Phenotypic integration in the feeding system of the eastern diamondback rattlesnake (Crotalus adamanteus)

Abstract

Selection can vary geographically across environments and temporally over the lifetime of an individual. Unlike geographic contexts, where different selective regimes can act on different alleles, age-specific selection is constrained to act on the same genome by altering age-specific expression. Snake venoms are exceptional traits for studying ontogeny because toxin expression variation directly changes the phenotype; relative amounts of venom components determine, in part, venom efficacy. Phenotypic integration is the dependent relationship between different traits that collectively produce a complex phenotype and, in venomous snakes, may include traits as diverse as venom, head shape and fang length. We examined the feeding system of the eastern diamondback rattlesnake (Crotalus adamanteus) across environments and over the lifetime of individuals and used a genotype-phenotype map approach, protein expression data and morphological data to demonstrate that: (i) ontogenetic effects explained more of the variation in toxin expression variation than geographic effects, (ii) both juveniles and adults varied geographically, (iii) toxin expression variation was a result of directional selection and (iv) different venom phenotypes covaried with morphological traits also associated with feeding in temporal (ontogenetic) and geographic (functional) contexts. These data are the first to demonstrate, to our knowledge, phenotypic integration between multiple morphological characters and a biochemical phenotype across populations and age classes. We identified copy number variation as the mechanism driving the difference in the venom phenotype associated with these morphological differences, and the parallel mitochondrial, venom and morphological divergence between northern and southern clades suggests that each clade may warrant classification as a separate evolutionarily significant unit.

Expression Differentiation Is Constrained to Low-Expression Proteins over Ecological Timescales

“…Polymorphisms at a much larger genomic scale, such as gene duplications and deletions (Stranger et al 2007), also can alter the expression level of a particular protein (Nguyen et al 2006). The correlation between gene copy-number differences and changes in gene expression has been documented previously (Cheng et al 2005;Freeman et al 2006;Nair et al 2008), including in venoms (Margres et al 2015b), and venom protein families are believed to be the result of gene duplication and positive selection (Casewell et al 2011) via the birth-anddeath model of protein evolution (Fry et al 2008). The significant expression variation we detected therefore could be the result of variation in copy number, assuming that variation in copy number would affect low-expression (and presumably low-copy) genes more than high-expression, high-copy genes (e.g., the difference between 10 and 12 copies for a particular protein may not be significant, but the difference between 2 and 4 copies may be).…”

“…lack of neutral differentiation and the significant phenotypic divergence across S. miliarius and C. adamanteus populations suggest that the observed protein expression differentiation was a result of directional selection (Margres et al 2015b), potentially despite gene flow, although founder effects currently cannot be ruled out. Expression variation is typically attributed to cis-regulatory mutations (Carroll 2008).…”

“…A fragment of cytochrome b, 841 and 1003 bp, respectively, was amplified in 25-ml PCR runs using the H16064 and L14910 primers and thermal cycling protocol described by Burbrink et al (2000). An 1018-bp fragment for C. adamanteus (accession numbers KJ730289, KJ730300, KJ730314, KJ730321, KJ730327, KJ730342, KJ730344, KJ730345, KJ730351, KJ730357, KJ730358, KJ730362, KJ730364, KJ730370, KJ730377, KJ730383, KJ730389, KJ730394, and KJ730396) and C. horridus (outgroup; accession number KJ730366) were taken from Margres et al (2015b) following the same approach. PCR products were purified using the QIAGEN QIAquick PCR Purification Kit, and sequencing was on the Applied Biosystems 3730 Genetic Analyzer.…”

“…We did not specifically test for plasticity in this study because venom expression differences have been shown repeatedly to be under genetic control and not environmentally induced (Daltry et al 1996;Gibbs et al 2011;Holding et al 2015;Margres et al 2015b), and the feeding ecology of venomous snakes makes adaptive plasticity unlikely. Because venom is stored for long periods of time (e.g., over winter and between infrequent feeding events) and previous meals may not be robust predictors of future meals, plasticity would be unlikely to provide any adaptive advantage for this trait.…”

“…We recorded sex, snout-vent length, and total length for each individual. We limited our analyses to adults to avoid the potentially confounding effects of ontogenetic protein expression variation, which has been documented previously (Mackessy 1988;Calvete et al 2010;Durban et al 2013;Margres et al 2015b …”