Variation in venom composition is a ubiquitous phenomenon in venomous snakes at all taxonomic levels, from temporal variation within individuals to higher levels. [1
]. The often extensive compositional variation between conspecific populations or between closely related species has been of particular interest, partly due to its medical consequences [3
] and partly due to its potential as a model system for understanding adaptive evolution at the molecular level (e.g., [6
]): many species display extreme intraspecific geographic variation in venom composition, and this variation may bear little relationship to population genetic structure or organismal phylogeny [9
]. Natural selection for optimization of venom to the diet of snakes has been identified as a likely key driver of venom evolution in several groups [9
]. However, these examples consist primarily of snakes with extreme dietary variation and/or disjunct distributions (e.g., Calloselasma rhodostoma
), or groups of well-differentiated species (e.g., Micrurus
), and the forces underlying venom variation in other cases remain poorly understood [10
]. Moreover, the genetic mechanisms underlying variation in snake venom composition, and the distribution of individual toxins among populations and species, remain largely unknown.
Rattlesnakes (genera Crotalus
) constitute an excellent group of model organisms for the study of venom variation, as they display extensive inter- and intraspecific variation in venom composition [16
]. An underlying theme in Crotalus
appears to be the presence of alternative and often largely mutually exclusive envenoming strategies: type I venoms [16
] contain large amounts of snake venom metalloproteinases (SVMPs), whereas type II venoms contain a high concentration of presynaptically neurotoxic, heterodimeric PLA2
toxins such as crotoxin and Mojave toxin (MTX) [17
], and are typically considerably more lethal in the mouse model than their type I counterparts.
Remarkably, the distribution of these different strategies among rattlesnakes shows little congruence with phylogeny or even species limits. Both strategies can be found across the full phylogenetic breadth of rattlesnakes. Species showing intraspecific variation, with different conspecific snakes secreting either type I and type II venoms [5
], can be found in all major rattlesnake clades [16
]. This intraspecific variation can be ontogenetic, such as in C. simus
, where the venom changes from type II to type I during ontogeny [25
], or geographic, in species such as C. scutulatus
and C. horridus
, where both venom types occur in different parts of their distributional range [14
]. In at least some cases, such as C. scutulatus
, this variation appears to be related to the presence or absence of the genes encoding these toxins rather than gene expression [22
Mapping these apparently homologous toxins onto the phylogeny of rattlesnakes would require remarkable numbers of gene loss events, or, even less plausibly, astonishingly numerous instances of convergent evolution. An alternative explanation for these patterns, which bypasses this difficulty, is adaptive hybridization. Hybridization has long been flagged as a potential source of adaptive variation and innovations [31
]. Extensive studies of hybrid zones between closely related species or differentiated conspecific populations have demonstrated that selectively advantageous genes are able to cross hybrid zones and spread into the other species, provided they are not linked to deleterious alleles at other loci [33
]. This applies even with slight reductions in hybrid fitness, which cause these zones to act as sinks for selectively neutral alleles [33
The hypothesis of a hybridogenic origin of the startling parallel patterns of intraspecific venom variation in several rattlesnakes holds considerable intuitive appeal: by effectively providing a shortcut for gene transfer between the branches of rattlesnake phylogeny, introgressive hybridization would explain geographic variation in the presence of particular gene orthologs in multiple unrelated species more parsimoniously than any hypothesis based solely on phylogenetic relationships. Glenn and Straight [37
] suggested that the presence of MTX-like toxins in some individuals of Crotalus viridis
(Prairie rattlesnake) from southwestern New Mexico was due to hybridization with nearby populations of type II venom C. scutulatus
. Aird et al.
] noted the resemblance between the venoms of C. atrox
and type I venom C. scutulatus
, and suggested that the type I venoms of some C. scutulatus
populations may be due to past hybridization between the two species. More recently, the presence of neurotoxic PLA2
toxins in some populations of C. horridus
has been variously attributed to past hybridization with Crotalus scutulatus
] and Sisturus catenatus
]. Similar arguments have been made to explain the presence of neurotoxic PLA2
toxins in some European vipers [40
]. However, none of these studies provided any independent evidence of interspecific hybridization having taken place. Moreover, despite this interest in hybridization as a possible mechanism for venom variation, we are not aware of any published study rigorously examining venom composition across a well characterized hybrid zone in any venomous animal.
Interspecific hybridization, potentially as a result of anthropogenic disturbance, has also been invoked in the popular literature to explain a claimed increase in the clinical severity of rattlesnake bites in the USA [42
]. The evidence for this hypothesized increase was robustly deconstructed by Hayes and Mackessy [43
]. However, the intriguing question remains whether occasional hybridization events could result in the rapid spread of novel, selectively advantageous toxin genes through the gene pool of a different species. Given their radical effect on venom function and lethality, one could hypothesize that highly lethal toxins such as MTX might be especially prone to this form of introgression and subsequent selective sweeps.
It is an implicit assumption of any hypothesis of hybridogenic introgression of venom toxin genes that the introgressing genes confer a selective advantage to the receiving gene pool [33
]. By the same token, any true hybrid zone between rattlesnake species with different venom compositions would thus provide a test of the hypothesis that particular toxins could be highly selectively advantageous and could spread rapidly across species limits after hybridization. Since individual variation in venom composition can lead to differential venom effectiveness against different prey species [44
], and thus to potential differences in individual fitness, this scenario seems potentially feasible.
Although numerous individual hybrids between different species and even genera of rattlesnakes have been documented [45
], the frequency and importance of hybridization have been disputed [51
]. The present study was prompted by the discovery of multiple specimens phenotypically intermediate between the Mohave rattlesnake (Crotalus scutulatus
) and the Prairie rattlesnake (C. viridis
) in a contact zone along the eastern slope of the Peloncillo Mountains, Hidalgo County, New Mexico, where the two species are largely parapatric [52
]. This region corresponds approximately to the location from which Glenn and Straight [37
] reported MTX-secreting specimens of Crotalus viridis
, which they interpreted as evidence of hybridization with neighboring populations of type II venom C. scutulatus
A third large rattlesnake species, the western diamondback (C. atrox
), occurs sympatrically with both species in Arizona and New Mexico, and also across the hybrid zone. Hybridization between C. scutulatus
and C. atrox
has been suspected of shaping venom composition in the former [37
]. Moreover, a few individuals in the putative C. scutulatus
hybrid zone were visually intermediate between C. scutulatus
and C. atrox
rather than C. scutulatus
and C. viridis
. We therefore included all three large, sympatric rattlesnake species from the area in this assessment of hybridization and its effects on venom composition.
Here, we use this apparent hybrid zone to test the hypothesis that highly lethal neurotoxic PLA2 toxins are likely to introgress into the gene pool of species lacking them. We analyze morphological data, mitochondrial DNA and single-copy nuclear gene sequences to test for evidence of hybridization between C. scutulatus and C. viridis or C. atrox. We then test for the presence of the genes encoding the acidic and basic subunits of the Mojave toxin using a PCR-based assay and sequencing, and relate the presence or absence of the toxin to the hybrid status of the snakes. Finally, we verified the presence of MTX in the venom by reverse-phase high performance liquid chromatography (RP-HPLC) to establish a link between hybrid status, toxin genotype and venom phenotype.
In this study, we set out to test the hypothesis that interspecific hybridization would facilitate introgression by highly lethal presynaptic neurotoxin genes such as Mojave toxin, and thus lead to evolutionary changes in the venom composition of the receiving species. Our results do not support this hypothesis in this instance.
Our genetic analyses unequivocally demonstrate the existence of a narrow hybrid zone between Crotalus scutulatus
along the eastern slope of the Peloncillo Mountains in Hidalgo County, southwestern New Mexico. Many, but not all, the specimens in the main hybrid area (Highway 80, within 18 km South of Road Forks, Hidalgo Co.) are recovered as genetically admixed in Structure analyses. Equally, many are morphologically intermediate between C
. Our single copy nuclear gene data show a continuum of levels of admixture between C
and C. viridis
, indicating that many of the specimens are the result of backcrosses with either parental species. F1 hybrids between the two species are thus fertile and able to breed with either parent stock. Despite some morphological indications to the contrary, we found little genetic evidence of hybridization between C. atrox
and either C. scutulatus
or C. viridis
. Individual specimens that were morphologically intermediate between C. scutulatus
and C. atrox
showed no genetic evidence of admixture from C. atrox
, but appear to be C. scutulatus
hybrids or backcrosses. Their phenetic resemblance to C. atrox
is due to a tail pattern involving bands of greater width than found in C. viridis
and equal width of dark and light bands, as found in C. atrox
. So far, this is only the second genetically characterized hybrid zone between two venomous snake species (after the Vipera aspis
hybrid zone in northern Spain, [55
Our results shed new light on the genetics of the MTX subunits. Contrary to Wooldridge et al.
], we invariably found the genes encoding the basic and acidic subunits of MTX to be either both detectable or both absent in all specimens. Wooldridge et al.
] reported that type I venom snakes lack only the gene encoding the acidic subunit, but retain the gene for the basic subunit. We were unable to replicate that result in our much larger sample. We suspect that their detection of the basic subunit gene in type I venom specimens may have been due to non-specific cross-amplification of other PLA2
toxins by their primer set: we obtained sequences of PLA2
-like genes differing from the MTX basic subunit when using Wooldridge et al.
’s basic subunit primers in specimens from the type I venom zone. Our redesigned primers (see methods) did not incur this problem, and all sequence-confirmed positive PCRs were identical to within a few base pairs with the published MTX sequences [53
]. These data suggest that the two subunits are tightly linked in the genome of C. scutulatus
, presumably due to close proximity on the same chromosome.
Furthermore, our results demonstrate an absolute genotype-phenotype link for the two MTX subunit genes: every single tested specimen with positive PCR results for the MTX subunits yielded HPLC profiles with the two characteristic peaks corresponding to the MTX subunits [5
], whereas no specimen without the genes yielded those peaks. This applied independently of genetic profile or hybrid status, and demonstrates that genotypic differences are reflected in the phenotype, and thus provide potential targets for natural selection. This situation contrasts with that found in other viperids, e.g., Echis
, in which selective transcription and translation as well as post-translational modifications play a prominent part in shaping venom composition [56
Both subunits of MTX were present in most C. scutulatus
(except from the previously documented area of type I venom snakes in central Arizona, [21
]), but absent from most C. viridis
. In southwestern New Mexico, only genetically “pure” C. scutulatus
or individuals with clearly admixed genotypes were positive for both MTX subunits, with the exception of three specimens identified as genetically pure C. viridis
based on scnDNA markers that were positive for the MTX subunits. Two showed evidence of admixture in the shape of C. scutulatus
ND4 haplotypes, and all three were from the immediate vicinity of the hybrid zone and surrounded by other admixed specimens. Overall, there is no evidence of introgression of MTX genes into the genome of C. viridis
beyond the hybrid zone and its immediate vicinity. We suspect that the snakes identified as Mojave toxin-bearing C. viridis
by Glenn and Straight [37
] were in reality hybrids from the zone documented here, although this cannot be verified in the absence of precise locality information in that paper.
The absence of either subunit of MTX in any of our C. atrox
is consistent with the lack of admixture between this species and the others, as well as most of the literature on the species, although Minton and Weinstein [57
] did report low concentrations of MTX from a few specimens of C. atrox
. The lack of admixture between C. atrox
and C. scutulatus
in our data also argues against a role for hybridization between these two species in generating the type I venom population of C. scutulatus
in Central Arizona, as hypothesized by Aird et al.
]. None of our MTX-negative C. scutulatus
showed evidence of admixture from C. atrox
, and neither did any other C. scutulatus
in our sample, a result consistent with previous analyses [51
The failure of the MTX genes to spread into the range of C. viridis
argues against the hypothesis that highly lethal neurotoxins necessarily represent a strong adaptive advantage for rattlesnakes. The parapatry between these two closely related species of rattlesnakes represents a best-case scenario for adaptive introgression of toxin-encoding genes: other things being equal, closely related species are more likely to be reproductively compatible than distantly related species, hybrids are likely to incur a lower loss of fitness than hybrids between more distantly related species, and it is less likely that linked, selectively disadvantageous loci will slow the spread of advantageous toxin genes [34
]. The imperviousness of the gene pool of C. viridis
to penetration by the MTX genes thus suggests that possession of these highly lethal neurotoxins is not necessarily a strong selective advantage. Clearly, we cannot exclude the possibility of a different outcome under other circumstances, such as different selective regimes or different constellations of linked genes. Moreover, since we do not know the age of this contact zone, we cannot reject the possibility that, given sufficient time, a degree of introgression of MTX genes may occur. However, given the results presented here, we conclude that the C. scutulatus
hybrid zone does not provide evidence favoring the hypothesis that limited hybridization may be enough to facilitate the wider and relatively rapid spread of toxin genes in a species in which they were previously missing.
Hybridization has been invoked as a cause of venom variation in multiple groups of snakes [30
], but with little evidence beyond incongruence between phylogeny and venom composition. The rigorous genetic identification of additional hybrid zones between venomous snake species could make a significant contribution to our understanding of venom evolution and the role of adaptive hybridization therein. Until then, based on the results presented herein, we suggest that hybridization should not be invoked as an explanation for unexpected patterns of inter- and intraspecific variation in snake venom composition or for unusual cases of clinical snakebite envenoming without compelling evidence.