The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system

Abstract

Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection.

Keywords: genomics; phylogenetics; serpentes.

Fig. 1.

The king cobra venom system with venom and accessory gland expression profiles. Pie charts display the normalized percentage abundance of toxin transcripts recovered from each tissue transcriptome. Three-finger toxins are the most abundant toxin family in the venom gland (66.73% of all toxin transcripts and 4.37% in the accessory gland), and they are represented in the genome by at least 21 loci. Lectins are the most abundant toxin family in the accessory gland (42.70% of all toxin transcripts and 0.03% in the venom gland), and they are represented in the genome by at least six loci. Asterisks indicate toxin gene families annotated in the genome. 3FTx, three-finger toxin; AchE, acetylcholinesterase; CRISP, cysteine-rich secretory protein; CVF, cobra venom factor; IGF-like, insulin-like growth factor; kallikrein, kallikrein serine proteases; kunitz, kunitz-type protease inhibitors; LAAO, l-amino acid oxidase; NGF, nerve growth factor; PDE, phosphodiesterase; PLA2, phospholipase A₂; PLB, phospholipase-B; SVMP, snake venom metalloproteinase. Drawing made based on a photo by F.J.V.

Fig. 2.

MiRNA expression profiles of the king cobra venom gland and accessory gland and miRNA expression patterns by in situ hybridization. (A) The 10 most abundant miRNAs in the venom gland show similarities with the known expression profile of the vertebrate pancreas (shown here for human; microRNA.org). (B) In situ hybridization of miR-375 in a C. radiatus embryo 27 d postoviposition with expression detected in the pancreas (arrow). (C) In situ hybridization of miR-375 in an N. siamensis embryo 32 d postoviposition, showing expression in the islet cell masses of the pancreas and the intrasplenic islet tissue. (D) In situ hybridization of miR-375 in a tissue section of the venom system of an adult O. hannah showing expression in the main venom gland. (Inset) Boundary of the venom gland (expression) and accessory gland (no expression) (SI Appendix, Fig. S6). AG, accessory gland; G, gallbladder; P, pancreas; S, spleen; VG, venom gland.

Fig. 3.

Histological section of the complete venom apparatus of the king cobra and spatial expression of lectin genes in the accessory gland. (A) Longitudinal section of the venom system reveals the two regions of the accessory gland: the proximal portion (PAG) and the distal portion (DAG; consistent with a previous morphological study) (26). The venom system is stained by alcian blue and periodic acid–Schiff, in which the secretory epithelial cells and secretion of the venom gland are periodic acid–Schiff-positive and the seromucous acini of the PAG and the mucous acini comprising the DAG are stained with alcian blue. (B) In situ hybridization of lectin gene Oh-516 (genome ID s8808 gene 2) shows that lectin expression is restricted to the PAG. DAG shows no staining. (C) Detail of the PAG shown in B showing strong granular staining in the epithelium of the PAG (SI Appendix, Fig. S14). VD, venom duct; VG, venom gland.

Fig. 4.

Contrasting evolutionary histories of king cobra toxin gene families. (A) The vast majority of toxin family gene duplication events occurred in the king cobra lineage compared with the Burmese python and their common ancestor. (B) Comparisons of venom gland expression, venom-related gene duplication events, and rate of evolution of main toxin families (red) and ancillary toxin families (green). (C) Massive expansion of the three-finger toxin gene family and (D) moderate expansion of other pathogenic toxin families by duplication of venom-expressed genes after the split of the Burmese python from the advanced snakes. (E) Ancillary toxin families show reduced evidence of gene duplication. Colored lines indicate gene loci, with line splits representing gene duplication events and dotted lines indicating gene loss. Venom gene duplications are defined as duplications that occurred after the split of the Burmese python from the advanced snakes (king cobra). ω represents the dN/dS ratio identified for venomous gene clades. The boundary for directional selection is indicated by a bold line. Note the logarithmic scale in the normalized venom gland expression graph. 3FTx, three-finger toxin; CRISP, cysteine-rich secretory protein; Hyal, hyaluronidase; kallikrein, kallikrein serine proteases; LAAO, l-amino acid oxidase; NGF, nerve growth factor; PLA2, phospholipase A₂; SVMP, snake venom metalloproteinase.