Evolutionary physiology at 30+: Has the promise been fulfilled?: Advances in Evolutionary Physiology: Advances in Evolutionary Physiology.

Three decades ago, interactions between evolutionary biology and physiology gave rise to evolutionary physiology. This caused comparative physiologists to improve their research methods by incorporating evolutionary thinking. Simultaneously, evolutionary biologists began focusing more on physiological mechanisms that may help to explain constraints on and trade-offs during microevolutionary processes, as well as macroevolutionary patterns in physiological diversity. Here we argue that evolutionary physiology has yet to reach its full potential, and propose new avenues that may lead to unexpected advances. Viewing physiological adaptations in wild animals as potential solutions to human diseases offers enormous possibilities for biomedicine. New evidence of epigenetic modifications as mechanisms of phenotypic plasticity that regulate physiological traits may also arise in coming years, which may also represent an overlooked enhancer of adaptation via natural selection to explain physiological evolution. Synergistic interactions at these intersections and other areas will lead to a novel understanding of organismal biology.

First successful artificial insemination of the endangered Louisiana pinesnake, Pituophis ruthveni.

The Louisiana pinesnake (Pituophis ruthveni) is considered one of the rarest snakes in North America and was federally listed under the Endangered Species Act in 2018. Captive breeding and reintroduction of zoo-bred hatchlings has been successful, however, limited founders in the captive population and the inability to bring new, wild genes into the captive colony presents a major concern for the conservation of this species. The use of artificial insemination (AI) was first applied to snakes in the 1980s but further development of the technique has since received little attention. Our goal was to develop a method of AI for use in breeding Louisiana pinesnakes to facilitate gene flow from wild to captive populations. We inseminated two captive Louisiana pinesnakes with semen collected from one donor male, novel to both females. Timing of AI occurred following the emergence of females from brumation, and when large, distinct follicles were detected using digital palpation. Females were inseminated four and five times over a period of 14 and 19 days, respectively, using fresh and 2-day refrigerator stored semen. One female laid seven eggs, which resulted in four fertile eggs and two viable hatchlings, while the second female produced three fertile of seven eggs laid but no viable hatchlings. Genetic analyses confirmed the donor male was the sire of hatchlings. This is the first successful AI of an endangered snake species and provides a framework for the use and optimization of assisted reproductive technologies for use in conservation breeding programs.

Limited effects of inbreeding on breeding coloration.

Animal color signals may function as indicators of fighting ability when males compete for access to females. This allows opponents to settle aggressive interactions before they escalate into physical combat and injury. Thus, there may be strong directional selection on these traits, toward enhanced signal quality. This renders sexually selected traits particularly susceptible to inbreeding depression, due to relatively low ratios of additive genetic variance to dominance variance. We measured the effects of inbreeding on an intrasexually selected color signal (the badge) in a population of Swedish sand lizards (Lacerta agilis) using the Rhh software based on 17 to 21 microsatellites. Males of this sexually dichromatic species use the badge during aggressive interactions to display, and assess, fighting ability. We found negative effects of homozygosity on badge size, saturation, and brightness. However, no such effects were observed on color hue. Pairwise correlations between badge size, hue, and saturation were all statistically significant. Thus, the sand lizard "badge" is a multicomponent signal with variation explained by covariation in badge size, saturation, and color hue. Body mass corrected for skeletal size (body condition) positively predicted badge size and saturation, encouraging future research on the extent that sexual signals may convey information on multigene targets (i.e. "genic capture").

Reduced Mitochondrial Respiration in Hybrid Asexual Lizards.

AbstractThe scarcity of asexual reproduction in vertebrates alludes to an inherent cost. Several groups of asexual vertebrates exhibit lower endurance capacity (a trait predominantly sourced by mitochondrial respiration) compared with congeneric sexual species. Here we measure endurance capacity in five species of Aspidoscelis lizards and examine mitochondrial respiration between sexual and asexual species using mitochondrial respirometry. Our results show reduced endurance capacity, reduced mitochondrial respiration, and reduced phenotypic variability in asexual species compared with parental sexual species, along with a positive relationship between endurance capacity and mitochondrial respiration. Results of lower endurance capacity and lower mitochondrial respiration in asexual Aspidoscelis are consistent with hypotheses involving mitonuclear incompatibility.

Postnatal expression of IGF2 is the norm in amniote vertebrates.

The insulin and insulin-like signalling (IIS) network plays an important role in mediating several life-history traits, including growth, reproduction and senescence. Although insulin-like growth factors (IGFs) 1 and 2 are both key hormones in the vertebrate IIS network, research on IGF2 in juveniles and adults has been largely neglected because early biomedical research on rodents found negligible IGF2 postnatal expression. Here, we challenge this assumption and ask to what degree IGF2 is expressed during postnatal life across amniotes by quantifying the relative gene expression of IGF1 and IGF2 using publicly available RNAseq data for 82 amniote species and quantitative polymerase chain reaction on liver cDNA at embryonic, juvenile and adult stages for two lizard, bird and mouse species. We found that (i) IGF2 is expressed postnatally across amniote species and life stages-often at a higher relative expression than IGF1, contradicting rodent models; (ii) the lack of rodent postnatal IGF2 expression is due to phylogenetic placement, not inbreeding or artificial selection; and (iii) adult IGF2 expression is sex-biased in some species. Our results demonstrate that IGF2 expression is typical for amniotes throughout life, suggesting that a comprehensive understanding of the mechanisms mediating variation in life-history traits will require studies that measure both IGFs.

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.

Multiple Paternity in Garter Snakes With Evolutionarily Divergent Life Histories.

Many animal species exhibit multiple paternity, defined as multiple males genetically contributing to a single female reproductive event, such as a clutch or litter. Although this phenomenon is well documented across a broad range of taxa, the underlying causes and consequences remain poorly understood. For example, it is unclear how multiple paternity correlates with life-history strategies. Furthermore, males and females may differ in mating strategies and these patterns may shift with ecological context and life-history variation. Here, we take advantage of natural life-history variation in garter snakes (Thamnophis elegans) to address these questions in a robust field setting where populations have diverged along a slow-to-fast life-history continuum. We determine both female (observed) and male (using molecular markers) reproductive success in replicate populations of 2 life-history strategies. We find that despite dramatic differences in annual female reproductive output: 1) females of both life-history ecotypes average 1.5 sires per litter and equivalent proportions of multiply-sired litters, whereas 2) males from the slow-living ecotype experience greater reproductive skew and greater variance in reproductive success relative to males from the fast-living ecotype males despite having equivalent average reproductive success. Together, these results indicate strong intrasexual competition among males, particularly in the fast-paced life-history ecotype. We discuss these results in the context of competing hypotheses for multiple paternity related to population density, resource variability, and life-history strategy.

Gene expression of the IGF hormones and IGF binding proteins across time and tissues in a model reptile.

The insulin and insulin-like signaling (IIS) network regulates cellular processes including pre- and postnatal growth, cellular development, wound healing, reproduction, and longevity. Despite their importance in the physiology of vertebrates, the study of the specific functions of the top regulators of the IIS network, insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs), has been mostly limited to a few model organisms. To expand our understanding of this network, we performed quantitative gene expression of IGF hormones in liver and qualitative expression of IGFBPs across tissues and developmental stages in a model reptile, the brown anole lizard (Anolis sagrei). We found that lizards express IGF2 across all life stages (preoviposition embryos to adulthood) and at a higher level than IGF1, which is opposite to patterns seen in laboratory rodents but similar to those seen in humans and other vertebrate models. IGFBP expression was ubiquitous across tissues (brain, gonad, heart, liver, skeletal muscle, tail, and regenerating tail) in adults, apart from IGFBP5, which was variable. These findings provide an essential foundation for further developing the anole lizard as a physiological and biomedical reptile model, as well as expanding our understanding of the function of the IIS network across species.

Characterization of two MHC II genes (DOB, DRB) in white-tailed deer (Odocoileus virginianus).

BACKGROUND: The major histocompatibility complex (MHC) is responsible for detecting and addressing foreign pathogens inside the body. While the general structure of MHC genes is relatively well conserved among mammalian species, it is notably different among ruminants due to a chromosomal inversion that splits MHC type II genes into two subregions (IIa, IIb). Recombination rates are reportedly high between these subregions, and a lack of linkage has been documented in domestic ruminants. However, no study has yet examined the degree of linkage between these subregions in a wild ruminant. The white-tailed deer (Odocoileus virginianus), a popular ruminant of the Cervidae family, is habitually plagued by pathogens in its natural environment (e.g. Haemonchus contortus, Elaeophora). Due to the association between MHC haplotypes and disease susceptibility, a deeper understanding of MHC polymorphism and linkage between MHC genes can further aid in this species' successful management. We sequenced MHC-DRB exon 2 (IIa) and MHC-DOB exon 2 (IIb) on the MiSeq platform from an enclosed white-tailed deer population located in Alabama. RESULTS: We identified 12 new MHC-DRB alleles, and resampled 7 alleles, which along with other published alleles brings the total number of documented alleles in white-tailed deer to 30 for MHC-DRB exon 2. The first examination of MHC-DOB in white-tailed deer found significantly less polymorphism (11 alleles), as was expected of a non-classical MHC gene. While MHC-DRB was found to be under positive, diversifying selection, MHC-DOB was found to be under purifying selection for white-tailed deer. We found no significant linkage disequilibrium between MHC-DRB and MHC-DOB, suggesting that these loci are unlikely to be closely linked. CONCLUSIONS: Overall, this study identified 12 new MHC-DRB exon 2 alleles and characterized a new, non-classical, MHC II gene (MHC-DOB) for white-tailed deer. We also found a lack of significant linkage between these two loci, which supports previous findings of a chromosomal inversion within the MHC type II gene region in ruminants, and suggests that white-tailed deer may have a recombination hotspot between these MHC regions similar to that found for Bos taurus.

Five months of voluntary wheel running downregulates skeletal muscle LINE-1 gene expression in rats.

Transposable elements (TEs) are mobile DNA and constitute approximately half of the human genome. LINE-1 (L1) is the only active autonomous TE in the mammalian genome and has been implicated in a number of diseases as well as aging. We have previously reported that skeletal muscle L1 expression is lower following acute and chronic exercise training in humans. Herein, we used a rodent model of voluntary wheel running to determine whether long-term exercise training affects markers of skeletal muscle L1 regulation. Selectively bred high-running female Wistar rats (n = 11 per group) were either given access to a running wheel (EX) or not (SED) at 5 wk of age, and these conditions were maintained until 27 wk of age. Thereafter, mixed gastrocnemius tissue was harvested and analyzed for L1 mRNA expression and DNA content along with other L1 regulation markers. We observed significantly (P < 0.05) lower L1 mRNA expression, higher L1 DNA methylation, and less L1 DNA in accessible chromatin regions in EX versus SED rats. We followed these experiments with 3-h in vitro drug treatments in L6 myotubes to mimic transient exercise-specific signaling events. The AMP-activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR; 4 mM) significantly decreased L1 mRNA expression in L6 myotubes. However, this effect was not facilitated through increased L1 DNA methylation. Collectively, these data suggest that long-term voluntary wheel running downregulates skeletal muscle L1 mRNA, and this may occur through chromatin modifications. Enhanced AMPK signaling with repetitive exercise bouts may also decrease L1 mRNA expression, although the mechanism of action remains unknown.

Shoo fly don't bother me: Flies track social primates and carry viable anthrax.

Flies-a small name for an enormous taxonomic group of over 110,000 described species that have unique ecological roles. Nonbiting flies ingest organic material in faecal matter or carrion, which is rich in microbes and nutrients that benefit both adults and their offspring (maggots). These are often referred to as "filth flies" because they are often pests in human settlements and responsible for the spread of enteric pathogens. Filth flies associate with human populations; however, whether this association is simply due to the presence of organic waste produced, or if flies move with social groups remains unknown. In this issue of Molecular Ecology, Gogarten et al. (2019) use a unique combination of field methods and molecular tools to show that filth flies (predominantly Muscidae [house flies] and Calliphoridae [blow flies]) associate and move with social nonhuman primate (NHP) groups (mangabeys and chimpanzees) for up to 12 days and over 1 km. Filth flies captured near these groups were found to have pathogen DNA on them from the causative agents of sylvatic anthrax and yaws. Furthermore, the authors were able to show that the anthrax bacteria on the flies was viable. Previous research emphasized sylvatic anthrax as a major conservation threat to wildlife at this field site (Hoffmann et al., 2017), highlighting the significance of filth flies as potential vectors of anthrax. The authors present a suite of methods and approaches that utilize flies to better understand rainforest biodiversity, pathogen transmission potential, and filth fly-host associations. This work represents new directions and opportunities to integrate entomology into field research and exploit the natural history of flies to understand the pathogen landscape and address outstanding questions in ecology and evolution.

How accurately do behavioural observations predict reproductive success in free-ranging lizards?

Behavioural ecologists often use data on patterns of male-female association to infer reproductive success of free-ranging animals. For example, a male seen with several females during the mating season is predicted to father more offspring than a male not seen with any females. We explored the putative correlation between this behaviour and actual paternity (as revealed by microsatellite data) from a long-term study on sand lizards ( Lacerta agilis), including behavioural observations of 574 adult males and 289 adult females, and paternity assignment of more than 2500 offspring during 1998-2007. The number of males that contributed paternity to a female's clutch was correlated with the number of males seen accompanying her in the field, but not with the number of copulation scars on her body. The number of females that a male accompanied in the field predicted the number of females with whom he fathered offspring, and his annual reproductive success (number of progeny). Although behavioural data explained less than one-third of total variance in reproductive success, our analysis supports the utility of behavioural-ecology studies for predicting paternity in free-ranging reptiles.

Rapid molecular evolution across amniotes of the IIS/TOR network.

The insulin/insulin-like signaling and target of rapamycin (IIS/TOR) network regulates lifespan and reproduction, as well as metabolic diseases, cancer, and aging. Despite its vital role in health, comparative analyses of IIS/TOR have been limited to invertebrates and mammals. We conducted an extensive evolutionary analysis of the IIS/TOR network across 66 amniotes with 18 newly generated transcriptomes from nonavian reptiles and additional available genomes/transcriptomes. We uncovered rapid and extensive molecular evolution between reptiles (including birds) and mammals: (i) the IIS/TOR network, including the critical nodes insulin receptor substrate (IRS) and phosphatidylinositol 3-kinase (PI3K), exhibit divergent evolutionary rates between reptiles and mammals; (ii) compared with a proxy for the rest of the genome, genes of the IIS/TOR extracellular network exhibit exceptionally fast evolutionary rates; and (iii) signatures of positive selection and coevolution of the extracellular network suggest reptile- and mammal-specific interactions between members of the network. In reptiles, positively selected sites cluster on the binding surfaces of insulin-like growth factor 1 (IGF1), IGF1 receptor (IGF1R), and insulin receptor (INSR); whereas in mammals, positively selected sites clustered on the IGF2 binding surface, suggesting that these hormone-receptor binding affinities are targets of positive selection. Further, contrary to reports that IGF2R binds IGF2 only in marsupial and placental mammals, we found positively selected sites clustered on the hormone binding surface of reptile IGF2R that suggest that IGF2R binds to IGF hormones in diverse taxa and may have evolved in reptiles. These data suggest that key IIS/TOR paralogs have sub- or neofunctionalized between mammals and reptiles and that this network may underlie fundamental life history and physiological differences between these amniote sister clades.

Here and there, but not everywhere: repeated loss of uncoupling protein 1 in amniotes.

Endothermy is an evolutionary innovation in eutherian mammals and birds. In eutherian mammals, UCP1 is a key protein in adaptive nonshivering thermogenesis (NST). Although ucp1 arose early in the vertebrate lineage, the loss of ucp1 was previously documented in several reptile species (including birds). Here we determine that ucp1 was lost at the base of the reptile lineage, as we fail to find ucp1 in every major reptile lineage. Furthermore, though UCP1 plays a key role in mammalian NST, we confirm that pig has lost several exons from ucp1 and conclude that pig is not a sole outlier as the only eutherian mammal lineage to do so. Through similarity searches and synteny analysis, we show that ucp1 has also been lost/pseudogenized in Delphinidae (dolphin, orca) and potentially Xenarthra (sloth, armadillo) and Afrotheria (hyrax). These lineages provide models for investigating alternate mechanisms of thermoregulation and energy metabolism in the absence of functional UCP1. Further, the repeated losses of a functional UCP1 suggest the pervasiveness of NST via UCP1 across the mammalian lineage needs re-evaluation.

Insulin-like signaling (IIS) responses to temperature, genetic background, and growth variation in garter snakes with divergent life histories.

The insulin/insulin-like signaling pathway (IIS) has been shown to mediate life history trade-offs in mammalian model organisms, but the function of this pathway in wild and non-mammalian organisms is understudied. Populations of western terrestrial garter snakes (Thamnophis elegans) around Eagle Lake, California, have evolved variation in growth and maturation rates, mortality senescence rates, and annual reproductive output that partition into two ecotypes: "fast-living" and "slow-living". Thus, genes associated with the IIS network are good candidates for investigating the mechanisms underlying ecological divergence in this system. We reared neonates from each ecotype for 1.5years under two thermal treatments. We then used qPCR to compare mRNA expression levels in three tissue types (brain, liver, skeletal muscle) for four genes (igf1, igf2, igf1r, igf2r), and we used radioimmunoassay to measure plasma IGF-1 and IGF-2 protein levels. Our results show that, in contrast to most mammalian model systems, igf2 mRNA and protein levels exceed those of igf1 and suggest an important role for igf2 in postnatal growth in reptiles. Thermal rearing treatment and recent growth had greater impacts on IGF levels than genetic background (i.e., ecotype), and the two ecotypes responded similarly. This suggests that observed ecotypic differences in field measures of IGFs may more strongly reflect plastic responses in different environments than evolutionary divergence. Future analyses of additional components of the IIS pathway and sequence divergence between the ecotypes will further illuminate how environmental and genetic factors influence the endocrine system and its role in mediating life history trade-offs.

Dissecting molecular stress networks: identifying nodes of divergence between life-history phenotypes.

The complex molecular network that underlies physiological stress response is comprised of nodes (proteins, metabolites, mRNAs, etc.) whose connections span cells, tissues and organs. Variable nodes are points in the network upon which natural selection may act. Thus, identifying variable nodes will reveal how this molecular stress network may evolve among populations in different habitats and how it might impact life-history evolution. Here, we use physiological and genetic assays to test whether laboratory-born juveniles from natural populations of garter snakes (Thamnophis elegans), which have diverged in their life-history phenotypes, vary concomitantly at candidate nodes of the stress response network, (i) under unstressed conditions and (ii) in response to an induced stress. We found that two common measures of stress (plasma corticosterone and liver gene expression of heat shock proteins) increased under stress in both life-history phenotypes. In contrast, the phenotypes diverged at four nodes both under unstressed conditions and in response to stress: circulating levels of reactive oxygen species (superoxide, H(2)O(2)); liver gene expression of GPX1 and erythrocyte DNA damage. Additionally, allele frequencies for SOD2 diverge from neutral markers, suggesting diversifying selection on SOD2 alleles. This study supports the hypothesis that these life-history phenotypes have diverged at the molecular level in how they respond to stress, particularly in nodes regulating oxidative stress. Furthermore, the differences between the life-history phenotypes were more pronounced in females. We discuss the responses to stress in the context of the associated life-history phenotype and the evolutionary pressures thought to be responsible for divergence between the phenotypes.

Effect of MHC and inbreeding on disassortative reproduction: A data revisit, extension and inclusion of fertilization in sand lizards.

The harmful effects of close inbreeding have been recognized for centuries and, with the rise of Mendelian genetics, was realized to be an effect of homozygosis. This historical background led to great interest in ways to quantify inbreeding, its depression effects on the phenotype and flow-on effects on mate choice and other aspects of behavioral ecology. The mechanisms and cues used to avoid inbreeding are varied and include major histocompatibility complex (MHC) molecules and the peptides they transport as predictors of the degree of genetic relatedness. Here, we revisit and complement data from a Swedish population of sand lizards (Lacerta agilis) showing signs of inbreeding depression to assess the effects of genetic relatedness on pair formation in the wild. Parental pairs were less similar at the MHC than expected under random mating but mated at random with respect to microsatellite relatedness. MHC clustered in groups of RFLP bands but no partner preference was observed with respect to partner MHC cluster genotype. Male MHC band patterns were unrelated to their fertilization success in clutches selected for analysis on the basis of showing mixed paternity. Thus, our data suggest that MHC plays a role in pre-copulatory, but not post-copulatory partner association, suggesting that MHC is not the driver of fertilization bias and gamete recognition in sand lizards.

Rates of molecular evolution vary in vertebrates for insulin-like growth factor-1 (IGF-1), a pleiotropic locus that regulates life history traits.

Insulin-like growth factor-1 (IGF-1) is a member of the vertebrate insulin/insulin-like growth factor/relaxin gene family necessary for growth, reproduction, and survival at both the cellular and organismal level. Its sequence, protein structure, and function have been characterized in mammals, birds, and fish; however, a notable gap in our current knowledge of the function of IGF-1 and its molecular evolution is information in ectothermic reptiles. To address this disparity, we sequenced the coding region of IGF-1 in 11 reptile species-one crocodilian, three turtles, three lizards, and four snakes. Complete sequencing of the full mRNA transcript of a snake revealed the Ea-isoform, the predominant isoform of IGF-1 also reported in other vertebrate groups. A gene tree of the IGF-1 protein-coding region that incorporated sequences from diverse vertebrate groups showed similarity to the species phylogeny, with the exception of the placement of Testudines as sister group to Aves, due to their high nucleotide sequence similarity. In contrast, long-branch lengths indicate more rapid divergence in IGF-1 among lizards and snakes. Additionally, lepidosaurs (i.e., lizards and snakes) had higher rates of non-synonymous:synonymous substitutions (dN/dS) relative to archosaurs (i.e., birds and crocodilians) and turtles. Tests for positive selection on specific codons within branches and evaluation of the changes in the amino acid properties, suggested positive selection in lepidosaurs on the C domain of IGF-1, which is involved in binding affinity to the IGF-1 receptor. Predicted structural changes suggest that major alterations in protein structure and function may have occurred in reptiles. These data propose new insights into the molecular co-evolution of IGF-1 and its receptors, and ultimately the evolution of IGF-1's role in regulating life-history traits across vertebrates.

Tails of reproduction: Regeneration leads to increased reproductive investment.

Trade-offs between life-history traits are due to limited resources or constraints in the regulation of genetic and physiological networks. Tail autotomy, with subsequent regeneration, is a common anti-predation mechanism in lizards and is predicted to trade-off with life-history traits, such as reproduction. We utilize the brown anole lizard with its unusual reproductive pattern of single-egg clutches every 7-10 days to test for a trade-off in reproductive investment over 8 weeks of tail regeneration on a limited diet. In contrast to predictions, we found that investing in tissue regeneration had a positive effect on reproduction in terms of egg size (11.7% relative to controls) and hatchling size (11.5% relative to controls), and no effect on egg number or survival, with the increase in reproduction starting at peak regeneration. We discuss mechanistic hypotheses that the process of regeneration may cause increased energetic efficiency or utilized shared physiological pathways with reproductive investment.