Testing the Toxicofera: comparative reptile transcriptomics casts doubt on the single, early evolution of the reptile venom system

Abstract

Background The identification of apparently conserved gene complements in the venom and salivary glands of a diverse set of reptiles led to the development of the Toxicofera hypothesis – the idea that there was a single, early evolution of the venom system in reptiles. However, this hypothesis is based largely on relatively small scale EST-based studies of only venom or salivary glands and toxic effects have been assigned to only some of these putative Toxcoferan toxins in some species. We set out to investigate the distribution of these putative venom toxin transcripts in order to investigate to what extent conservation of gene complements may reflect a bias in previous sampling efforts. Results We have carried out the first large-scale test of the Toxicofera hypothesis and found it lacking in a number of regards. Our quantitative transcriptomic analyses of venom and salivary glands and other body tissues in five species of reptile, together with the use of available RNA-Seq datasets for additional species shows that the majority of genes used to support the establishment and expansion of the Toxicofera are in fact expressed in multiple body tissues and most likely represent general maintenance or “housekeeping” genes. The apparent conservation of gene complements across the Toxicofera therefore reflects an artefact of incomplete tissue sampling. In other cases, the identification of a non-toxic paralog of a gene encoding a true venom toxin has led to confusion about the phylogenetic distribution of that venom component. Conclusions Venom has evolved multiple times in reptiles. In addition, the misunderstanding regarding what constitutes a toxic venom component, together with the misidentification of genes and the classification of identical or near-identical sequences as distinct genes has led to an overestimation of the complexity of reptile venoms in general, and snake venom in particular, with implications for our understanding of (and development of treatments to counter) the molecules responsible for the physiological consequences of snakebite.

A Critique of the Toxicoferan Hypothesis

“…However, the downside of these qualities is that it has also avoided scrutiny and testing. There have recently been several studies which have cast doubt on the Toxicofera hypothesis (Hargreaves et al 2014a;Reyes-Velasco et al 2015), although their interpretation has led to alternative conclusions. Several phylogenetic analyses incorporating non-venom gland transcriptomic data have shown that non-toxin sequences nest within clades of toxin genes, and it has been acknowledged that such findings provide ".…”

“…A large scale test of the robustness of this hypothesis found that many Evolution of Venomous Animals and Their Toxins DOI 10.1007/978-94-007-6727-0_4-1 # Springer Science+Business Media Dordrecht 2015 of the genes used to support the single, early evolution of venom in squamates are in fact expressed in multiple body tissues including the salivary gland of a non-Toxicoferan lizard, the leopard gecko (Eublepharis macularius) (Hargreaves et al 2014a). No evidence has been found of either a venomspecific splice variant or significantly elevated expression level in the venom or salivary gland.…”

“…Therefore these genes have been used to support shared ancestral toxicity, without actually functioning as toxins. Additionally, it now seems certain that many of the proposed shared venom toxins within the Toxicofera actually result from the confusion of orthologs and paralogs, where non-toxic relatives of toxin genes have been identified (Hargreaves et al 2014a). For example, genes encoding complement c3 and nerve growth factor have been shown to have undergone an Elapid-specific gene duplication (Sunagar et al 2013;Hargreaves et al 2014a, b) to give rise to the putatively toxic "cobra venom factor" and nerve growth factor b (Hargreaves et al 2014b).…”

Evolution: Fangtastic Venoms Underpin Parasitic Mimicry

“…Thus, in the Toxicofera, the venom appears to have evolved before the delivery system (although see [6] for an alternative view of venom evolution in reptiles).…”

An investigation of the evolution of the anguimorph lizard venom system

“…Thus recent reviews that relied on the Hsiang et al trees to reconstruct the evolutionary history of the organisms and also the associated venom systems inherit the problems of the original study (Mackessy et al, 2016;Sweet, 2016). Both Mackessy and Sweet also relied heavily upon Hargreaves tissue expression paper (Hargreaves et al, 2014) for their interpretation. For the exact differences between the traditional and Toxicofera phylogenetic approaches we recommend looking into dedicated studies (Streicher and Weins, 2016;Mackessy et al, 2016;Jackson et al, 2016;Zheng and Weins, 2016;Vidal and Hedges, 2009;Townsend et al, 2004).…”