Journal of Molecular Evolution

, Volume 75, Issue 5–6, pp 168–183 | Cite as

Structural and Molecular Diversification of the Anguimorpha Lizard Mandibular Venom Gland System in the Arboreal Species Abronia graminea

  • Ivan Koludarov
  • Kartik Sunagar
  • Eivind A. B. Undheim
  • Timothy N. W. Jackson
  • Tim Ruder
  • Darryl Whitehead
  • Alejandro C. Saucedo
  • G. Roberto Mora
  • Alejandro C. Alagon
  • Glenn King
  • Agostinho Antunes
  • Bryan G. Fry


In the past, toxinological research on reptiles has focused principally on clinically important species. As a result, our understanding of the evolution of the reptile venom system is limited. Here, for the first time, we describe the structural and molecular evolutionary features of the mandibular toxin-secreting gland of Abronia graminea, a representative of one of the poorly known and entirely arboreal lineages of anguimorph lizards. We show that the mandibular gland is robust and serous, characters consistent with those expected of a toxin-secreting gland in active use. A wide array of transcripts were recovered that were homologous to those encoded by the indisputably venomous helodermatid lizards. We show that some of these toxin transcripts are evolving under active selection and show evidence of rapid diversification. Helokinestatin peptides in particular are revealed to have accumulated residues that have undergone episodic diversifying selections. Conversely, the natriuretic peptides have evolved under tremendous evolutionary constraints despite being encoded in tandem with helokinestatins by the same gene precursor. Of particular note is the sequencing for the first time of kunitz peptides from a lizard toxin-secreting gland. Not only are kunitz peptides shown to be an ancestral toxicoferan toxin, the ancestral state of this peptide is revealed to be a dual domain encoding precursor. This research provides insight into the evolutionary history of the ancient toxicoferan reptile venom system. In addition, it shows that even ‘clinically irrelevant’ species can be a rich source of novel venom components, worthy of investigation for drug design and biomedical research.


Venom Phylogeny Molecular evolution 



BGF was funded by the Australian Research Council and the University of Queensland. EABU would like to acknowledge funding from the University of Queensland (International Postgraduate Research Scholarship, UQ Centennial Scholarship, and UQ Advantage Top-Up Scholarship) and the Norwegian State Education Loans Fund. This research was supported in part by the Portuguese Foundation for Science and Technology (FCT) through the Ph.D. grant conferred to KS (SFRH/BD/61959/2009) and the project PTDC/AAC-AMB/121301/2010 (FCOMP-01-0124-FEDER-019490) to AA.

Conflict of interest



  1. Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  2. Anisimova M, Bielawski JP, Yang ZH (2002) Accuracy and power of Bayes prediction of amino acid sites under positive selection. Mol Biol Evol 19:950–958PubMedCrossRefGoogle Scholar
  3. Beck DD (2005) Biology of gila monsters and beaded lizards. University of California Press, BerkeleyGoogle Scholar
  4. Bogert CM, del Campo RM (1956) The gila monster and its allies. The relationships, habits, and behavior of the lizards of the family Helodermatidae. Bull Am Mus Nat Hist 109:1–238Google Scholar
  5. Bouabboud CF, Kardassakis DG (1988) Acute myocardial-infarction following a gila monster (Heloderma suspectum cinctum) bite. West J Med 148:577–579Google Scholar
  6. Cantrell FL (2003) Envenomation by the Mexican beaded lizard: a case report. J Toxicol Clin Toxicol 41:241–244PubMedCrossRefGoogle Scholar
  7. Casewell NR, Huttley GA, Wuester W (2012) Dynamic evolution of venom proteins in squamate reptiles. Nat Commun 3:1066. doi: 10.1038/ncomms2065
  8. Clemetson KJ, Lu QM, Clemetson JM (2005) Snake C-type lectin-like proteins and platelet receptors. Pathophysiol Haemost Thromb 34:150–155PubMedCrossRefGoogle Scholar
  9. Conesa A, Gotz S (2008) Blast2GO: a comprehensive suite for functional analysis in plant genomics. Int J Plant Genomics 2008:619832PubMedGoogle Scholar
  10. Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676PubMedCrossRefGoogle Scholar
  11. Datta G, Tu AT (1997) Structure and other chemical characterizations of gila toxin, a lethal toxin from lizard venom. J Pept Res 50:443–450PubMedCrossRefGoogle Scholar
  12. Delport W, Poon AFY, Frost SDW, Pond SLK (2010) Datamonkey 2010: a suite of phylogenetic analysis tools for evolutionary biology. Bioinformatics 26:2455–2457PubMedCrossRefGoogle Scholar
  13. Doley R, Nguyen NBT, Reza MA, Kini RM (2008) Unusual accelerated rate of deletions and insertions in toxin genes in the venom glands of the pygmy copperhead (Austrelaps labialis) from Kangaroo island. BMC Evol Biol 8:1–13CrossRefGoogle Scholar
  14. Doron-Faigenboim A, Stern A, Bacharach E, Pupko T (2005) Selecton: a server for detecting evolutionary forces at a single amino-acid site. Bioinformatics 21:2101–2103PubMedCrossRefGoogle Scholar
  15. Drickamer K (1992) Engineering galactose-binding activity into a c-type mannose-binding protein. Nature 360:183–186PubMedCrossRefGoogle Scholar
  16. Fry BG, Wickramaratana JC, Lemme S, Beuve A, Garbers D, Hodgson WC, Alewood P (2005) Novel natriuretic peptides from the venom of the inland taipan (Oxyuranus microlepidotus): isolation, chemical and biological characterisation. Biochem Biophys Res Commun 327:1011–1015PubMedCrossRefGoogle Scholar
  17. Fry BG, Vidal N, Norman JA, Vonk FJ, Scheib H, Ramjan SF, Kuruppu S, Fung K, Hedges SB, Richardson MK, Hodgson WC, Ignjatovic V, Summerhayes R, Kochva E (2006) Early evolution of the venom system in lizards and snakes. Nature 439:584–588PubMedCrossRefGoogle Scholar
  18. Fry BG, Scheib H, van der Weerd L, Young B, McNaughtan J, Ramjan SF, Vidal N, Poelmann RE, Norman JA (2008) Evolution of an arsenal: structural and functional diversification of the venom system in the advanced snakes (Caenophidia). Mol Cell Proteomics 7:215–246PubMedGoogle Scholar
  19. Fry BG, Vidal N, van der Weerd L, Kochva E, Renjifo C (2009a) Evolution and diversification of the Toxicofera reptile venom system. J Proteomics 72:127–136PubMedCrossRefGoogle Scholar
  20. Fry BG, Wroe S, Teeuwisse W, van Osch MJ, Moreno K, Ingle J, McHenry C, Ferrara T, Clausen P, Scheib H, Winter KL, Greisman L, Roelants K, van der Weerd L, Clemente CJ, Giannakis E, Hodgson WC, Luz S, Martelli P, Krishnasamy K, Kochva E, Kwok HF, Scanlon D, Karas J, Citron DM, Goldstein EJ, McNaughtan JE, Norman JA (2009b) A central role for venom in predation by Varanus komodoensis (Komodo Dragon) and the extinct giant Varanus (Megalania) priscus. Proc Natl Acad Sci USA 106:8969–8974PubMedCrossRefGoogle Scholar
  21. Fry BG, Roelants K, Winter K, Hodgson WC, Griesman L, Kwok HF, Scanlon D, Karas J, Shaw C, Wong L, Norman JA (2010a) Novel venom proteins produced by differential domain-expression strategies in beaded lizards and gila monsters (genus Heloderma). Mol Biol Evol 27:395–407PubMedCrossRefGoogle Scholar
  22. Fry BG, Winter K, Norman JA, Roelants K, Nabuurs RJ, van Osch MJ, Teeuwisse WM, van der Weerd L, McNaughtan JE, Kwok HF, Scheib H, Greisman L, Kochva E, Miller LJ, Gao F, Karas J, Scanlon D, Lin F, Kuruppu S, Shaw C, Wong L, Hodgson WC (2010b) Functional and structural diversification of the Anguimorpha lizard venom system. Mol Cell Proteomics 9:2369–2390PubMedCrossRefGoogle Scholar
  23. Fry BG, Casewell NR, Wüster W, Vidal N, Young B, Jackson TNW (2012) The structural and functional diversification of the Toxicofera reptile venom system. Toxicon 9(11):2369–2390Google Scholar
  24. Goetz S, Arnold R, Sebastian-Leon P, Martin-Rodriguez S, Tischler P, Jehl M-A, Dopazo J, Rattei T, Conesa A (2011) B2G-FAR, a species-centered GO annotation repository. Bioinformatics 27:919–924CrossRefGoogle Scholar
  25. Gotz S, Garcia-Gomez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, Robles M, Talon M, Dopazo J, Conesa A (2008) High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res 36:3420–3435PubMedCrossRefGoogle Scholar
  26. Hooker KR, Caravati EM (1994) Gila monster envenomation. Ann Emerg Med 24:731–735PubMedCrossRefGoogle Scholar
  27. Huang TF, Chiang HS (1994) Effect on human platelet-aggregation of phospholipase a(2) purified from Heloderma horridum (beaded lizard) venom. Biochim Biophys Acta 1211:61–68PubMedCrossRefGoogle Scholar
  28. Komori Y, Nikai T, Sugihara H (1988) Purification and characterization of a lethal toxin from the venom of Heloderma horridum horridum. Biochem Biophys Res Commun 154:613–619PubMedCrossRefGoogle Scholar
  29. Kwok HF, Chen T, O’Rourke M, Ivanyi C, Hirst D, Shaw C (2008) Helokinestatin: a new bradykinin B-2 receptor antagonist decapeptide from lizard venom. Peptides 29:65–72PubMedCrossRefGoogle Scholar
  30. Li M, Fry BG, Kini RM (2005a) Eggs-only diet: its implications for the toxin profile changes and ecology of the marbled sea snake (Aipysurus eydouxii). J Mol Evol 60:81–89PubMedCrossRefGoogle Scholar
  31. Li M, Fry BG, Kini RM (2005b) Putting the brakes on snake venom evolution: the unique molecular evolutionary patterns of Aipysurus eydouxii (Marbled sea snake) phospholipase A(2) toxins. Mol Biol Evol 22:934–941PubMedCrossRefGoogle Scholar
  32. Ma CB, Wang H, Wu YX, Zhou M, Lowe G, Wang L, Zhang YQ, Chen TB, Shaw C (2012) Helokinestatin-7 peptides from the venoms of Heloderma lizards. Peptides 35:300–305PubMedCrossRefGoogle Scholar
  33. Mebs D (1969a) Isolation and properties of kallikrein from venom of gila monster (Heloderma suspectum). Hoppe Seylers Z Physiol Chem 350:821–826PubMedCrossRefGoogle Scholar
  34. Mebs D (1969b) Purification and properties of a kinin liberating enzyme from venom of Heloderma suspectum. Naunyn Schmiedebergs Arch Pharmakol 264:280–281PubMedCrossRefGoogle Scholar
  35. Mochcamorales J, Martin BM, Possani LD (1990) Isolation and characterization of helothermine, a novel toxin from Heloderma horridum horridum (mexican beaded lizard) venom. Toxicon 28:299–309CrossRefGoogle Scholar
  36. Morita T (2005) Structures and functions of snake venom CLPs (C-type lectin-like proteins) with anticoagulant-, procoagulant-, and platelet-modulating activities. Toxicon 45:1099–1114PubMedCrossRefGoogle Scholar
  37. Morrissette J, Elhayek R, Possani L, Coronado R (1994) Isolation and characterization of ryanodine receptor toxins from Heloderma horridum (mexican beaded lizard) venom. Biophys J 66:A415CrossRefGoogle Scholar
  38. Morrissette J, Kratzschmar J, Haendler B, Elhayek R, Mochcamorales J, Martin BM, Patel JR, Moss RL, Schleuning WD, Coronado R, Possani LD (1995) Primary structure and properties of helothermine, a peptide toxin that blocks ryanodine receptors. Biophys J 68:2280–2288PubMedCrossRefGoogle Scholar
  39. Nielsen R, Yang ZH (1998) Likelihood models for detecting positively selected amino acid sites and applications to the HIV-1 envelope gene. Genetics 148:929–936PubMedGoogle Scholar
  40. Nikai T, Imai K, Sugihara H, Tu AT (1988) Isolation and characterization of horridum toxin with arginine ester hydrolase activity from Heloderma horridum (beaded lizard) venom. Arch Biochem Biophys 264:270–280PubMedCrossRefGoogle Scholar
  41. Nikai T, Imai K, Komori Y, Sugihara H (1992) Isolation and characterization of arginine ester hydrolase from Heloderma horridum (beaded lizard) venom. Int J Biochem 24:415–420PubMedCrossRefGoogle Scholar
  42. Nobile M, Magnelli V, Lagostena L, Mochcamorales J, Possani LD, Prestipino G (1994) The toxin helothermine affects potassium currents in newborn rat cerebellar granule cells. J Membr Biol 139:49–55PubMedGoogle Scholar
  43. Nobile M, Noceti F, Prestipino G, Possani LD (1996) Helothermine, a lizard venom toxin, inhibits calcium current in cerebellar granules. Exp Brain Res 110:15–20PubMedCrossRefGoogle Scholar
  44. Piskurek O, Austin CC, Okada N (2006) Sauria SINEs: novel short, interspersed retroposable elements that are widespread in reptile, genomes. J Mol Evol 62:630–644PubMedCrossRefGoogle Scholar
  45. Pond SLK, Frost SDW (2005a) Not so different after all: a comparison of methods for detecting amino acid sites under selection. Mol Biol Evol 22:1208–1222CrossRefGoogle Scholar
  46. Pond SLK, Frost SDW (2005b) A genetic algorithm approach to detecting lineage-specific variation in selection pressure. Mol Biol Evol 22:478–485PubMedCrossRefGoogle Scholar
  47. Pond SLK, Frost SDW, Muse SV (2005) HyPhy: hypothesis testing using phylogenies. Bioinformatics 21:676–679PubMedCrossRefGoogle Scholar
  48. Pond SLK, Posada D, Gravenor MB, Woelk CH, Frost SDW (2006) Automated phylogenetic detection of recombination using a genetic algorithm. Mol Biol Evol 23:1891–1901CrossRefGoogle Scholar
  49. Pond SLK, Murrell B, Fourment M, Frost SDW, Delport W, Scheffler K (2011) A random effects branch-site model for detecting episodic diversifying selection. Mol Biol Evol 28:3033–3043CrossRefGoogle Scholar
  50. Posada D, Crandall KA (2002) The effect of recombination on the accuracy of phylogeny estimation. J Mol Evol 54:396–402PubMedGoogle Scholar
  51. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574PubMedCrossRefGoogle Scholar
  52. Stern A, Doron-Faigenboim A, Erez E, Martz E, Bacharach E, Pupko T (2007) Selecton 2007: advanced models for detecting positive and purifying selection using a Bayesian inference approach. Nucleic Acids Res 35:W506–W511PubMedCrossRefGoogle Scholar
  53. Strimple PD, Tomassoni AJ, Otten EJ, Bahner D (1997) Report on envenomation by a Gila monster (Heloderma suspectum) with a discussion of venom apparatus, clinical findings, and treatment. Wilderness Environ Med 8:111–116PubMedCrossRefGoogle Scholar
  54. Sunagar K, Johnson WE, O’Brien SJ, Vasconcelos V, Antunes A (2012) Evolution of CRISPs associated with toxicoferan-reptilian venom and mammalian reproduction. Mol Biol Evol 29:1807–1822PubMedCrossRefGoogle Scholar
  55. Suzuki Y, Nei M (2004) False-positive selection identified 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
  56. Tu AT, Hendon RR (1983) Characterization of lizard venom hyaluronidase and evidence for its action as a spreading factor. Comp Biochem Physiol B 76:377–383PubMedCrossRefGoogle Scholar
  57. Utaisincharoen P, Mackessy SP, Miller RA, Tu AT (1993) Complete primary structure and biochemical-properties of gilatoxin, a serine-protease with kallikrein-like and angiotensin-degrading activities. J Biol Chem 268:21975–21983PubMedGoogle Scholar
  58. Vidal N, Hedges SB (2005) The phylogeny of squamate reptiles (lizards, snakes, and amphisbaenians) inferred from nine nuclear protein-coding genes. C R Biol 328:1000–1008PubMedCrossRefGoogle Scholar
  59. Wiens JJ, Kuczynski CA, Townsend T, Reeder TW, Mulcahy DG, Sites JW Jr (2010) Combining phylogenomics and fossils in higher-level squamate reptile phylogeny: molecular data change the placement of fossil taxa. Syst Biol 59:674–688PubMedCrossRefGoogle Scholar
  60. Wiens JJ, Hutter CR, Mulcahy DG, Noonan BP, Townsend TM, Sites JW, Reeder TW (2012) Resolving the, phylogeny of lizards, and snakes (squamata), with extensive sampling, of genes and species. Biol Lett 8(6):1043–1046Google Scholar
  61. Woolley S, Johnson J, Smith MJ, Crandall KA, McClellan DA (2003) TreeSAAP: selection on amino acid properties using phylogenetic trees. Bioinformatics 19:671–672PubMedCrossRefGoogle Scholar
  62. Yang Z (2007) PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol 24:1586–1591PubMedCrossRefGoogle Scholar
  63. Yang ZH, Nielsen R (2002) Codon-substitution models for detecting molecular adaptation at individual sites along specific lineages. Mol Biol Evol 19:908–917PubMedCrossRefGoogle Scholar
  64. Yang ZH, Wong WSW, Nielsen R (2005) Bayes empirical Bayes inference of amino acid sites under positive selection. Mol Biol Evol 22:1107–1118PubMedCrossRefGoogle Scholar
  65. Zhang JZ, Nielsen R, Yang ZH (2005) Evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level. Mol Biol Evol 22:2472–2479PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Ivan Koludarov
    • 1
  • Kartik Sunagar
    • 3
    • 4
  • Eivind A. B. Undheim
    • 1
    • 2
  • Timothy N. W. Jackson
    • 1
  • Tim Ruder
    • 1
  • Darryl Whitehead
    • 5
  • Alejandro C. Saucedo
    • 6
  • G. Roberto Mora
    • 6
  • Alejandro C. Alagon
    • 6
  • Glenn King
    • 2
  • Agostinho Antunes
    • 3
    • 4
  • Bryan G. Fry
    • 1
  1. 1.Venom Evolution LaboratorySchool of Biological Sciences, University of QueenslandSt. LuciaAustralia
  2. 2.Institute for Molecular Biosciences, University of QueenslandSt. LuciaAustralia
  3. 3.CIMAR/CIIMAR, Centro Interdisciplinar de Investigação Marinha e AmbientalUniversidade do PortoPortoPortugal
  4. 4.Departamento de BiologiaFaculdade de Ciências, Universidade do PortoPortoPortugal
  5. 5.School of Biomedical Sciences, University of QueenslandSt. LuciaAustralia
  6. 6.Departamento de Medicina Molecular y BioprocesosInstituto de Biotecnología, Universidad Nacional Autónoma de MéxicoCuernavacaMexico

Personalised recommendations