Abstract
Three-finger toxins (3FTx) represent one of the most abundantly secreted and potently toxic components of colubrid (Colubridae), elapid (Elapidae) and psammophid (Psammophiinae subfamily of the Lamprophidae) snake venom arsenal. Despite their conserved structural similarity, they perform a diversity of biological functions. Although they are theorised to undergo adaptive evolution, the underlying diversification mechanisms remain elusive. Here, we report the molecular evolution of different 3FTx functional forms and show that positively selected point mutations have driven the rapid evolution and diversification of 3FTx. These diversification events not only correlate with the evolution of advanced venom delivery systems (VDS) in Caenophidia, but in particular the explosive diversification of the clade subsequent to the evolution of a high pressure, hollow-fanged VDS in elapids, highlighting the significant role of these toxins in the evolution of advanced snakes. We show that Type I, II and III α-neurotoxins have evolved with extreme rapidity under the influence of positive selection. We also show that novel Oxyuranus/Pseudonaja Type II forms lacking the apotypic loop-2 stabilising cysteine doublet characteristic of Type II forms are not phylogenetically basal in relation to other Type IIs as previously thought, but are the result of secondary loss of these apotypic cysteines on at least three separate occasions. Not all 3FTxs have evolved rapidly: κ-neurotoxins, which form non-covalently associated heterodimers, have experienced a relatively weaker influence of diversifying selection; while cytotoxic 3FTx, with their functional sites, dispersed over 40% of the molecular surface, have been extremely constrained by negative selection. We show that the a previous theory of 3FTx molecular evolution (termed ASSET) is evolutionarily implausible and cannot account for the considerable variation observed in very short segments of 3FTx. Instead, we propose a theory of Rapid Accumulation of Variations in Exposed Residues (RAVER) to illustrate the significance of point mutations, guided by focal mutagenesis and positive selection in the evolution and diversification of 3FTx.
Highlights
Venoms are key evolutionary innovations in the Kingdom Animalia and are complex concoctions of biologically active proteins, salts, and organic molecules such as polyamines, amino acids and neurotransmitters [1]
A notable finding was that the novel Type II α-ntx sequences from Oxyuranus and Pseudonaja, which lack the Type II α-ntx characteristic cysteine doublet (−2C) between plesiotypic cysteines 5 and 6, were not phylogenetically basal to the other Type II, nor were they monophyletic, but rather were nested within the regular Type II forms (+2C) (Figure 2)
Various selection analyses employed in this study indicated that these contrasting prey capture strategies and the evolution of the advanced venom-delivery apparatus in Caenophidia have significantly influenced the evolution of the plesiotypic α-ntxs
Summary
Venoms are key evolutionary innovations in the Kingdom Animalia and are complex concoctions of biologically active proteins (from polypeptide globular enzymes to small peptides), salts, and organic molecules such as polyamines, amino acids and neurotransmitters [1]. Venom components originate via toxin recruitment events during which ordinary protein-encoding genes, typically those involved in key regulatory processes (such as hemostasis or neurotransmission) are duplicated, and the new copies are selectively expressed in the venom gland [1,2,3,4,5,6,7,8,9,10,11,12,13] These novel paralogs can further duplicate and give rise to multigene families, following the “birth and death” mode of evolution, where the rapid evolution of these families results in extensive neofunctionalization of some copies, while the other non-functional forms are lost through degradation or get transformed into pseudogenes [14]. Three-finger toxins (3FTx) are one of the most abundantly secreted non-enzymatic components of elapid (Elapidae), colubrid (Colubridae) and psammophiide (Psammophiinae subfamily withing the Lamprophidae) snake venom They are characterised by a broad diversity of functional forms (Table 1). The discovery of α-colubritoxin, revealed this potent toxin type to be widespread in “non-front-fanged”
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