time, that transcript and peptide abundances are correlated. This means that MS can be used for quantitative, non-invasive venom profiling, which will be beneficial for studies of endangered species.