3D printed models are an accurate, cost-effective, and reproducible tool for quantifying terrestrial thermal environments.

Predicting ecological responses to rapid environmental change has become one of the greatest challenges of modern biology. One of the major hurdles in forecasting these responses is accurately quantifying the thermal environments that organisms experience. The distribution of temperatures available within an organism's habitat is typically measured using data loggers called operative temperature models (OTMs) that are designed to mimic certain properties of heat exchange in the focal organism. The gold standard for OTM construction in studies of terrestrial ectotherms has been the use of copper electroforming which creates anatomically accurate models that equilibrate quickly to ambient thermal conditions. However, electroformed models require the use of caustic chemicals, are often brittle, and their production is expensive and time intensive. This has resulted in many researchers resorting to the use of simplified OTMs that can yield substantial measurement errors. 3D printing offers the prospect of robust, easily replicated, morphologically accurate, and cost-effective OTMs that capture the benefits but alleviate the problems associated with electroforming. Here, we validate the use of OTMs that were 3D printed using several materials across eight lizard species of different body sizes and living in habitats ranging from deserts to tropical forests. We show that 3D printed OTMs have low thermal inertia and predict the live animal's equilibration temperature with high accuracy across a wide range of body sizes and microhabitats. Finally, we developed a free online repository and database of 3D scans (https://www.3dotm.org/) to increase the accessibility of this tool to researchers around the world and facilitate ease of production of 3D printed models. 3D printing of OTMs is generalizable to taxa beyond lizards. If widely adopted, this approach promises greater accuracy and reproducibility in studies of terrestrial thermal ecology and should lead to improved forecasts of the biological impacts of climate change.

Variable stoichiometric and macronutrient responses to lizard predation in Ozark glade grasshopper communities.

The General Stress Paradigm (GSP) predicts that prey body compositions should shift under chronic predation as prey increase body carbon and decrease body nitrogen content through dietary changes, heightened metabolism, reduced dietary efficiency, and the breakdown of nitrogen rich tissues to make labile carbohydrates available. In our study, we explored how the elemental and macronutrient content along with the morphology of three abundant Ozark glade grasshopper species differed between glades with and without predatory collared lizard (Crotaphytus collaris) populations. Our results indicated that lichen grasshoppers (Trimerotropis saxatilis) increased body C:N ratios in response to predators. Scudder's short-wing grasshoppers (Melanoplus scudderi) increased both body %C and %protein content, while the handsome grasshoppers (Syrbula admirabilis) did not significantly respond to the presence of collared lizards. None of the three grasshopper species showed morphological responses to predation. We also found that elemental and macronutrient content of grasshoppers was not always significantly correlated and was not associated with the same environmental factors, indicating a need to incorporate both perspectives in future research and utilize more accurate macromolecular assays. Overall, we found support for some aspects of the GSP in field-active animals and add to the growing body of evidence that predator-induced changes in prey body composition are more complex than predicted by the original GSP.

A chromosome-level genome assembly for the eastern fence lizard (Sceloporus undulatus), a reptile model for physiological and evolutionary ecology.

BACKGROUND: High-quality genomic resources facilitate investigations into behavioral ecology, morphological and physiological adaptations, and the evolution of genomic architecture. Lizards in the genus Sceloporus have a long history as important ecological, evolutionary, and physiological models, making them a valuable target for the development of genomic resources. FINDINGS: We present a high-quality chromosome-level reference genome assembly, SceUnd1.0 (using 10X Genomics Chromium, HiC, and Pacific Biosciences data), and tissue/developmental stage transcriptomes for the eastern fence lizard, Sceloporus undulatus. We performed synteny analysis with other snake and lizard assemblies to identify broad patterns of chromosome evolution including the fusion of micro- and macrochromosomes. We also used this new assembly to provide improved reference-based genome assemblies for 34 additional Sceloporus species. Finally, we used RNAseq and whole-genome resequencing data to compare 3 assemblies, each representing an increased level of cost and effort: Supernova Assembly with data from 10X Genomics Chromium, HiRise Assembly that added data from HiC, and PBJelly Assembly that added data from Pacific Biosciences sequencing. We found that the Supernova Assembly contained the full genome and was a suitable reference for RNAseq and single-nucleotide polymorphism calling, but the chromosome-level scaffolds provided by the addition of HiC data allowed synteny and whole-genome association mapping analyses. The subsequent addition of PacBio data doubled the contig N50 but provided negligible gains in scaffold length. CONCLUSIONS: These new genomic resources provide valuable tools for advanced molecular analysis of an organism that has become a model in physiology and evolutionary ecology.

Role of phenotypic plasticity in morphological differentiation between watersnake populations.

An individual's morphology is shaped by the environmental pressures it experiences, and the resulting morphological response is the culmination of both genetic factors and environmental (non-genetic) conditions experienced early in its life (i.e. phenotypic plasticity). The role that phenotypic plasticity plays in shaping phenotypes is important, but evidence for its influence is often mixed. We exposed female neonate diamond-backed watersnakes (Nerodia rhombifer) from populations experiencing different prey-size regimes to different feeding treatments to test the influence of phenotypic plasticity in shaping trophic morphology. We found that snakes in a large-prey treatment from a population frequently encountering large prey exhibited a higher growth rate in body size than individuals in a small-prey treatment from the same population. This pattern was not observed in snakes from a population that regularly encounters small prey. We also found that regardless of treatment, snakes from the small-prey population were smaller at birth than snakes from the large-prey population and remained so throughout the study. These results suggest that the ability to plastically respond to environmental pressures may be population-specific. These results also indicate a genetic predisposition towards larger body sizes in a population where large prey items are more common.

Covariation between Thermally Mediated Color and Performance Traits in a Lizard.

Physiological changes in response to environmental cues are not uncommon. Temperature has strong, predictable effects on many traits, such that many traits in ectotherms follow stereotyped thermal performance curves in response to increasing temperature. The prairie lizard-an abundant lizard throughout the central United States-has thermally sensitive, blue abdominal and throat patches. Currently, the role of these patches is not well understood. In this study, we set out to investigate whether individual plasticity in patch color paralleled individual plasticity in sprint speed (do they covary), and if the plasticity in these two patches signal redundant or independent information, testing competing hypotheses suggested for the evolution of multiple signals. We found that both abdominal and throat patch hue follow classical thermal performance curves, suggesting that at the species level hue is a good predictor of sprinting ability. At the individual level, we found that color and performance were statistically repeatable, so individuals with relatively high phenotypic values maintain relatively high phenotypic values across all temperatures. Additionally, we found that abdominal and patch hue covary with sprinting speed at the individual level. Together, these results suggest that the bluest individuals are the fastest individuals across temperatures. However, we found that abdominal and throat patch hue do not covary with each other at the individual level, suggesting that these signals may have independent functions. The importance of examining the function of individual variation cannot be overstated, and overall, more work is needed to better understand both the proximate and ultimate mechanisms underlying signal plasticity in this species and others.

Population level differences in thermal sensitivity of energy assimilation in terrestrial salamanders.

Thermal adaptation predicts that thermal sensitivity of physiological traits should be optimized to thermal conditions most frequently experienced. Furthermore, thermodynamic constraints predict that species with higher thermal optima should have higher performance maxima and narrower performance breadths. We tested these predictions by examining the thermal sensitivity of energy assimilation between populations within two species of terrestrial-lungless salamanders, Plethodon albagula and P. montanus. Within P. albagula, we examined populations that were latitudinally separated by >450km. Within P. montanus, we examined populations that were elevationally separated by >900m. Thermal sensitivity of energy assimilation varied substantially between populations of P. albagula separated latitudinally, but did not vary between populations of P. montanus separated elevationally. Specifically, in P. albagula, the lower latitude population had a higher thermal optimum, higher maximal performance, and narrower performance breadth compared to the higher latitude population. Furthermore, across all individuals as thermal optima increased, performance maxima also increased, providing support for the theory that "hotter is better".

A test of energetic trade-offs between growth and immune function in watersnakes.

Energy budgets explain how organisms allocate energetic intake to accomplish essential processes. A likely life history trade-off occurs between growth and immune response in juvenile organisms, where growth is important to avoid predation or obtain larger prey and immune response is essential to survival in the presence of environmental pathogens. We examined the innate (wound healing) and adaptive (lymphoid tissue, thymus and spleen) components of immune response along with growth in two populations of the diamond-backed watersnake Nerodia rhombifer raised in a common environment. We found that neonate snakes born to females from populations characterized by different predator and prey environments did not differ in energetic intake, but snakes from the population containing large prey grew significantly faster than those from a population containing small prey. Thymus mass, when corrected for body mass, was larger in snakes from the small prey population than in snakes from the large prey population. Additionally, the snakes from the population containing small prey healed significantly faster than those from the population containing large prey. Thus, we detected a negative correlation between growth (over a 4-month period) and wound healing across populations that is suggestive of an energetic trade-off between growth and immune response. The differences observed in growth and immune response among these two populations appear to suggest different energy allocation strategies to maximize fitness in response to differing conditions experienced by snakes in the two populations.

Variation in thermal tolerance of North American ants.

Changing climates are predicted to alter the distribution of thermal niches. Small ectotherms such as ants may be particularly vulnerable to heat injury and death. We quantified the critical thermal maxima of 92 ant colonies representing 14 common temperate ant species. The mean CTmax for all measured ants was 47.8 °C (±0.27; range=40.2-51.2 °C), and within-colony variation was lower than among-colony variation. Critical thermal maxima differed among species and were negatively correlated with body size. Results of this study illustrate the importance of accounting for mass, among and within colony variation, and interspecific differences in diel activity patterns, which are often neglected in studies of ant thermal physiology.

Individual (co)variation in standard metabolic rate, feeding rate, and exploratory behavior in wild-caught semiaquatic salamanders.

Repeatability is an important concept in evolutionary analyses because it provides information regarding the benefit of repeated measurements and, in most cases, a putative upper limit to heritability estimates. Repeatability (R) of different aspects of energy metabolism and behavior has been demonstrated in a variety of organisms over short and long time intervals. Recent research suggests that consistent individual differences in behavior and energy metabolism might covary. Here we present new data on the repeatability of body mass, standard metabolic rate (SMR), voluntary exploratory behavior, and feeding rate in a semiaquatic salamander and ask whether individual variation in behavioral traits is correlated with individual variation in metabolism on a whole-animal basis and after conditioning on body mass. All measured traits were repeatable, but the repeatability estimates ranged from very high for body mass (R = 0.98), to intermediate for SMR (R = 0.39) and food intake (R = 0.58), to low for exploratory behavior (R = 0.25). Moreover, repeatability estimates for all traits except body mass declined over time (i.e., from 3 to 9 wk), although this pattern could be a consequence of the relatively low sample size used in this study. Despite significant repeatability in all traits, we find little evidence that behaviors are correlated with SMR at the phenotypic and among-individual levels when conditioned on body mass. Specifically, the phenotypic correlations between SMR and exploratory behavior were negative in all trials but significantly so in one trial only. Salamanders in this study showed individual variation in how their exploratory behavior changed across trials (but not body mass, SMR, and feed intake), which might have contributed to observed changing correlations across trials.

The effects of temperature and activity on intraspecific scaling of metabolic rates in a lungless salamander.

The scaling of metabolic rate with body mass holds substantial predictive power as many biological processes depend on energy. A significant body of theory has been developed based on the assumption that metabolic rate scales with body mass as a power function with an exponent of 0.75, and that this scaling relationship is independent of temperature. Here we test this hypothesis at the intraspecific level in a lungless salamander using data on both standard and maximal metabolic rates (SMR and MMR, respectively). We also address a recently proposed alternative explanation that predicts systematic variation in this mass-scaling exponent, the metabolic level boundaries hypothesis (MLB). Consistent with predictions of the metabolic theory of ecology the mass scaling of SMR and MMR were independent of temperature, however, we find evidence that the mass-scaling exponent for SMR and MMR differ significantly from 0.75. Further, our data do not provide strong support for MLB. Mass-scaling exponents for MMR generally exceed those for SMR, although these differences are rarely statistically significant.

Limited capacity for acclimation of thermal physiology in a salamander, Desmognathus brimleyorum.

Habitats vary in temperature both spatially and temporally. Variation in thermal habitat introduces challenges to organisms and may reduce fitness unless organisms can physiologically adjust to such changes. Theory predicts that thermal variability should influence the capacity for acclimation such that increased variation should favor a reduction in the thermal sensitivity of physiological traits. In this study, we investigated acclimation to constant and variable conditions in populations of the salamander Desmognathus brimleyorum from the Ouachita Mountains of Arkansas, USA. We exposed salamanders to constant and variable temperature regimes for 8 weeks in the laboratory. We then tested salamanders for acclimation of thermal tolerance, and the thermal sensitivities of swimming performance and standard metabolic rate. Our results indicate limited capacity for thermal acclimation to constant and variable conditions in D. brimleyorum. Instead, variation in physiological traits is dominated by differences among populations. Population differences do not appear to be correlated with observed variation in the thermal conditions of the streams, but are likely a consequence of structural and ecological differences. Due to the mixed support for theoretical predictions for acclimation to alternative environments, further consideration should be given to revising and expanding current theoretical models.

In situ genetic differentiation in a Hispaniolan lizard (Ameiva chrysolaema): a multilocus perspective.

A previous phylogeographic study of mitochondrial haplotypes for the Hispaniolan lizard Ameiva chrysolaema revealed deep genetic structure associated with seawater inundation during the late Pliocene/early Pleistocene and evidence of subsequent population expansion into formerly inundated areas. We revisit hypotheses generated by our previous study using increased geographic sampling of populations and analysis of three nuclear markers (alpha-enolase intron 8, alpha-cardiac-actin intron 4, and beta-actin intron 3) in addition to mitochondrial haplotypes (ND2). Large genetic discontinuities correspond spatially and temporally with historical barriers to gene flow (sea inundations). NCPA cross-validation analysis and Bayesian multilocus analyses of divergence times (IMa and MCMCcoal) reveal two separate episodes of fragmentation associated with Pliocene and Pleistocene sea inundations, separating the species into historically separate Northern, East-Central, West-Central, and Southern population lineages. Multilocus Bayesian analysis using IMa indicates asymmetrical migration from the East-Central to the West-Central populations following secondary contact, consistent with expectations from the more pervasive sea inundation in the western region. The West-Central lineage has a genetic signature of population growth consistent with the expectation of geographic expansion into formerly inundated areas. Within each lineage, significant spatial genetic structure indicates isolation by distance at comparable temporal scales. This study adds to the growing body of evidence that vicariant speciation may be the prevailing source of lineage accumulation on oceanic islands. Thus, prior theories of island biogeography generally underestimate the role and temporal scale of intra-island vicariant processes.

Ecological speciation in Gambusia fishes.

Although theory indicates that natural selection can facilitate speciation as a by-product, demonstrating ongoing speciation via this by-product mechanism in nature has proven difficult. We examined morphological, molecular, and behavioral data to investigate ecology's role in incipient speciation for a post-Pleistocene radiation of Bahamas mosquitofish (Gambusia hubbsi) inhabiting blue holes. We show that adaptation to divergent predator regimes is driving ecological speciation as a by-product. Divergence in body shape, coupled with assortative mating for body shape, produces reproductive isolation that is twice as strong between populations inhabiting different predator regimes than between populations that evolved in similar ecological environments. Gathering analogous data on reproductive isolation at the interspecific level in the genus, we find that this mechanism of speciation may have been historically prevalent in Gambusia. These results suggest that speciation in nature can result as a by-product of divergence in ecologically important traits, producing interspecific patterns that persist long after speciation events have completed.

Partial island submergence and speciation in an adaptive radiation: a multilocus analysis of the Cuban green anoles.

Sympatric speciation is often proposed to account for species-rich adaptive radiations within lakes or islands, where barriers to gene flow or dispersal may be lacking. However, allopatric speciation may also occur in such situations, especially when ranges are fragmented by fluctuating water levels. We test the hypothesis that Miocene fragmentation of Cuba into three palaeo-archipelagos accompanied species-level divergence in the adaptive radiation of West Indian Anolis lizards. Analysis of morphology, mitochondrial DNA (mt DNA) and nuclear DNA in the Cuban green anoles (carolinensis subgroup) strongly supports three pre dictions made by this hypothesis. First, three geographical sets of populations, whose ranges correspond with palaeo-archipelago boundaries, are distinct and warrant recognition as independent evolutionary lineages or species. Coalescence of nuclear sequence fragments sampled from these species and the large divergences observed between their mtDNA haplotypes suggest separation prior to the subsequent unification of Cuba ca. 5 Myr ago. Second, molecular phylogenetic relationships among these species reflect historical geographical relationships rather than morphological similarity. Third, all three species remain distinct despite extensive geographical contact subsequent to island unification, occasional hybridization and introgression of mtDNA haplotypes. Allopatric speciation initiated during partial island submergence may play an important role in speciation during the adaptive radiation of Anolis lizards.

Population structure and history of a phenotypically variable teiid lizard (Ameiva chrysolaema) from Hispaniola: the influence of a geologically complex island.

Ameiva chrysolaema is distributed across the island of Hispaniola in the West Indies. The species is restricted to dry lowlands between major mountain ranges and along the southern and eastern coasts. Phylogenetic and phylogeographic analyses of mtDNA sequence variation from 14 sampling localities identify at least three independent evolutionary lineages, separated from one another by major mountain ranges. Nested clade phylogeographic analysis (NCPA) suggests a complex history of population fragmentation, consistent with geological evidence of seawater incursions into the Azua and Enriquillo basins during the Pliocene/Pleistocene (approximately 1.6 mya). Significantly negative Fu's F(S) values and parameters of mismatch distributions suggest that formerly fragmented populations have recently expanded their ranges. Significantly large average population clade distances (APCD) for two sampling localities in the Azua basin suggest secondary contact at these localities of previously separated populations. The distribution of haplotypes among polymorphic populations of A. chrysolaema suggests that variation in dorsal pattern represents a polymorphism within evolutionary lineages. Ameiva leberi is ecologically indistinguishable from and syntopic with A. chrysolaema. Genetic data suggest that A. leberi is a junior synonym of A. chrysolaema.