UNCORRECTED PROOF Functional basis of a molecular adaptation: Prey-specific toxic effects of venom from Sistrurus rattlesnakes H. Lisle Gibbs a, * , Stephen P. Mackessy b a Department of Evolution, Ecology and Organismal Biology, Ohio State University, 300 Aronoff Laboratory, 318 W.12th Ave., Columbus, OH 43210-1293, USA b School of Biological Sciences, University of Northern Colorado, 50120th St., CB 92, Greeley, CO 80639-0017, USA article info Article history: Received 3 December 2008 Received in revised form 26 January 2009 Accepted 27 January 2009 Available online xxxx Keywords: Sistrurus rattlesnakes Snake venom Prey-specific LD 50 s Comparative analysis Venom evolution abstract Understanding the molecular bases of adaptations requires assessing the functional significance of phenotypic variation at the molecular level. Here we conduct such an assessment for an adaptive trait (snake venom proteins) which shows high levels of interspecific variation at the molecular level. We tested the toxicity of venom from four taxa of Sistrurus rattlesnakes with different diets towards 3 representative preys (mice, lizards and frogs). There were significant differences among prey in their overall suscep- tibility to Sistrurus venom, with frogs being an order of magnitude more resistant than mice or lizards. However, only in mice there was substantial variation in the toxicity of venom from different Sistrurus taxa, with the variation being roughly correlated with the incidence of mammals in the snake’s diet. A comparative analysis using published data of the toxicity of rattlesnake and outgroup (Agkistrodon) venoms to mice confirms that both the gain and loss of toxicity to mammals were major modes of venom evolution in Sis- trurus catenatus and Sistrurus miliarius. Our findings identify toxicity to mammals as a major axis along which venom evolution has occurred among Sistrurus rattlesnakes, with little evidence for evolutionary changes in toxicity towards the other preys tested. They also emphasize the need to consider ecological and evolutionary factors other than diet alone as causes of variation in venom toxicity. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Identifying the molecular basis of adaptations in natural populations is an important yet largely unrealized goal in evolutionary biology, despite its potential to address fundamental questions about the role of different types of selective and genetic mechanisms as the basis for adaptive variation in phenotype (Golding and Dean, 1998; Orr, 2005). A key step in this research approach is identifying the functional significance of phenotypic variation at the molecular level. This will be most successful in systems where the possible function of the variation can be narrowly defined due to the nature of the adaptation. In this sense, predator–prey systems offer such a clearly defined phenotypic interface because the functional goals of the traits directly involved in killing the prey by the predator or resisting predation by the prey are clear (Brodie and Brodie, 1999). Venoms produced by snakes in the Colubroidea are an example of a trait in a predator which shows high levels of variation at the molecular level and which also has a clearly defined function, namely the capture and digestion of prey. Venomous snakes such as rattlesnakes produce a complex mixture of up to 40 distinct proteins of several different families (Mackessy, 2008). Specialized venom glands located in the upper jaw synthesize and store venom which is then injected into prey via long, hollow fangs. Detailed and comprehensive characterization of the genes that underlie this variation and of the proteins they encode are becoming increasingly common for snakes (e.g. Sanz et al., * Corresponding author. Fax: þ1 614 292 2030. E-mail address: gibbs.128@osu.edu (H.L. Gibbs). Contents lists available at ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon ARTICLE IN PRESS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 0041-0101/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2009.01.034 Toxicon xxx (2009) 1–8 TOXCON3427_proof  12 February 2009  1/8 Please cite this article in press as: H. Lisle Gibbs, Stephen P. Mackessy, Functional basis of a molecular adaptation: Prey-specific toxic effects of venom from Sistrurus rattlesnakes, Toxicon (2009), doi:10.1016/j.toxicon.2009.01.034