Stable isotopes reveal sex- and context-dependent amino acid routing in green anole lizards (Anolis carolinensis).

Allocation of acquired resources to phenotypic traits is affected by resource availability and current selective context. While differential investment in traits is well documented, the mechanisms driving investment at lower levels of biological organization, which are not directly related to fitness, remain poorly understood. We supplemented adult male and female Anolis carolinensis lizards with an isotopically labelled essential amino acid (13C-leucine) to track routing in four tissues (muscle, liver, gonads and spleen) under different combinations of resource availability (high- and low-calorie diets) and exercise training (sprint training and endurance capacity). We predicted sprint training should drive routing to muscle, and endurance training to liver and spleen, and that investment in gonads should be of lower priority in each of the cases of energetic stress. We found complex interactions between training regime, diet and tissue type in females, and between tissue type and training, and tissue type and diet in males, suggesting that males and females adjust their 13C-leucine routing strategies differently in response to similar environmental challenges. Importantly, our data show evidence of increased 13C-leucine routing in training contexts not to muscle as we expected, but to the spleen, which turns over blood cells, and to the liver, which supports metabolism under differing energetic scenarios. Our results reveal the context-specific nature of long-term trade-offs associated with increased chronic activity. They also illustrate the importance of considering the costs of locomotion in studies of life-history strategies.

Conserved and convergent mechanisms underlying performance-life-history trade-offs.

Phenotypic trade-offs are inevitable in nature, but the mechanisms driving them are poorly understood. Movement and oxygen are essential to all animals, and as such, the common ancestor to all living animals passed on mechanisms to acquire oxygen and contract muscle, sometimes at the expense of other activities or expression of traits. Nevertheless, convergent pathways have also evolved to deal with critical trade-offs that are necessary to survive ubiquitous environmental challenges. We discuss how whole-animal performance traits, such as locomotion, are important to fitness, yet costly, resulting in trade-offs with other aspects of the phenotype via specific conserved and convergent mechanistic pathways across all animals. Specifically, we discuss conserved pathways involved in muscle structure and signaling, insulin/insulin-like signaling, sirtuins, mitochondria and hypoxia-inducible factors, as well as convergent pathways involved in energy regulation, development, reproductive investment and energy storage. The details of these mechanisms are only known from a few model systems, and more comparative studies are needed. We make two main recommendations as a framework for future studies of animal form and function. First, studies of performance should consider the broader life-history context of the organism, and vice versa, as performance expression can require a large portion of acquired resources. Second, studies of life histories or mechanistic pathways that measure performance should do so in meaningful and standardized ways. Understanding proximate mechanisms of phenotypic trade-offs will not only better explain the phenotypes of the organisms we study, but also allow predictions about phenotypic variation at the evolutionary scale.

Sex-specific multivariate morphology/performance relationships in Anolis carolinensis.

Animals rely on their ability to perform certain tasks sufficiently well to survive, secure mates and reproduce. Performance traits depend on morphology, and so morphological traits should predict performance, yet this relationship is often confounded by multiple competing performance demands. Males and females experience different selection pressures on performance, and the consequent sexual conflict over performance expression can either constrain performance evolution or drive sexual dimorphism in both size and shape. Furthermore, change in a single morphological trait may benefit some performance traits at the expense of others, resulting in functional trade-offs. Identifying general or sex-specific relationships between morphology and performance at the organismal level thus requires a multivariate approach, as individuals are products of both an integrated phenotype and the ecological environment in which they have developed and evolved. We estimated the multivariate morphology→performance gradient in wild-caught, green anoles (Anolis carolinensis) by measuring external morphology and forelimb and hindlimb musculature, and mapping these morphological traits to seven measured performance traits that cover the broad range of ecological challenges faced by these animals (sprint speed, endurance, exertion distance, climbing power, jump power, cling force and bite force). We demonstrate that males and females differ in their multivariate mapping of traits on performance, indicating that sex-specific ecological demands likely shape these relationships, but do not differ in performance integration.

Endurance training does not affect maximum exertion/distance capacity in Anolis carolinensis lizards.

Locomotor performance abilities are key predictors of survival and reproductive success in animals and understanding how selection targets them can provide insights into how morphology and physiology relate to fitness. But despite the large body of work on performance traits, along with well-established protocols to measure them, performance can be challenging to measure. Endurance, for instance, is commonly measured by recording how long an animal can run at a set pace until exhaustion, which is time consuming and requires dedicated equipment. Consequently, exertion or distance capacity, measured as distance run until exhaustion when chased, is often used as a proxy for endurance, but the relationship between these two metrics has never been assessed even though they likely rely on different underlying physiological mechanisms. We tested experimentally for a relationship between endurance and exertion by training green anole lizards for sprinting and endurance and measuring whether exertion capacity responds to either type of training. Prior to training and across treatments, males displayed a mean (±s.d.) exertion capacity of 14.08±0.29 m and females 12.03±3.52 m; after training, this was 14.78±3.57 m and 12.19±2.21 m, respectively. We found that exertion capacity was unaffected by either type of training in green anoles. We also show that a positive relationship between endurance and exertion capacity pre-training exists only in females and that this relationship is inconsistent among studies. Exertion should be studied as a locomotor trait in its own right and not as a proxy for endurance.

Sprint training interacts with body mass to affect hepatic insulin-like growth factor expression in female green anoles (Anolis carolinensis).

Locomotor performance is a key predictor of fitness in many animal species. As such, locomotion integrates the output of a number of morphological, physiological, and molecular levels of organization, yet relatively little is known regarding the major molecular pathways that bolster locomotor performance. One potentially relevant pathway is the insulin and insulin-like signaling (IIS) network, a significant regulator of physiological processes such as reproduction, growth, and metabolism. Two primary hormones of this network, insulin-like growth factor 1 (IGF1) and insulin-like growth factor 2 (IGF2) are important mediators of these processes and, consequently, of life-history strategies. We sprint-trained green anole (Anolis carolinensis) females to test the responsiveness of IGF1 and IGF2 hepatic gene expression to exercise training. We also tested how sprint training would affect glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and eukaryotic elongation factor 2 (EEF2). The former is a crucial enzyme for glycolytic function in a cell, and the latter is necessary for protein synthesis. Resistance exercise forces animals to increase investment of resources towards skeletal muscle growth. Because IGF1 and IGF2 are important hormones for growth, and GAPDH and EEF2 are crucial for proper cellular function, we hypothesized that these four genes would be affected by sprint training. We found that sprint training affects IGF and EEF2 expression, such that larger sprint-trained lizards express hepatic IGF1, IGF2, and EEF2 to a lesser extent than similarly sized untrained lizards. These results demonstrate that the IIS, and pathways connected to it, can react in a size-dependent manner and are implicated in the exercise response in reptiles.

Expression of insulin-like growth factors depends on both mass and resource availability in female green anoles (Anolis carolinensis).

The insulin and insulin-like signaling (IIS) network is an important mediator of cellular growth and metabolism in animals, and is sensitive to environmental conditions such as temperature and resource availability. The two main hormones of the IIS network, insulin-like growth factor 1 (IGF1) and insulin-like growth factor 2 (IGF2), are present in all vertebrates, yet little is known regarding the responsiveness of IGF2 in particular to external stimuli in non-mammalian animals. We manipulated diet (low or high quantity of food: low and high diet group, respectively) in adult green anole (Anolis carolinensis) females to test the effect of energetic state on hepatic gene expression of IGF1 and IGF2. The absolute expression of IGF2 in female green anoles was 100 times higher than that of IGF1 regardless of diet treatment, and IGF1 and IGF2 expression interacted with post-treatment body mass and treatment, as did the expression of the purported housekeeping genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and eukaryotic elongation factor 2 (EEF2). The low diet group showed a negative relationship between body mass and gene expression for all genes, whereas the relationships between body mass and gene expression in the high diet group were either absent (in the case of IGF1) or positive (for all other genes). After accounting for total change in mass, the low diet group expressed IGF2, GAPDH and EEF2 at higher levels compared with individuals in the high diet group of a similar change in mass. These results illustrate that expression of IGF1 and IGF2, and of the housekeeping genes is affected by energe-tic status in reptiles.

Experimentally enhanced performance decreases survival in nature.

Superior locomotor performance confers advantages in terms of male combat success, survival and fitness in a variety of organisms. In humans, investment in increased performance via the exercise response is also associated with numerous health benefits, and aerobic capacity is an important predictor of longevity. Although the response to exercise is conserved across vertebrates, no studies have tested whether non-human animals that invest in increased athletic performance through exercise realize a survival advantage in nature. Green anole lizards respond to exercise training, and enhanced performance drives trade-offs with reproduction and immunocompetence. We released sprint-trained, endurance-trained and untrained-control male and female green anole lizards into an isolated, urban island in New Orleans, LA, USA and monitored their survival. Sedentary controls realized a significant survivorship advantage compared to trained lizards. Our results suggest that locomotor capacity is currently optimized to maximize survival in green anoles, and that forcing additional investment in performance moves them into a suboptimal phenotypic space relative to their current environmental demands.

Energetic costs of performance in trained and untrained Anolis carolinensis lizards.

The energetic costs of performance constitute a non-trivial component of animals' daily energetic budgets. However, we currently lack an understanding of how those costs are partitioned among the various stages of performance development, maintenance and production. We manipulated individual investment in performance by training Anolis carolinensis lizards for endurance or sprinting ability. We then measured energetic expenditure both at rest and immediately following exercise to test whether such training alters the maintenance and production costs of performance. Trained lizards had lower resting metabolic rates than controls, suggestive of a maintenance saving associated with enhanced performance as opposed to a cost. Production costs also differed, with sprint-trained lizards incurring a larger energetic performance cost and experiencing longer recovery times compared with endurance trained and control animals. Although performance training modifies metabolism, production costs are probably the key drivers of trade-offs between performance and other life-history traits in this species.

The Integrative Life history of Maternal Effects.

Context-dependent allocation of resources drives trade-offs among fitness-related traits and other phenotypes to which those traits are linked. In addition, the amount and type of acquired resources can also affect the phenotypes of other organisms through indirect genetic effects, as exemplified by the maternal provisioning of offspring. Despite a large literature on maternal effects, we lack a comprehensive understanding of the extent to which mothers might affect the phenotypes of their offspring, as well as the various mechanisms by which they do so, particularly with regard to many functional traits that are key determinants of survival and reproduction. Our goals in this paper are to review the various approaches to measuring and understanding maternal effects, and to highlight some promising avenues for integration of maternal effects with some other key areas of evolutionary ecology. We focus especially on nutritional geometry; maternal age; and traits proximate to fitness such as whole-organism performance. Finally, we discuss the logistic and practical limits of quantifying these effects in many animal systems, and emphasize the value of integrative approaches in understanding the mechanisms underlying maternal influence on offspring phenotypes.

The maternal energetic environment affects both egg and offspring phenotypes in green anole lizards (Anolis carolinensis).

Animals exist in dynamic environments that may affect both their own fitness and that of their offspring. Maternal effects might allow mothers to prepare their offspring for the environment in which they will be born via several mechanisms, not all of which are well understood. Resource scarcity and forced resource allocation are two scenarios that could affect maternal investment by altering the amount and type of resources available for investment in offspring, albeit in potentially different ways. We tested the hypothesis that maternal dietary restriction and sprint training have different consequences for the offspring phenotype in an oviparous lizard (Anolis carolinensis). To do this, we collected and reared eggs from adult diet-manipulated females (low-diet [LD] or high-diet [HD]) and sprint-trained females (sprint trained [ST] or untrained [UT]) and measured both egg characteristics and hatchling morphology. ST and LD mothers laid both the fewest and heaviest eggs, and ST, UT, and LD eggs also had significantly longer incubation periods than the HD group. Hatchlings from the diet experiment (LD and HD offspring) were the heaviest overall. Furthermore, both body mass of the mother at oviposition and change in maternal body mass over the course of the experiment had significant and sometimes different effects on egg and offspring phenotypes, highlighting the importance of maternal energetic state to the allocation of maternal resources.

A performance-based cost to honest signalling in male green anole lizards (Anolis carolinensis).

Sexual signals are considered costly to produce and maintain under the handicap paradigm, and the reliability of signals is in turn thought to be maintained by these costs. Although previous studies have investigated the costly nature of signal production, few have considered whether honesty might be maintained not by the costliness of the signal itself, but by the costs involved in producing the signalled trait. If such a trait is itself costly to produce, then the burden of energetic investment may fall disproportionately on that trait, in addition to any costs of signal maintenance that may also be operating. Under limited resource conditions, these costs may therefore be great enough to disrupt an otherwise reliable signal-to-trait relationship. We present experimental evidence showing that dietary restriction decouples the otherwise honest relationship between a signal (dewlap size) and a whole-organism performance trait (bite force) in young adult male Anolis carolinensis lizards. Specifically, while investment in dewlap size is sustained under low-resource condition relative to the high-resource treatment, investment in bite force is substantially lower. Disruption of the otherwise honest dewlap size to bite force relationship is therefore driven by costs associated with the expression of performance rather than the costs of signal production in A. carolinensis.

Machine learning accurately predicts the multivariate performance phenotype from morphology in lizards.

Completing the genotype-to-phenotype map requires rigorous measurement of the entire multivariate organismal phenotype. However, phenotyping on a large scale is not feasible for many kinds of traits, resulting in missing data that can also cause problems for comparative analyses and the assessment of evolutionary trends across species. Measuring the multivariate performance phenotype is especially logistically challenging, and our ability to predict several performance traits from a given morphology is consequently poor. We developed a machine learning model to accurately estimate multivariate performance data from morphology alone by training it on a dataset containing performance and morphology data from 68 lizard species. Our final, stacked model predicts missing performance data accurately at the level of the individual from simple morphological measures. This model performed exceptionally well, even for performance traits that were missing values for >90% of the sampled individuals. Furthermore, incorporating phylogeny did not improve model fit, indicating that the phenotypic data alone preserved sufficient information to predict the performance based on morphological information. This approach can both significantly increase our understanding of performance evolution and act as a bridge to incorporate performance into future work on phenomics.

Defining individual quality over lifetimes and selective contexts.

Individual quality has been measured as a variety of different traits and in several different contexts. However, the implications of such measurements in terms of overall fitness are less straightforward than has generally been appreciated. Here we outline some key issues in this regard that have yet to be addressed. Specifically, we consider the importance of both variation in selection on individual and multivariate suites of traits, and of context-specific plasticity in allocation strategies. We argue that an explicit life-history perspective is crucial for understanding variation in quality, as both the strength and direction of selection and an individual's response to it can vary within a breeding season. Hence, 'quality' is not a static characteristic that can be measured by taking longitudinal measures of single traits across a population, but rather a dynamic, multivariate suite of traits that is dependent not only on the selective context, but also on the nature and intensity of selection operating at any given time. We highlight these points by considering recent research on selection and plasticity.

Size but not relatedness drives the spatial distribution of males within an urban population of Anolis carolinensis lizards.

The way that individuals are spatially organized in their environment is a fundamental population characteristic affecting social structure, mating system, and reproductive ecology. However, for many small or cryptic species, the factors driving the spatial distribution of individuals within a population are poorly understood and difficult to quantify. We combined microsatellite data, remote sensing, and mark-recapture techniques to test the relative importance of body size and relatedness in determining the spatial distribution of male Anolis carolinensis individuals within a focal population over a five-year period. We found that males maintain smaller home ranges than females. We found no relationship between male body size and home range size, nor any substantial impact of relatedness on the geographic proximity. Instead, the main driver of male spatial distribution in this population was differences in body size. We also found no evidence for offspring inheritance of their parent's territories. Males were never sampled within their father's territory providing strong support for male-biased dispersal. This study introduces a novel approach by combining standard mark release capture data with measures of pairwise relatedness, body size, and GPS locations to better understand the factors that drive the spatial distribution of individuals within a population.

Immune activation affects whole-organism performance in male but not female green anole lizards (Anolis carolinensis).

Immune responses are intuitively beneficial, but they can incur a variety of costs, many of which are poorly understood. The nature and extent of trade-offs between immune activity and other components of the integrated phenotype can vary, and depend on the type of immune challenge, as well as the energetic costs of simultaneously expressing other traits. There may also be sex differences in both immune activity and immunity-induced trade-offs, particularly in the case of trade-offs involving functional traits such as whole-organism performance capacities that might be of different fitness value to males and females. We tested the response of three performance traits (sprinting, endurance, and biting) to two different immune challenges (LPS injection and wound healing) in both male and female Anolis carolinensis lizards. We found clear differences in how male and female performance capacities were affected by immune activation. LPS injection and wound healing had interactive effects on all three performance traits in males, but immune activation did not affect female performance. We also found that the degree of wound healing exhibited complex interactive effects involving sex and type of immune activation that varied depending on the performance trait in question. These results demonstrate that male and female green anoles experience different consequences of immune responses, and also that the type and extent of that activation can drive trait-specific performance trade-offs.

Performance is no proxy for genetic quality: trade-offs between locomotion, attractiveness, and life history in crickets.

The genetic relationships among traits contributing to overall fitness are an important subject of inquiry because such relationships influence how suites of traits respond to selection. Within the field of sexual selection, these relationships are also of interest for assessing whether any given trait can be used as a proxy for total fitness. A growing number of studies have demonstrated close links between whole-organism performance traits and determinants of individual fitness; however, an understanding of the genetic relationships between performance and important aspects of genetic quality is currently lacking. We present the results of a quantitative genetic study in which we estimate covariation between a locomotor performance trait (maximal jumping ability), calling effort, sexual attractiveness, and life-history traits in male Teleogryllus commodus crickets. We show that the major axis of genetic variation (gmax) is characterized by a contrast between jump performance and life-history traits associated with mating success. Moreover, two additional axes of significant multivariate genetic variation exist, each characterized by strong contrasts among traits. These results argue against the existence of a single axis representing genetic quality, favoring instead the idea that resource allocation strategies shape multiple dimensions of genetic quality through trade-offs among key life-history traits, including performance.

Sprint speed is unaffected by dietary manipulation in trained male Anolis carolinensis lizards.

Performance traits are energetically costly, and their expression and use can drive trade-offs with other energetically costly life-history traits. However, different performance traits incur distinct costs and may be sensitive to both resource limitation and to the types of resources that are accrued. Protein is likely to be especially important for supporting burst performance traits such as sprint speed, but the effect of varying diet composition on sprint training in lizards, an emerging model system for exercise training, is unknown. We tested the hypothesis that the response to sprint training is sensitive to both the type and amount of resources in Anolis carolinensis. We also measured bite force across all treatments as a control whole-organism performance trait that should be unaffected by locomotor training. Both mass and bite force are reduced by dietary restriction over the course of 9 weeks of sprint training, but sprint speed is unaffected by either training or dietary restriction relative to controls. Furthermore, protein supplementation does not rescue a decline in either mass or bite force in trained, diet-restricted males. These results contrast with those for endurance training, and suggest that sprint speed is more canalized than either endurance or bite force in green anoles.

Conflict, compensation, and plasticity: Sex-specific, individual-level trade-offs in green anole (Anolis carolinensis) performance.

Trade-offs in performance expression occur because animals must perform multiple whole-organism performance tasks that place conflicting demands on shared underlying morphology. Although not always detectable within populations, such trade-offs may be apparent when analyzed at the level of the individual, particularly when all of the available data are taken into account as opposed to only maximum values. Detection of performance trade-offs is further complicated in species where sexual dimorphism drives performance differences between males and females, leading potentially to differing patterns of trade-offs within each sex. We tested for within- and between-individual trade-offs among three whole-organism performance traits (sprint speed, endurance, and bite force) in adult male and female Anolis carolinensis lizards using all of the measured performance data. Sprinting and endurance did not trade-off among individuals in either sex, but we found a significant negative among-individual relationship between sprint speed and bite force in females only, likely driven by the mechanical burden of larger than optimal heads imposed on females through intralocus sexual conflict. We also found evidence for marked within-individual plasticity in male bite force, but no within-individual trade-offs between any traits in either sex. These data offer new insight into the sex-specific nature of performance trade-offs and plasticity and, ultimately, into the constraints on multivariate performance evolution.

The evolution of performance-based male fighting ability in Caribbean Anolis lizards.

Despite the empirical and theoretical attention paid to the role of sexual signals in resolving agonistic interactions between conspecific males, few studies have applied a comparative perspective, particularly across species that vary in combat intensity. We investigated the relative roles of a male sexual signal (dewlap size) and whole-organism performance capacity (bite force) on male combat outcomes in nine species of Caribbean Anolis lizards that differ markedly in territoriality, as indicated by sexual size dimorphism. We found that (1) dewlap size was generally an honest signal of bite force in dimorphic but not less dimorphic species; (2) maximum bite force consistently predicted male combat success in dimorphic but not less dimorphic species; (3) in contrast to a priori predictions, dewlap size significantly predicted male combat success in less dimorphic but not dimorphic species; and (4) the incidence of biting but not dewlapping increases as species become more dimorphic. These findings suggest that more dimorphic (and hence more territorial) species escalate to biting during fights more readily compared with less territorial species. The ecological and behavioral qualities of species may therefore modify both the shape and the size of sexually selected traits as well as the nature of the information those traits convey.

How Do We Measure the Cost of Whole-Organism Performance Traits?

SYNOPSIS: Whole-organism performance traits, such as maximal speed and endurance capacity are undoubtedly costly, but we know little about how or when all of the costs associated with performance are paid to individuals or how to measure them. To understand how performance traits might be involved in trade-offs with other life-history traits it is critical to determine the development, production, and maintenance costs of performance traits, as well as how each of these changes with increased or decreased use of the performance trait. We discuss the advantages and disadvantages of several potential phenotypic measures of dynamic whole-organism performance that may be used in life-history studies, including direct performance measures; metabolic rates; ecological cost of transport; and changes in metabolic rate after training. We use the first approach, direct performance measures, to show trade-offs between endurance capacity and several traditional life history variables in phrynosomatid lizards. The largest problem currently in determining the costs of performance traits and how those costs might lead to life-history trade-offs is that there are estimates of performance costs in very few taxa, and when there are, those species typically are not studied with respect to "traditional" life-history traits.