Journal of Molecular Evolution

, Volume 66, Issue 2, pp 151–166 | Cite as

Rapid Evolution by Positive Selection and Gene Gain and Loss: PLA2 Venom Genes in Closely Related Sistrurus Rattlesnakes with Divergent Diets

  • H. Lisle Gibbs
  • Wayne Rossiter


Rapid evolution of snake venom genes by positive selection has been reported previously but key features of this process such as the targets of selection, rates of gene turnover, and functional diversity of toxins generated remain unclear. This is especially true for closely related species with divergent diets. We describe the evolution of PLA2 gene sequences isolated from genomic DNA from four taxa of Sistrurus rattlesnakes which feed on different prey. We identified four to seven distinct PLA2 sequences in each taxon and phylogenetic analyses suggest that these sequences represent a rapidly evolving gene family consisting of both paralogous and homologous loci with high rates of gene gain and loss. Strong positive selection was implicated as a driving force in the evolution of these protein coding sequences. Exons coding for amino acids that make up mature proteins have levels of variation two to three times greater than those of the surrounding noncoding intronic sequences. Maximum likelihood models of coding sequence evolution reveal that a high proportion (∼30%) of all codons in the mature protein fall into a class of codons with an estimated d N /d S (ω) ratio of at least 2.8. An analysis of selection on individual codons identified nine residues as being under strong (p < 0.01) positive selection, with a disproportionately high proportion of these residues found in two functional regions of the PLA2 protein (surface residues and putative anticoagulant region). This is direct evidence that diversifying selection has led to high levels of functional diversity due to structural differences in proteins among these snakes. Overall, our results demonstrate that both gene gain and loss and protein sequence evolution via positive selection are important evolutionary forces driving adaptive divergence in venom proteins in closely related species of venomous snakes.


Sistrurus rattlesnakes Snake venom genes PLA2 venom proteins Positive selection Gene family evolution Functional diversity in proteins 



We thank Steve Mackessy for generously providing the S. c. edwardsii DNA sample, Jose Diaz, Giancarlo Lopez-Martinez, and David Denlinger for assistance with the lab work, Doug Wynn and Dan Harvey for help in the field, Joe Bielawski, Juan Calvete, James Cotton, Matt Dean, Brian Golding, Jeff Good, Dusan Kordis, Michael Nachman, Tom Waite, and Tom Wilson for advice and discussion, Laura Kubatko for help with the phylogenetic analyses, and the Gibbs Lab Group, Greg Booton, Brian Fry, and three anonymous reviewers for comments. Funding for this study was provided by the Columbus Zoo and Ohio State University.


  1. Brunie S, Bolin J, Gewirth D et al (1985) The refined crystal structure of dimeric phospholipase A2 at 25 Å. J Biol Chem 260:9742–9749PubMedGoogle Scholar
  2. Carredano E, Westerlund B, Persson B et al (1998) The threedimensional structures of two toxins from snake venom throw light on the anticoagulant and neurotoxic sites of phospholipase A2. Toxicon 36:75–92PubMedCrossRefGoogle Scholar
  3. Cereb N, Hughes AL, Yang SY (1997) Locus-specific conservation of the HLA class I introns by intra-locus homogenization. Immunogenetics 47:30–36PubMedCrossRefGoogle Scholar
  4. Chen Y, Wang Y, Hseu M et al (2004) Molecular evolution and structure-function relationships of crototoxin-like and asparagines-6-containing phosphlolipase A2 in pit viper venoms. Biochem J 381:25–34PubMedCrossRefGoogle Scholar
  5. Chijiwa T, Deshimaru M, Nobuhisha I et al (2000) Regional evolution of venom-gland phospholipase A2 isoenzymes of Trimeresurus flavoviridis snakes in the southwestern islands of Japan. Biochem J 347:491–499PubMedCrossRefGoogle Scholar
  6. Chijiwa T, Yamaguchi Y, Ogawa T et al (2003) Interisland evolution of Trimeresurus flavoviridis venom phospholipase A2 isozymes. J Mol Evol 56:286–293PubMedCrossRefGoogle Scholar
  7. Conant R, Collins JT (1998) A field guide to reptiles & amphibians of eastern & central North America. 3rd ed. Houghton Mifflin, New YorkGoogle Scholar
  8. Cotton JA, Page RDM (2005) Rates and patterns of gene duplication and loss in the human genome. Proc Roy Soc Lond 272:277–283CrossRefGoogle Scholar
  9. Creer S, Malhotra A, Thorpe RS, et al (2003) Genetic and ecological correlates of intraspecific variation in pitviper venom composition detected using matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and isoelectric focusing. J Mol Evol 56:317–329PubMedCrossRefGoogle Scholar
  10. Daltry JC, Wuster W, Thorpe RS (1996) Diet and snake venom evolution. Nature 379:537–540PubMedCrossRefGoogle Scholar
  11. Danse JM, Gasparini S, Menez A (1997) Molecular biology of snake venom phospholipase A2. In: Kini RM (ed) Venom phospholipse A2 enzymes: structure, function, and mechanism. J. Wiley and Sons, Chichester, UK, pp 29–71Google Scholar
  12. Demuth JP, Bie TD, Stajich JE et al (2006) The evolution of mammalian gene families. PLoS ONE 1:e85 doi: 101371/journal.pone.0000085
  13. Douglas ME, Douglas MR, Schuett GW et al (2006) Evolution of rattlesnakes (Viperidae; Crotalus) in the warm deserts of western North America shaped by Neogene vicariance and Quaternary climate change. Mol Ecol 15:3353–3374PubMedCrossRefGoogle Scholar
  14. Feder ME, Mitchell-Olds T (2003) Evolutionary and ecological functional genomics. Nature Rev Genet 4:649–655CrossRefGoogle Scholar
  15. Fry BG (2005) From genome to ‘venome’: molecular origin and evolution of the snake venom proteome inferred from phylogenetic analysis of toxin sequences and related body proteins. Genome Res 15:403–420PubMedCrossRefGoogle Scholar
  16. Fry BG, Wüster W (2004) Assembling an arsenal: origin and evolution ofthe snake venom proteome inferred from phylogenetic analysis of toxin sequences. Mol Biol Evol 21:870–883PubMedCrossRefGoogle Scholar
  17. Fry BG, Wüster W, Kini RM et al (2003) Molecular evolution of elapid snake venom three finger toxins. J Mol Evol 57:110–129PubMedCrossRefGoogle Scholar
  18. Fry BG, Vidal N, Norman JA et al (2006) Early evolution of the venom system in lizards and snakes. Nature 439:584–588PubMedCrossRefGoogle Scholar
  19. Gasteiger E, Hoogland C, Gattiker A et al (2005) Protein identification and analysis tools on the ExPASy server. In: Walker JM (ed) The proteomics protocols handbook. Humana Press, New York, pp 571–607Google Scholar
  20. Gillespie JH (1994). The causes of molecular evolution. Oxford University Press, OxfordGoogle Scholar
  21. Golding GB, Dean AM (1998) The structural basis of molecular adaptation. Mol Biol Evol 15:355–369PubMedGoogle Scholar
  22. Gubensek F, Kordis D (1997) Venom phospholipase A2 genes and their molecular evolution. In: Kini RM (ed) Venom phospholipse A2 enzymes: structure, function, and mechanism. J. Wiley and Sons, Chichester, UK, pp 73–95Google Scholar
  23. Hasegawa M, Kishino H (1989) Confidence limits on the maximum-likelihood estimate of the hominoid tree from mitochondrial-DNA sequences. Evolution 43:627–677CrossRefGoogle Scholar
  24. Heatwole H, Powell J (1998) Resistance of eels (Gymnothorax) to the venom of sea kraits (Laticauda colubrina): a test of coevolution. Toxicon 36:619–625PubMedCrossRefGoogle Scholar
  25. Hoekstra HE, Coyne J (2007) The locus of evolution: evo devo and the genetics of adaptation. Evolution 61:995–1016PubMedCrossRefGoogle Scholar
  26. Holycross AT, Mackessey SP (2002) Variation in the diet of Sistrurus catenatus (massasauga) with emphasis on Sistrurus catenatus edwardsii (desert massasauga). J Herpetol 36:454–464Google Scholar
  27. Hughes AL, Yeager M (1997) Comparative evolutionary rates of introns and exons in murine rodents. J Mol Evol 45:125–130PubMedCrossRefGoogle Scholar
  28. John TR, Smith L, Kaiser I (1994) Genomic sequences encoding the acidic and basic subunits of Mojave toxin:unusually high sequence identity of non-coding regions. Gene 139:229–234PubMedCrossRefGoogle Scholar
  29. Jorge da Silva NJ, Aird SD (2001) Prey specificity, comparative lethality and compositional differences of coral snake venoms. Comp Biochem Physiol C Toxicol Pharmacol 128:425–456PubMedCrossRefGoogle Scholar
  30. Junqueira-de-Azevedo ILM, Ho PL (2002) A survey of gene expression and diversity in the venom glands of the pitviper snake Bothrops insularis through the generation of expressed sequence tags (ESTs). Gene 299:279–291CrossRefGoogle Scholar
  31. Junqueira-de-Azevedo ILM, Ching ATC, Carvalho E et al (2006) Lachesis muta (Viperidae) cDNAs reveal diverging pit viper molecules and scaffolds typical of Cobra (Elapidae) venoms: implications for snake toxin repertoire evolution. Genetics 173:877–889PubMedCrossRefGoogle Scholar
  32. Kini RM, ed. (1997) Venom phospholipase A2 enzymes: structure, function, and mechanism. J. Wiley and Sons, Chichester, UKGoogle Scholar
  33. Kini RM (2003) Excitement ahead: structure, function, and mechanism of snake venom phospholipase A2 enzymes. Toxicon 42:827–840PubMedCrossRefGoogle Scholar
  34. Kini RM (2005) Structure-function relationships and mechanism of anticoagulant phospholipase A2 enzymes from snake venoms. Toxicon 45:1147–1161PubMedCrossRefGoogle Scholar
  35. Kini RM, Chan YM (1999) Accelerated evolution and molecular surface of venom phospholipase A2 enzymes. J Mol Evol 48:125–132PubMedCrossRefGoogle Scholar
  36. Kini RM, Evans HJ (1989) A model to explain the pharmacological effects of snake venom phospholipases A2. Toxicon 27:613–635PubMedCrossRefGoogle Scholar
  37. Kordis D, Bdolah A, Gubensek F (1998) Positive Darwinian selection in Vipera palestinae phospholipase genes is unexpectantly limited to the third exon. Biochem Biophys Res Commun 251:613–619PubMedCrossRefGoogle Scholar
  38. Kordis D, Krizaj I, Gubensek F (2002) Funtional diversification of animal toxins by adaptive evolution. In: Menez A (ed) Perspectives in molecular toxinology. J. Wiley and Sons, New York, pp 401–419Google Scholar
  39. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform 5:150–163PubMedCrossRefGoogle Scholar
  40. Li M, Fry BG, Kini RM (2005) Eggs only diet: the shift in preferred prey by the marbled sea snake (Aipysurus eydouxii) resulting in a loss of postsynaptic neurotoxicity. J Mol Evol 60:81–9PubMedCrossRefGoogle Scholar
  41. Li M, Fry BG, Kini RM (2005) Putting the brakes on snake venom evolution: the unique molecular evolutionary patterns of Aipysurus eydouxii (marbled sea snake) phospholipase A2 toxins. Mol Biol Evol 22:934–941PubMedCrossRefGoogle Scholar
  42. Lomonte B, Moreno E, Tarkowski A et al (1994) Neutralizing interaction between heparins and myotoxin II, a lysine 49 phospholipase A2 from Bothrops asper snake venom. Identification of a heparin-binding and cytolytic toxin region by the use of synthetic peptides and molecular modeling. J Biol Chem 269:29867–29873PubMedGoogle Scholar
  43. Lynch VJ (2007) Inventing an arsenal: adaptive evolution and neofunctionalization of snake venom phospholipase A2 genes. BMC Evol Biol 7:2PubMedCrossRefGoogle Scholar
  44. Lynch M, Conery JS (2000) The evolutionary fate and consequences of duplicate genes. Science 293:1151–1155CrossRefGoogle Scholar
  45. Lynch M, Conery JS (2003) The evolutionary demography of duplicate genes. J Struct Funct Genom 3:35–44CrossRefGoogle Scholar
  46. Menez A (2002) Perspectives in molecular toxinology. J. Wiley & Sons, New YorkGoogle Scholar
  47. Metz EC, Robles-Sikisaka R, Vacquier VD (1998) Nonsynonymous substitution in abalone sperm fertilization genes exceeds substitution in introns and mitochondrial DNA. Proc Natl Acad Sci USA 95:10676–10681PubMedCrossRefGoogle Scholar
  48. Nakashima K, Nobuhisa I, Deshimaru M et al (1993) Accelerated evolution of Trimeresurus flavoviridis venom gland phospholipase A2 isozymes. Proc Natl Acad Sci USA 90:5964–5968PubMedCrossRefGoogle Scholar
  49. Nakashima K, Nobuhisa I, Deshimaru M et al (1995) Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes. Proc Natl Acad Sci USA 92:5605–5609PubMedCrossRefGoogle Scholar
  50. Nei M, Rooney AP (2005) Concerted and birth-and-death evolution of multigene families. Annu Rev Genet 39:121–152PubMedCrossRefGoogle Scholar
  51. Nobuhisa I, Nakashima K, Deshimaru M et al (1996) Accelerated evolution of Trimeresurus okinavensis venom gland phospholipase A2 isozyme-encoding genes. Gene 172:267–272PubMedCrossRefGoogle Scholar
  52. Ohno M, Chijiwa T, Oda-Ueda N et al (2003) Molecular evolution of myotixic phospholipases A2 from snake venom. Toxicon 42:841–854PubMedCrossRefGoogle Scholar
  53. Page RDM (1998). GeneTree:comparing gene and species phylogenies using reconciled trees. Bioinformatics 14:819–820PubMedCrossRefGoogle Scholar
  54. Peitsch MC (1996) ProMod and Swiss-Model: Internet-based tools for automated comparative protein modelling. Biochem Soc Trans 24:274–279PubMedGoogle Scholar
  55. Petan T, Krizaj I, Gubensek F et al (2002) Phenylalanine-24 in the N-terminal region of ammodytoxins is important for both enzymic activity and presynaptic toxicity. Biochem. J 363:353–358PubMedCrossRefGoogle Scholar
  56. Posada D, Crandall KA (1998) Modeltest: testing the model of DNA substitution. Bioinformatics 14:817–818PubMedCrossRefGoogle Scholar
  57. Prijatelj P, Copic A, Krizaj I et al (2000) Charge reversal of ammodytoxin A, a phospholipase A2-toxin, does not abolish its neurotoxicity. Biochem J 352:251–255PubMedCrossRefGoogle Scholar
  58. Prijatelj P, Krizaj I, Kralj B et al (2002) The C-terminal region of ammodytoxins is important but not sufficient for neurotoxicity. Eur J Biochem 269:5759–5764PubMedCrossRefGoogle Scholar
  59. Rozas J, Sánchez-DelBarrio J C, Messeguer X et al (2003) DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19:2496–2497PubMedCrossRefGoogle Scholar
  60. Samanta U, Bahadur RP, Chakrabarti P (2002) Quantifying the accessible surface area of protein residues in their local environment. Protein Eng 15:659–667PubMedCrossRefGoogle Scholar
  61. Sanz L, Gibbs HL, Mackessy SP et al (2006) Venom proteomes of closely related Sistrurus rattlesnakes with divergent diets. J Proteome Res 5:2098–2112PubMedCrossRefGoogle Scholar
  62. Senepathy P, Shapiro MB, Harris NL (1990) Splice junctions, branch point sites, and exons: sequence statistics, identification, and applications to genome project. Methods Enzymol 183:252–278CrossRefGoogle Scholar
  63. Singh G, Gourinath S, Sharma S et al (2001) Sequence and crystal structure determination of a basic phospholipase A2 from common krait (Bungarus caeruleus) at 24 Å resolution: identification and characterization of its pharmacological sites. J Mol Biol 307:1049–1059PubMedCrossRefGoogle Scholar
  64. Slowinski JB, Knight A, Rooney AP (1997) Inferring species trees from gene trees: a phylogenetic analysis of the elapidae (Serpentes) based on the amino acid sequences of venom proteins. Mol Phylogenet Evol 8:349–362PubMedCrossRefGoogle Scholar
  65. Stern DL (2000) Evolutionary developmental biology and the problem of variation. Evolution 54:1079–1091PubMedGoogle Scholar
  66. Suzuki Y, Nei M (2004) False-positive selection identifed by ML-based methods: examples from the Sig1 gene of the diatom Thalassiosira weissflogii and the tax gene of a human T-cell lymphotropic virus. Mol Biol Evol 21:914–921PubMedCrossRefGoogle Scholar
  67. Swofford DL (2003) PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods). Version 4. Sinauer Associates, Sunderland, MAGoogle Scholar
  68. Tatsuya A, Shiina T, Kimura N et al (2003) Comparative sequencing of human and chimpanzee MHC class I regions unveils insertions/deletions as the major path to genomic divergence. Proc Natl Acad Sci USA 100:7708–7713CrossRefGoogle Scholar
  69. Tsai I-H, Wang Y-M, Chen Y-H et al (2003) Geographic variations, cloning, and functional analyses of the venom acidic phospholipases A2 of Crotalus viridis viridis. Arch Biochem Biophys 411:289–296PubMedCrossRefGoogle Scholar
  70. Tsai I, Wang Y, Chen Y et al (2004) Venom phospholipase A2 of bamboo viper (Trimeresurus stejnegeri): molecular characterization, geographic variations and evidence for multiple ancestries. Biochem J 377:215–223PubMedCrossRefGoogle Scholar
  71. Wong WSW, Yang Z, Goldman N et al (2004) Accuracy and power of statistical methods for detecting adaptive evolution in protein coding sequences and for identifying positively selected sites. Genetics Z (1997) PAML: a program package for phylogenetic analysis by maximum likelihood. Comput Appl BioSci 13:555–556Google Scholar
  72. Yang Z (2002) Inference of selection from multiple species alignments. Curr Opin Genet Dev 12:688–694PubMedCrossRefGoogle Scholar
  73. Yang Z, Wong WSW, Nielsen R (2005) Bayes empirical Bayes inference of amino acid sites under positive selection. Mol Biol Evol 22:1107–1118PubMedCrossRefGoogle Scholar
  74. Zhao K, Zhou Y, Lin Z (2000) Structure of basic phospholipase A2 from Agkistrodon halys Pallas: implications for its association, hemolytic and anticoagulant activities. Toxicon 38:901–916PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  1. 1.Department of EvolutionEcology and Organismal Biology, Ohio State UniversityColumbusUSA
  2. 2.Department of Ecology, Evolution, & Natural ResourcesRutgers UniversityNew BrunswickUSA

Personalised recommendations