Quantitative genetics of sexual plasticity: the environmental threshold model and genotype-by-environment interaction for phallus development in the snail Bulinus truncatus

Sexual polymorphisms are model systems for analyzing the evolution of reproductive strategies. However, their plasticity and other binary traits have rarely been studied, with respect to environmental variables. A possible reason is that, although threshold models offer an adequate quantitative genetics framework for binary traits in a single environment, analyzing their plasticity requires more refined empirical and theoretical approaches. The statistical framework proposed here, based on the environmental threshold model (ETM), should partially fill this gap. This methodology is applied to an empirical dataset on a plastic sexual polymorphism, aphally, in the snail Bulinus truncatus. Aphally is characterized by the co-occurrence of regular hermaphrodites (euphallics) together with hermaphrodites deprived of the male copulatory organ (aphallics). Reaction norms were determined for 40 inbred lines, distributed at three temperatures, in a first experiment. A second experiment allowed us to rule out maternal effects. We confirmed the existence of high broad-sense heritabilities as well as a positive effect of high temperatures on aphally. However a significant genotype-by-environment interaction was detected for the first time, suggesting that sexual plasticity itself can respond to selection. A nested series of four ETM-like models was developed for estimating genetical effects on both mean aphally rate and plasticity. These models were tested using a maximum-likelihood procedure and fitted to aphally data. Although no perfect fit of models to data was observed, the refined versions of ETM models conveniently reduce the analysis of complex reaction norms of binary traits into standard quantitative genetics parameters, such as genetic values and environmental variances.     

The Genetical and Environmental Determination of Phally Polymorphism in the Freshwater Snail Bulinus Truncatus

In some species of self-fertile pulmonate snails, two sexual morphs co-occur in natural populations: regular individuals and aphallic individuals that cannot transmit sperm to other snails. Purely aphallic populations therefore reproduce obligatorily by selfing. Understanding the evolution of aphally and selfing in these snails requires a precise knowledge of phally determination. In this paper, we investigate the genetic and environmental determination of aphally in Bulinus truncatus by a survey of the family (offspring) aphally ratio of 233 individuals originating from seven natural populations and a study of the reaction norm of the family aphally ratio to temperature using 60 individuals from 10 selfed lineages of one population. Our results indicate a high genetic variability for the determination of aphally between populations and within some populations, associated with a high level of genetic determination. Our second experiment indicates a significant temperature and lineage effect though no interaction between these two effects. We discuss our results in the framework of threshold models developed for dimorphic traits with polygenic inheritance. We propose that the sexual morph of an individual at a given temperature is determined by a temperature threshold value depending on both the individual genotype and probabilistic processes.     

Segregating variation in the polycomb group gene cramped alters the effect of temperature on multiple traits

The phenotype produced by a given genotype can be strongly modulated by environmental conditions. Therefore, natural populations continuously adapt to environment heterogeneity to maintain optimal phenotypes. It generates a high genetic variation in environment-sensitive gene networks, which is thought to facilitate evolution. Here we analyze the chromatin regulator crm, identified as a candidate for adaptation of Drosophila melanogaster to northern latitudes. We show that crm contributes to environmental canalization. In particular, crm modulates the effect of temperature on a genomic region encoding Hedgehog and Wingless signaling effectors. crm affects this region through both constitutive heterochromatin and Polycomb silencing. Furthermore, we show that crm European and African natural variants shift the reaction norms of plastic traits. Interestingly, traits modulated by crm natural variants can differ markedly between Drosophila species, suggesting that temperature adaptation facilitates their evolution.     

Reaction norm variants for male calling song in populations of Achroia grisella (Lepidoptera: Pyralidae): toward a resolution of the lek paradox

Significant additive genetic variance often occurs for male advertisement traits in spite of the directional selection imposed by female choice, a problem generally known in evolutionary biology as the lek paradox. One hypothesis, which has limited support from recent studies, for the resolution of this paradox is the role of genotype x environment interaction in which no one genotype exhibits the superior performance in all environments--a crossover of reaction norms. However, these studies have not characterized the actual variation of reaction norms present in natural populations, and the extent to which crossover maintains genetic variance remains unknown. Here, we present a study of genotype x environment interaction for the male calling song in populations of Achroia grisella (Lepidoptera: Pyralidae; lesser waxmoth). We report significant variance among reaction norms for male calling song in two North American populations of A. grisella as measured along temperature, food availability, and density gradients, and there is a relatively high incidence of crossover of the temperature reaction norms. This range of reaction norm variants and their crossover may reflect the co-occurrence of plastic and canalized genotypes, and we argue that the different responses of these variants along environmental gradients may contribute toward the maintenance of genetic variance for male song.     

Ontogenetic phenotypic plasticity during the reproductive phase in Arabidopsis thaliana (Brassicaceae)

While phenotypic plasticity has been the focus of much research and debate in the recent ecological and evolutionary literature, the developmental nature of the phenomenon has been mostly overlooked. A developmental perspective must ultimately be an integral part of our understanding of how organisms cope with heterogeneous environments. In this paper I use the rapid cycling Arabidopsis thaliana to address the following questions concerning developmental plasticity. (1) Are there genetic and/or environmental differences in parameters describing ontogenetic trajectories? (2) Is ontogenetic variation produced by differences in genotypes and/or environments for two crucial traits of the reproductive phase of the life cycle, stem elongation and flower production? (3) Is there ontogenetic variability for the correlation between the two characters? I found genetic variation, plasticity, and variation for plasticity affecting at least some of the growth parameters, indicating potential for evolution via heterochronic shifts in ontogenetic trajectories. Within-population differences among families are determined before the onset of the reproductive phase, while among-population variation is the result of divergence during the reproductive phase of the ontogeny. Finally, the ontogenetic profiles of character correlations are very distinct between the ecologically meaningful categories of early- and late-flowering “ecotypes” in this species, and show susceptibility to environmental change.     

Phenotypic plasticity of reproductive effort in a colonial ascidian,Botryllus schlosseri

Phenotypic plasticity is the capability of a genotype to produce different phenotypes in different environments. Previous studies have indicated phenotypic variability in asexual, male, and female reproduction in Botryllus schlosseri, a hermaphroditic, colonial ascidian, but not explicitly tested for genotype by environment interactions that indicate genetic variation in plastic responses. Consequently, clones derived from an estuarine population were deployed at their native site and a warmer, higher productivity site 10 km up-river. Male reproduction was assayed by testis size, female reproduction by the number of eggs produced, and asexual reproduction by colony growth rate. To test for ontogenetic effects, data were collected from two different generations of zooids born in the field. Analyses of variance indicated plasticity in asexual and female reproduction during the first zooid generation and plasticity in all three traits during the third zooid generation. Reaction norms varied significantly among genotypes in direction and magnitude for asexual reproduction at both times, implying that selection on asexual reproduction is weak. Sperm production during the third zooid generation was significantly lower at the nonnative site, but there was no genotype by environment interaction. The reaction norms for female reproduction varied significantly among genotypes in direction and magnitude during the first zooid generation, but only varied in magnitude during the third generation, with egg production being higher in all genotypes at the nonnative site. Comparisons of weighted frequency distributions between sites demonstrated that differences in egg production in the third generation were due to increases in the proportion of reproductive zooids within a colony. The greater emphasis on female reproduction at a site associated with higher food availability and temperature, and the greater emphasis on male reproduction at a colder, food-limited site, supports predictions from sex allocation theory.     

Size and architectural traits as ontogenetic determinants of fitness in a phenotypically plastic annual weed (Amaranthus albus)

Abstract Plasticity of size and architectural traits, and their importance to reproductive fitness, were estimated in relation to nutrient availability in the annual Amaranthus albus . Seeds from seven field-collected genotypes were used to rear a first generation under uniform conditions. Seed families (inbred lines) from four first-generation plants per genotype were used to rear a second generation in a glasshouse for 10 weeks. Nine plants per family were regularly fertilized, while nine others were unfertilized. Size was estimated at 5, 8, and 10 weeks; architectural traits were recorded at 8 weeks (no. branches) and 10 weeks (no. branches and branch length). Fitness was assessed by the number of seeds per plant. Because traits were intercorrelated, size-scaled architectural traits were generated and allometric analyses performed. Genetic variation was analyzed for the second generation among inbred lines of the original genotypes, and among families within each genotype. For fertilized and unfertilized groups, early size (volume occupied at 5 weeks) and branch length per mass were significant determinants of fitness. The number of branches per size explained a smaller proportion of the variance in fitness. Genotype by treatment interaction was apparent for some traits, indicating genetic variation for plasticity, but plasticity of size did not change over ontogeny. Significant effects of genotype on size and architectural traits, and fitness, were detected mostly in the unfertilized group. Thus, selection is most likely to differentiate among genotypes in nutrient-poor environments. Lengthening of multiple branches increases seed output throughout the season, and genotypes with longer branches per unit mass have a selective advantage. Because early size is correlated with seed output, ontogenetic plasticity in response to improved soil resources allows opportunistic increases in fitness.     

Intraspecific variation and plasticity in growth and foliar morphology along a climate gradient in the Canary Island pine

Foliar plasticity in response to ontogeny, location within the plant and environmental changes is widespread among long-lived organisms. To quantify the phenotypic variation in needle morphology and anatomy in response to a climate gradient, we compared contrasted populations of Pinus canariensis grown in five sites inside and outside the natural distribution area of the species. Most needle and growth traits were strongly affected by site. In general, site xericity increased the relative area of the dermal and transfusion tissues and decreased mesophyll and endodermis. Within each site, provenances from less productive locations tended to show longer needles, less relative area of dermal tissues but higher relative area of mesophyll and transfusion tissue than provenances from fertile origins. Although sclerophylly increased with aridity, no genetic differences were found for this trait thus apparently the ontogenetic delay of some provenances in xeric environments was not related with the formation of tougher needles. Several patterns of phenotypic response to different environments were shown. In general, all traits were plastic but the degree of plasticity was higher in traits related with growth than foliar traits. These results, combined with formerly published research, suggest that highly plastic populations rather than narrowly specialized ones have been selected in this species to cope with the complex interaction of environmental factors in its habitat.     

Growing up aspen: ontogeny and trade-offs shape growth,defence and reproduction in a foundation species

Background and AimsIntraspecific variation in foundation species of forest ecosystems can shape community and ecosystem properties, particularly when that variation has a genetic basis. Traits mediating interactions with other species are predicted by simple allocation models to follow ontogenetic patterns that are rarely studied in trees. The aim of this research was to identify the roles of genotype, ontogeny and genotypic trade-offs shaping growth, defence and reproduction in aspen.MethodsWe established a common garden replicating >500 aspen genets in Wisconsin, USA. Trees were measured through the juvenile period into the onset of reproduction, for growth, defence chemistry (phenolic glycosides and condensed tannins), nitrogen, extrafloral nectaries, leaf morphology (specific leaf area), flower production and foliar herbivory and disease. We also assayed the TOZ19 sex marker and heterozygosity at ten microsatellite loci.Key ResultsWe found high levels of genotypic variation for all traits, and high heritabilities for both the traits and their ontogenetic trajectories. Ontogeny strongly shaped intraspecific variation, and trade-offs among growth, defence and reproduction supported some predictions while contradicting others. Both direct resistance (chemical defence) and indirect defence (extrafloral nectaries) declined during the juvenile stage, prior to the onset of reproduction. Reproduction was higher in trees that were larger, male and had higher individual heterozygosity. Growth was diminished by genotypic allocation to both direct and indirect defence as well as to reproduction, but we found no evidence of trade-offs between defence and reproduction.ConclusionsKey traits affecting the ecological communities of aspen have high levels of genotypic variation and heritability, strong patterns of ontogeny and clear trade-offs among growth, defence and reproduction. The architecture of aspen’s community genetics – its ontogeny, trade-offs and especially its great variability – is shaped by both its broad range and the diverse community of associates, and in turn further fosters that diversity.     

Structural blueprint and ontogeny determine the adaptive value of the plastic response to competition in clonal plants: a modelling approach

Local competitive interactions strongly influence plant community dynamics. To maintain their performance under competition, clonal plants may plastically modify their network architecture to grow in the direction of least interference. The adaptive value of this plastic avoidance response may depend, however, on traits linked with the plant’s structural blueprint and ontogeny. We tested this hypothesis using virtual populations. We used an Individual Based Model to simulate competitive interactions among clones within a plant population. Clonal growth was studied under three competition intensities in plastic and non-plastic individuals. Plasticity buffered the negative impacts of competition at intermediate densities of competitors by promoting clone clumping. Success despite competition was promoted by traits linked with (1) the plant’s structural blueprint (weak apical dominance and sympodial growth) and (2) ontogenetic processes, with an increasing or a decreasing dependence of the elongation process on the branch generation level or length along the competition intensity gradient respectively. The adaptive value of the plastic avoidance response depended on the same traits. This response only modulated their importance for clone success. Our results show that structural blueprint and ontogeny can be primary filters of plasticity and can have strong implications for evolutionary ecology, as they may explain why clonal plants have developed many species-specific plastic avoidance behaviours.     

Ontogenetic changes in genetic variances of age-dependent plasticity along a latitudinal gradient

The expression of phenotypic plasticity may differ among life stages of the same organism. Age-dependent plasticity can be important for adaptation to heterogeneous environments, but this has only recently been recognized. Whether age-dependent plasticity is a common outcome of local adaptation and whether populations harbor genetic variation in this respect remains largely unknown. To answer these questions, we estimated levels of additive genetic variation in age-dependent plasticity in six species of damselflies sampled from 18 populations along a latitudinal gradient spanning 3600 km. We reared full sib larvae at three temperatures and estimated genetic variances in the height and slope of thermal reaction norms of body size at three points in time during ontogeny using random regression. Our data show that most populations harbor genetic variation in growth rate (reaction norm height) in all ontogenetic stages, but only some populations and ontogenetic stages were found to harbor genetic variation in thermal plasticity (reaction norm slope). Genetic variances in reaction norm height differed among species, while genetic variances in reaction norm slope differed among populations. The slope of the ontogenetic trend in genetic variances of both reaction norm height and slope increased with latitude. We propose that differences in genetic variances reflect temporal and spatial variation in the strength and direction of natural selection on growth trajectories and age-dependent plasticity. Selection on age-dependent plasticity may depend on the interaction between temperature seasonality and time constraints associated with variation in life history traits such as generation length.     

PHENOTYPIC VARIATION OF GROWTH TRAJECTORIES IN FINCHES

Evolution cannot proceed without phenotypic variation for selection to act on. This is particularly true of ontogenetic parameters because it is changes in these parameters that give rise to new phenotypes. I analyzed the amount and dimensionality of phenotypic variation on growth trajectories in early ontogeny in three species of finches (Fringillidae) using the recently developed infinite-dimensional model. For two species, eight traits were analyzed, and for a third, six traits. Growth data were analyzed only up to 6 d of age in two species and 8 d of age in the third. The results were very similar for all species and traits. A very large proportion of the phenotypic variation in growth trajectories was confined to a single dimension. This dimension corresponded to a simultaneous increase/decrease at all ages in early ontogeny. The eigenfunctions, each describing a family of similar-shaped growth trajectories, were highly collinear among traits. A high covariance existed among traits at the same and different ages. If some part of the phenotypic variation has an additive genetic basis, then any selection for a change in size at one age in one trait will lead to a response in a size at subsequent ages and in the other traits. This in turn suggests that morphological evolution frequently will move along a multivariate size axis, as has indeed been found in several taxa.     

Co-Gradient Variation in Growth Rate and Development Time of a Broadly Distributed Butterfly

Widespread species often show geographic variation in thermally-sensitive traits, providing insight into how species respond to shifts in temperature through time. Such patterns may arise from phenotypic plasticity, genetic adaptation, or their interaction. In some cases, the effects of genotype and temperature may act together to reduce, or to exacerbate, phenotypic variation in fitness-related traits across varying thermal environments. We find evidence for such interactions in life-history traits of Heteronympha merope, a butterfly distributed across a broad latitudinal gradient in south-eastern Australia. We show that body size in this butterfly is negatively related to developmental temperature in the laboratory, in accordance with the temperature-size rule, but not in the field, despite very strong temperature gradients. A common garden experiment on larval thermal responses, spanning the environmental extremes of H. merope''s distribution, revealed that butterflies from low latitude (warmer climate) populations have relatively fast intrinsic growth and development rates compared to those from cooler climates. These synergistic effects of genotype and temperature across the landscape (co-gradient variation) are likely to accentuate phenotypic variation in these traits, and this interaction must be accounted for when predicting how H. merope will respond to temperature change through time. These results highlight the importance of understanding how variation in life-history traits may arise in response to environmental change. Without this knowledge, we may fail to detect whether organisms are tracking environmental change, and if they are, whether it is by plasticity, adaptation or both.     

Early developmental plasticity and integrative responses in arctic charr (Salvelinus alpinus): effects of water velocity on body size and shape

Environmental conditions such as temperature and water velocity may induce changes among alternative developmental pathways, i.e. phenotypic responses, in vertebrates. However, the extent to which the environment induces developmental plasticity and integrated developmental responses during early ontogeny of fishes remains poorly documented. We analyzed the responses of newly hatched Arctic charr (Salvelinus alpinus) to four experimental water velocities during 100 days of development. To our knowledge, this work is the first to analyze developmental plasticity responses of body morphology to an experimental gradient of water velocities during early ontogeny of fish. Arctic charr body size and shape responses show first, that morphometric traits display significant differences between low and high water velocities, thus revealing directional changes in body traits. Secondly, trait variation allows the recognition of critical ontogenetic periods that are most responsive to environmental constraints (40-70 and 80-90 days) and exhibit different levels of developmental plasticity. This is supported by the observation of asynchronous timing of variation peaks among treatments. Third, morphological interaction of traits is developmentally plastic and time-dependent. We suggest that developmental responses of traits plasticity and interaction at critical ontogenetic periods are congruent with specific environmental conditions to maintain the functional integrity of the organism.     

Diverse genotypes of the amphibian‐killing fungus produce distinct phenotypes through plastic responses to temperature

Phenotypes are the target of selection and affect the ability of organisms to persist in variable environments. Phenotypes can be influenced directly by genes and/or by phenotypic plasticity. The amphibian‐killing fungus Batrachochytrium dendrobatidis (Bd) has a global distribution, unusually broad host range, and high genetic diversity. Phenotypic plasticity may be an important process that allows this pathogen to infect hundreds of species in diverse environments. We quantified phenotypic variation of nine Bd genotypes from two Bd lineages (Global Pandemic Lineage [GPL] and Brazil) and a hybrid (GPL‐Brazil) grown at three temperatures (12, 18 and 24°C). We measured five functional traits including two morphological traits (zoospore and zoosporangium sizes) and three life history traits (carrying capacity, time to fastest growth and exponential growth rate) in a phylogenetic framework. Temperature caused highly plastic responses within each genotype, with all Bd genotypes showing phenotypic plasticity in at least three traits. Among genotypes, Bd generally showed the same direction of plastic response to temperature: larger zoosporangia, higher carrying capacity, longer time to fastest growth and slower exponential growth at lower temperatures. The exception was zoospore size, which was highly variable. Our findings indicate that Bd genotypes have evolved novel phenotypes through plastic responses to temperature over very short timescales. High phenotypic variability likely extends to other traits and may facilitate the large host range and rapid spread of Bd.     

Two reproductive traits show contrasting genetic architectures in Plantago lanceolata

In many species, temperature‐sensitive phenotypic plasticity (i.e., an individual's phenotypic response to temperature) displays a positive correlation with latitude, a pattern presumed to reflect local adaptation. This geographical pattern raises two general questions: (a) Do a few large‐effect genes contribute to latitudinal variation in a trait? (b) Is the thermal plasticity of different traits regulated pleiotropically? To address the questions, we crossed individuals of Plantago lanceolata derived from northern and southern European populations. Individuals naturally exhibited high and low thermal plasticity in floral reflectance and flowering time. We grew parents and offspring in controlled cool‐ and warm‐temperature environments, mimicking what plants would encounter in nature. We obtained genetic markers via genotype‐by‐sequencing, produced the first recombination map for this ecologically important nonmodel species, and performed quantitative trait locus (QTL) mapping of thermal plasticity and single‐environment values for both traits. We identified a large‐effect QTL that largely explained the reflectance plasticity differences between northern and southern populations. We identified multiple smaller‐effect QTLs affecting aspects of flowering time, one of which affected flowering time plasticity. The results indicate that the genetic architecture of thermal plasticity in flowering is more complex than for reflectance. One flowering time QTL showed strong cytonuclear interactions under cool temperatures. Reflectance and flowering plasticity QTLs did not colocalize, suggesting little pleiotropic genetic control and freedom for independent trait evolution. Such genetic information about the architecture of plasticity is environmentally important because it informs us about the potential for plasticity to offset negative effects of climate change.     

Ontogenetic changes in tolerance to herbivory in Arabidopsis

Tolerance to herbivory—the ability of plants to maintain fitness despite herbivore damage—is expected to change during the life cycle of plants because the physiological mechanisms underlying tolerance to herbivory are linked to growth, and resource allocation to growth changes throughout ontogeny. We used the model plant Arabidopsis thaliana to test two hypotheses: that tolerance increases as plants grow, and that tolerance decreases at the onset of reproduction. We chose three accessions previously reported to vary for resistance to herbivory in order to explore whether tolerance and resistance are inversely related. Cabbage looper (Trichoplusia ni) larvae were allowed to feed on plants at either the four-leaf, six-leaf, or 1st-flower developmental stage until 50% of the leaf area was removed. Overall, we found a trend for increased tolerance with ontogenetic stage, but there were important differences among accessions in their response to herbivory at different stages. Tolerance did not decrease with the onset of flowering, nor did we find any correlation between resistance and tolerance levels. Three main plant traits correlated strongly with tolerance: stem mass, an earlier onset of reproduction and a longer fruiting period. This study suggests there may be considerable variation in ontogenetic patterns of tolerance in natural populations of A. thaliana, and warrants further investigations with more accessions or natural populations, and detailed measurements of traits purported to contribute to tolerance in our quest to understand the mechanisms of tolerance to herbivory.     

Keeping your options open: Maintenance of thermal plasticity during adaptation to a stable environment

Phenotypic plasticity may allow species to cope with environmental variation. The study of thermal plasticity and its evolution helps understanding how populations respond to variation in temperature. In the context of climate change, it is essential to realize the impact of historical differences in the ability of populations to exhibit a plastic response to thermal variation and how it evolves during colonization of new environments. We have analyzed the real‐time evolution of thermal reaction norms of adult and juvenile traits in Drosophila subobscura populations from three locations of Europe in the laboratory. These populations were kept at a constant temperature of 18ºC, and were periodically assayed at three experimental temperatures (13ºC, 18ºC, and 23ºC). We found initial differentiation between populations in thermal plasticity as well as evolutionary convergence in the shape of reaction norms for some adult traits, but not for any of the juvenile traits. Contrary to theoretical expectations, an overall better performance of high latitude populations across temperatures in early generations was observed. Our study shows that the evolution of thermal plasticity is trait specific, and that a new stable environment did not limit the ability of populations to cope with environmental challenges.     

Plasticity in Vegetative Growth over Contrasted Growing Sites of an F1 Olive Tree Progeny during Its Juvenile Phase

Climatic changes impact fruit tree growth and severely limit their production. Investigating the tree ability to cope with environmental variations is thus necessary to adapt breeding and management strategies in order to ensure sustainable production. In this study, we assessed the genetic parameters and genotype by environment interaction (GxE) during the early tree growth. One hundred and twenty olive seedlings derived from the cross ‘Olivière’ x ‘Arbequina’ were examined across two sites with contrasted environments, accounting for ontogenetic trends over three years. Models including the year of growth, branching order, environment, genotype effects, and their interactions were built with variance function and covariance structure of residuals when necessary. After selection of a model, broad sense heritabilities were estimated. Despite strong environmental effect on most traits, no GxE was found. Moreover, the internal structure of traits co-variation was similar in both sites. Ontogenetic growth variation, related to (i) the overall tree form and (ii) the growth and branching habit at growth unit scale, was not altered by the environment. Finally, a moderate to strong genetic control was identified for traits at the whole tree scale and at internode scale. Among all studied traits, the maximal internode length exhibited the highest heritability (H² = 0.74). Considering the determinant role of this trait in tree architecture and its stability across environments, this study consolidates its relevance for breeding.     

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

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.