Convergent evolution of a blood-red nectar pigment in vertebrate-pollinated flowers.

Nearly 90% of flowering plants depend on animals for reproduction. One of the main rewards plants offer to pollinators for visitation is nectar. Nesocodon mauritianus (Campanulaceae) produces a blood-red nectar that has been proposed to serve as a visual attractant for pollinator visitation. Here, we show that the nectar's red color is derived from a previously undescribed alkaloid termed nesocodin. The first nectar produced is acidic and pale yellow in color, but slowly becomes alkaline before taking on its characteristic red color. Three enzymes secreted into the nectar are either necessary or sufficient for pigment production, including a carbonic anhydrase that increases nectar pH, an aryl-alcohol oxidase that produces a pigment precursor, and a ferritin-like catalase that protects the pigment from degradation by hydrogen peroxide. Our findings demonstrate how these three enzymatic activities allow for the condensation of sinapaldehyde and proline to form a pigment with a stable imine bond. We subsequently verified that synthetic nesocodin is indeed attractive to Phelsuma geckos, the most likely pollinators of Nesocodon We also identify nesocodin in the red nectar of the distantly related and hummingbird-visited Jaltomata herrerae and provide molecular evidence for convergent evolution of this trait. This work cumulatively identifies a convergently evolved trait in two vertebrate-pollinated species, suggesting that the red pigment is selectively favored and that only a limited number of compounds are likely to underlie this type of adaptation.

Signaling dynamics in coexisting monoclonal cell subpopulations unveil mechanisms of resistance to anti-cancer compounds.

BACKGROUND: Tumor heterogeneity is a main contributor of resistance to anti-cancer targeted agents though it has proven difficult to study. Unfortunately, model systems to functionally characterize and mechanistically study dynamic responses to treatment across coexisting subpopulations of cancer cells remain a missing need in oncology. METHODS: Using single cell cloning and expansion techniques, we established monoclonal cell subpopulations (MCPs) from a commercially available epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer cell line. We then used this model sensitivity to the EGFR inhibitor osimertinib across coexisting cell populations within the same tumor. Pathway-centered signaling dynamics associated with response to treatment and morphological characteristics of the MCPs were assessed using Reverse Phase Protein Microarray. Signaling nodes differentially activated in MCPs less sensitive to treatment were then pharmacologically inhibited to identify target signaling proteins putatively implicated in promoting drug resistance. RESULTS: MCPs demonstrated highly heterogeneous sensitivities to osimertinib. Cell viability after treatment increased > 20% compared to the parental line in selected MCPs, whereas viability decreased by 75% in other MCPs. Reduced treatment response was detected in MCPs with higher proliferation rates, EGFR L858R expression, activation of EGFR binding partners and downstream signaling molecules, and expression of epithelial-to-mesenchymal transition markers. Levels of activation of EGFR binding partners and MCPs' proliferation rates were also associated with response to c-MET and IGFR inhibitors. CONCLUSIONS: MCPs represent a suitable model system to characterize heterogeneous biomolecular behaviors in preclinical studies and identify and functionally test biological mechanisms associated with resistance to targeted therapeutics.

Thermal tolerance plasticity and dynamics of thermal tolerance in Eublepharis macularius: Implications for future climate-driven heat stress.

The intensity and duration of heat waves, as well as average global temperatures, are expected to increase due to climate change. Heat waves can cause physiological stress and reduce fitness in animals. Species can reduce overheating risk through phenotypic plasticity, which allows them to raise their thermal tolerance limits over time. This mechanism could be important for ectotherms whose body temperatures are directly influenced by available environmental temperatures. Geckos are a large, diverse group of ectotherms that vary in their thermal habitats and times of daily activity, which could affect how they physiologically adjust to heat waves. Data on thermal physiology are scarce for reptiles, with only one study in geckos. Understanding thermal tolerance and plasticity, and their relationship, is essential for understanding how some species are able to adjust or adapt to changing temperatures. In this study, we estimated thermal tolerance and plasticity, and their interaction, in the crepuscular gecko, Eublepharis macularius, a species that is emerging as a model for reptile biology. After estimating basal thermal tolerance for 28 geckos, thermal tolerance was measured for each individual a second time at several timepoints (3, 6, or 24 h) to determine thermal tolerance plasticity. We found that thermal tolerance plasticity (1) does not depend on the basal thermal tolerance of the organism, (2) was highest after 6 h from initial heat shock, and (3) was negatively influenced by individual body mass. Our findings contribute to the increasing body of work focused on understanding the influence of biological and environmental factors on thermal tolerance plasticity in organisms and provide phenotypic data to further investigate the molecular basis of thermal tolerance plasticity in organisms.

The revised reference genome of the leopard gecko (Eublepharis macularius) provides insight into the considerations of genome phasing and assembly.

Genomic resources across squamate reptiles (lizards and snakes) have lagged behind other vertebrate systems and high-quality reference genomes remain scarce. Of the 23 chromosome-scale reference genomes across the order, only 12 of the ~60 squamate families are represented. Within geckos (infraorder Gekkota), a species-rich clade of lizards, chromosome-level genomes are exceptionally sparse representing only two of the seven extant families. Using the latest advances in genome sequencing and assembly methods, we generated one of the highest-quality squamate genomes to date for the leopard gecko, Eublepharis macularius (Eublepharidae). We compared this assembly to the previous, short-read only, E. macularius reference genome published in 2016 and examined potential factors within the assembly influencing contiguity of genome assemblies using PacBio HiFi data. Briefly, the read N50 of the PacBio HiFi reads generated for this study was equal to the contig N50 of the previous E. macularius reference genome at 20.4 kilobases. The HiFi reads were assembled into a total of 132 contigs, which was further scaffolded using HiC data into 75 total sequences representing all 19 chromosomes. We identified 9 of the 19 chromosomal scaffolds were assembled as a near-single contig, whereas the other 10 chromosomes were each scaffolded together from multiple contigs. We qualitatively identified that the percent repeat content within a chromosome broadly affects its assembly contiguity prior to scaffolding. This genome assembly signifies a new age for squamate genomics where high-quality reference genomes rivaling some of the best vertebrate genome assemblies can be generated for a fraction of previous cost estimates. This new E. macularius reference assembly is available on NCBI at JAOPLA010000000.

A new lineage of Galapagos giant tortoises identified from museum samples.

The Galapagos Archipelago is recognized as a natural laboratory for studying evolutionary processes. San Cristóbal was one of the first islands colonized by tortoises, which radiated from there across the archipelago to inhabit 10 islands. Here, we sequenced the mitochondrial control region from six historical giant tortoises from San Cristóbal (five long deceased individuals found in a cave and one found alive during an expedition in 1906) and discovered that the five from the cave are from a clade that is distinct among known Galapagos giant tortoises but closely related to the species from Española and Pinta Islands. The haplotype of the individual collected alive in 1906 is in the same clade as the haplotype in the contemporary population. To search for traces of a second lineage in the contemporary population on San Cristóbal, we closely examined the population by sequencing the mitochondrial control region for 129 individuals and genotyping 70 of these for both 21 microsatellite loci and >12,000 genome-wide single nucleotide polymorphisms [SNPs]. Only a single mitochondrial haplotype was found, with no evidence to suggest substructure based on the nuclear markers. Given the geographic and temporal proximity of the two deeply divergent mitochondrial lineages in the historical samples, they were likely sympatric, raising the possibility that the lineages coexisted. Without the museum samples, this important discovery of an additional lineage of Galapagos giant tortoise would not have been possible, underscoring the value of such collections and providing insights into the early evolution of this iconic radiation.

Isolating and quantifying the role of developmental noise in generating phenotypic variation.

Genotypic variation, environmental variation, and their interaction may produce variation in the developmental process and cause phenotypic differences among individuals. Developmental noise, which arises during development from stochasticity in cellular and molecular processes when genotype and environment are fixed, also contributes to phenotypic variation. While evolutionary biology has long focused on teasing apart the relative contribution of genes and environment to phenotypic variation, our understanding of the role of developmental noise has lagged due to technical difficulties in directly measuring the contribution of developmental noise. The influence of developmental noise is likely underestimated in studies of phenotypic variation due to intrinsic mechanisms within organisms that stabilize phenotypes and decrease variation. Since we are just beginning to appreciate the extent to which phenotypic variation due to stochasticity is potentially adaptive, the contribution of developmental noise to phenotypic variation must be separated and measured to fully understand its role in evolution. Here, we show that variation in the component of the developmental process corresponding to environmental and genetic factors (here treated together as a unit called the LALI-type) versus the contribution of developmental noise, can be distinguished for leopard gecko (Eublepharis macularius) head color patterns using mathematical simulations that model the role of random variation (corresponding to developmental noise) in patterning. Specifically, we modified the parameters of simulations corresponding to variation in the LALI-type to generate the full range of phenotypic variation in color pattern seen on the heads of eight leopard geckos. We observed that over the range of these parameters, variation in color pattern due to LALI-type variation exceeds that due to developmental noise in the studied gecko cohort. However, the effect of developmental noise on patterning is also substantial. Our approach addresses one of the major goals of evolutionary biology: to quantify the role of stochasticity in shaping phenotypic variation.

Mixed phylogenetic signal in fish toxicity data across chemical classes.

Chemical use in society is growing rapidly and is one of the five major pressures on biodiversity worldwide. Since empirical toxicity studies of pollutants generally focus on a handful of model organisms, reliable approaches are needed to assess sensitivity to chemicals across the wide variety of species in the environment. Phylogenetic comparative methods (PCM) offer a promising approach for toxicity extrapolation incorporating known evolutionary relationships among species. If phylogenetic signal in toxicity data is high, i.e., closely related species are more similarly sensitive as compared to distantly related species, PCM could ultimately help predict species sensitivity when toxicity data are lacking. Here, we present the largest ever test of phylogenetic signal in toxicity data by combining phylogenetic data from fish with acute mortality data for 42 chemicals spanning 10 different chemical classes. Phylogenetic signal is high for some chemicals, particularly organophosphate pesticides, but not necessarily for many chemicals in other classes (e.g., metals, organochlorines). These results demonstrate that PCM may be useful for toxicity extrapolation in untested species for those chemicals with clear phylogenetic signal. This study provides a framework for using PCM to understand the patterns and causes of variation in species sensitivity to pollutants.

Ecological and evolutionary influences on body size and shape in the Galápagos marine iguana (Amblyrhynchus cristatus).

Oceanic islands are often inhabited by endemic species that have undergone substantial morphological evolutionary change due to processes of multiple colonizations from various source populations, dispersal, and local adaptation. Galápagos marine iguanas are an example of an island endemic exhibiting high morphological diversity, including substantial body size variation among populations and sexes, but the causes and magnitude of this variation are not well understood. We obtained morphological measurements from marine iguanas throughout their distribution range. These data were combined with genetic and local environmental data from each population to investigate the effects of evolutionary history and environmental conditions on body size and shape variation and sexual dimorphism. Our results indicate that body size and shape are highly variable among populations. Sea surface temperature and island perimeter, but not evolutionary history as depicted by phylogeographic patterns in this species, explain variation in body size among populations. Conversely, evolutionary history, but not environmental parameters or island size, was found to influence variation in body shape among populations. Finally, in all populations except one, we found strong sexual dimorphism in body size and shape in which males are larger, with higher heads than females, while females have longer heads than males. Differences among populations suggest that plasticity and/or genetic adaptation may shape body size and shape variation in marine iguanas. This study will help target future investigations to address the contribution of plasticity versus genetic adaptation on size and shape variation in marine iguanas.

Amelotin: an enamel matrix protein that experienced distinct evolutionary histories in amphibians, sauropsids and mammals.

BACKGROUND: Amelotin (AMTN) is an ameloblast-secreted protein that belongs to the secretory calcium-binding phosphoprotein (SCPP) family, which originated in early vertebrates. In rodents, AMTN is expressed during the maturation stage of amelogenesis only. This expression pattern strongly differs from the spatiotemporal expression of other ameloblast-secreted SCPPs, such as the enamel matrix proteins (EMPs). Furthermore, AMTN was characterized in rodents only. In this study, we applied various approaches, including in silico screening of databases, PCRs and transcriptome sequencing to characterize AMTN sequences in sauropsids and amphibians, and compared them to available mammalian and coelacanth sequences. RESULTS: We showed that (i) AMTN is tooth (enamel) specific and underwent pseudogenization in toothless turtles and birds, and (ii) the AMTN structure changed during tetrapod evolution. To infer AMTN function, we studied spatiotemporal expression of AMTN during amelogenesis in a salamander and a lizard, and compared the results with available expression data from mouse. We found that AMTN is expressed throughout amelogenesis in non-mammalian tetrapods, in contrast to its expression limited to enamel maturation in rodents. CONCLUSIONS: Taken together our findings suggest that AMTN was primarily an EMP. Its functions were conserved in amphibians and sauropsids while a change occurred early in the mammalian lineage, modifying its expression pattern during amelogenesis and its gene structure. These changes likely led to a partial loss of AMTN function and could have a link with the emergence of prismatic enamel in mammals.

Phylogenetic signal in amphibian sensitivity to copper sulfate relative to experimental temperature.

The release of large quantities of chemicals into the environment represents a major source of environmental disturbance. In recent years, the focus of ecotoxicology has shifted from describing the effects of chemical contaminants on individual species to developing more integrated approaches for predicting and evaluating long term effects of chemicals across species and ecosystems. Traditional ecotoxicology is typically based on data of sensitivity to a contaminant of a few surrogate species and often considers little variability in chemical sensitivity within and among taxonomic groups. This approach assumes that evolutionary history and phylogenetic relatedness among species have little or no impact on species' sensitivity to chemical compounds. Few studies have tested this assumption. Using phylogenetic comparative methods and published data for amphibians, we show that sensitivity to copper sulfate, a commonly used pesticide, exhibits a strong phylogenetic signal when controlling for experimental temperature. Our results indicate that evolutionary history needs to be accounted for to make accurate predictions of amphibian sensitivity to this contaminant under different temperature scenarios. Since physiological and metabolic traits showing high phylogenetic signal likely underlie variation in species sensitivity to chemical stressors, future studies should evaluate and predict species vulnerability to pollutants using evolutionarily informed approaches.

Molecular evolution and functional divergence of the metallothionein gene family in vertebrates.

The metallothionein (MT) gene superfamily consists of metal-binding proteins involved in various metal detoxification and storage mechanisms. The evolution of this gene family in vertebrates has mostly been studied in mammals using sparse taxon or gene sampling. Genomic databases and available data on MT protein function and expression allow a better understanding of the evolution and functional divergence of the different MT types. We recovered 77 MT coding sequences from 20 representative vertebrates with annotated complete genomes. We found multiple MT genes, also in reptiles, which were thought to have only one MT type. Phylogenetic and synteny analyses indicate the existence of a eutherian MT1 and MT2, a tetrapod MT3, an amniote MT4, and fish MT. The optimal gene-tree/species-tree reconciliation analyses identified the best root in the fish clade. Functional analyses reveal variation in hydropathic index among protein domains, likely correlated with their distinct flexibility and metal affinity. Analyses of functional divergence identified amino acid sites correlated with functional divergence among MT types. Uncovering the number of genes and sites possibly correlated with functional divergence will help to design cost-effective MT functional and gene expression studies. This will permit further understanding of the distinct roles and specificity of these proteins and to properly target specific MT for different types of functional studies. Therefore, this work presents a critical background on the molecular evolution and functional divergence of vertebrate MTs to carry out further detailed studies on the relationship between heavy metal metabolism and tolerances among vertebrates.

Phylogenomic analyses support the position of turtles as the sister group of birds and crocodiles (Archosauria).

BACKGROUND: The morphological peculiarities of turtles have, for a long time, impeded their accurate placement in the phylogeny of amniotes. Molecular data used to address this major evolutionary question have so far been limited to a handful of markers and/or taxa. These studies have supported conflicting topologies, positioning turtles as either the sister group to all other reptiles, to lepidosaurs (tuatara, lizards and snakes), to archosaurs (birds and crocodiles), or to crocodilians. Genome-scale data have been shown to be useful in resolving other debated phylogenies, but no such adequate dataset is yet available for amniotes. RESULTS: In this study, we used next-generation sequencing to obtain seven new transcriptomes from the blood, liver, or jaws of four turtles, a caiman, a lizard, and a lungfish. We used a phylogenomic dataset based on 248 nuclear genes (187,026 nucleotide sites) for 16 vertebrate taxa to resolve the origins of turtles. Maximum likelihood and Bayesian concatenation analyses and species tree approaches performed under the most realistic models of the nucleotide and amino acid substitution processes unambiguously support turtles as a sister group to birds and crocodiles. The use of more simplistic models of nucleotide substitution for both concatenation and species tree reconstruction methods leads to the artefactual grouping of turtles and crocodiles, most likely because of substitution saturation at third codon positions. Relaxed molecular clock methods estimate the divergence between turtles and archosaurs around 255 million years ago. The most recent common ancestor of living turtles, corresponding to the split between Pleurodira and Cryptodira, is estimated to have occurred around 157 million years ago, in the Upper Jurassic period. This is a more recent estimate than previously reported, and questions the interpretation of controversial Lower Jurassic fossils as being part of the extant turtles radiation. CONCLUSIONS: These results provide a phylogenetic framework and timescale with which to interpret the evolution of the peculiar morphological, developmental, and molecular features of turtles within the amniotes.

The revised reference genome of the leopard gecko ( Eublepharis macularius ) provides insight into the considerations of genome phasing and assembly.

Genomic resources across squamate reptiles (lizards and snakes) have lagged behind other vertebrate systems and high-quality reference genomes remain scarce. Of the 23 chromosome-scale reference genomes across the order, only 12 of the ~60 squamate families are represented. Within geckos (infraorder Gekkota), a species-rich clade of lizards, chromosome-level genomes are exceptionally sparse representing only two of the seven extant families. Using the latest advances in genome sequencing and assembly methods, we generated one of the highest quality squamate genomes to date for the leopard gecko, Eublepharis macularius (Eublepharidae). We compared this assembly to the previous, short-read only, E. macularius reference genome published in 2016 and examined potential factors within the assembly influencing contiguity of genome assemblies using PacBio HiFi data. Briefly, the read N50 of the PacBio HiFi reads generated for this study was equal to the contig N50 of the previous E. macularius reference genome at 20.4 kilobases. The HiFi reads were assembled into a total of 132 contigs, which was further scaffolded using HiC data into 75 total sequences representing all 19 chromosomes. We identified that 9 of the 19 chromosomes were assembled as single contigs, while the other 10 chromosomes were each scaffolded together from two or more contigs. We qualitatively identified that percent repeat content within a chromosome broadly affects its assembly contiguity prior to scaffolding. This genome assembly signifies a new age for squamate genomics where high-quality reference genomes rivaling some of the best vertebrate genome assemblies can be generated for a fraction previous cost estimates. This new E. macularius reference assembly is available on NCBI at JAOPLA010000000. The genome version and its associated annotations are also available via this Figshare repository https://doi.org/10.6084/m9.figshare.20069273 .

Influence of RNA-Seq library construction, sampling methods, and tissue harvesting time on gene expression estimation.

RNA sequencing (RNA-Seq) is popular for measuring gene expression in non-model organisms, including wild populations. While RNA-Seq can detect gene expression variation among wild-caught individuals and yield important insights into biological function, sampling methods can also affect gene expression estimates. We examined the influence of multiple technical variables on estimated gene expression in a non-model fish, the westslope cutthroat trout (Oncorhynchus clarkii lewisi), using two RNA-Seq library types: 3' RNA-Seq (QuantSeq) and whole mRNA-Seq (NEB). We evaluated effects of dip netting versus electrofishing, and of harvesting tissue immediately versus 5 min after euthanasia on estimated gene expression in blood, gill, and muscle. We found no significant differences in gene expression between sampling methods or tissue collection times with either library type. When library types were compared using the same blood samples, 58% of genes detected by both NEB and QuantSeq showed significantly different expression between library types, and NEB detected 31% more genes than QuantSeq. Although the two library types recovered different numbers of genes and expression levels, results with NEB and QuantSeq were consistent in that neither library type showed differences in gene expression between sampling methods and tissue harvesting times. Our study suggests that researchers can safely rely on different fish sampling strategies in the field. In addition, while QuantSeq is more cost effective, NEB detects more expressed genes. Therefore, when it is crucial to detect as many genes as possible (especially low expressed genes), when alternative splicing is of interest, or when working with an organism lacking good genomic resources, whole mRNA-Seq is more powerful.

Melanistic coloration does not influence thermoregulation in the crepuscular gecko Eublepharis macularius.

Body coloration in ectotherms serves multiple biological functions, including avoiding predators, communicating with conspecific individuals, and involvement in thermoregulation. As ectotherms rely on environmental sources of heat to regulate their internal body temperature, stable melanistic body coloration or color change can be used to increase or decrease heat absorption and heat exchange with the environment. While melanistic coloration for thermoregulation functions to increase solar radiation absorption and consequently heating in many diurnal ectotherms, research on crepuscular and nocturnal ectotherms is lacking. Since crepuscular and nocturnal ectotherms generally absorb heat from the substrate, in these organisms melanistic coloration may have other primary functions beside thermoregulation. As such, in this work we hypothesized that the proportion of dorsal melanistic body coloration would not influence heating and cooling rates in the crepuscular gecko, Eublepharis macularius, and that changes in environmental temperature would not trigger color changes in this species. Temperature measurements of the geckos and of the environment were taken using infrared thermography and temperature loggers. Color data were obtained using objective photography and a newly developed custom software package. We found that body temperature reflected substrate temperatures, and that the proportion of melanistic coloration has no influence on heating or cooling rates or on color changes. These findings support that melanistic coloration in E. macularius may not be used for thermoregulation and strengthen the hypothesis that in animals active in low light conditions, melanistic coloration may be used instead for camouflage or other functions.

Dating cryptodiran nodes: origin and diversification of the turtle superfamily Testudinoidea.

The superfamily Testudinoidea is the most diverse and widely distributed clade of extant turtles. Surprisingly, despite an extensive fossil record, and increasing amount of molecular data available, the temporal origin of this group is still largely unknown. To address this issue, we used a comprehensive molecular dataset to perform phylogenetic and molecular dating analyses, as well as seven fossil constraints to calibrate the ages of the nodes in the phylogeny. The molecular dataset includes the complete mitochondrial genomes of 37 turtle species, including newly sequenced mitochondrial genomes of Phrynops hilarii, Emys orbicularis, Rhinoclemmys punctularia, and Chelonoidis nigra, and four nuclear markers. Our results revealed that the earliest divergences within crown testudinoids occurred around 95.0 Mya, in the early Late Cretaceous, earlier than previously reported, raising new questions about the historical biogeography of this group.

Hybridization and low genetic diversity in the endangered Alabama red-bellied turtle (Pseudemys alabamensis).

Pseudemys alabamensis is one of the most endangered freshwater turtle species in the United States due to its restricted geographic distribution in coastal Alabama and Mississippi. Populations of P. alabamensis are geographically isolated from one another by land and saltwater, which could act as barriers to gene flow. It is currently unknown how differentiated these populations are from one another and whether they have experienced reductions in population size. Previous work found morphological differences between Alabama and Mississippi populations, suggesting that they may be evolutionarily distinct. Other Pseudemys turtles such as P. concinna and P. floridana occur naturally within the same geographic area as P. alabamensis and are known to hybridize with each other. These more abundant species could threaten the unique genetic identity of P. alabamensis through introgression. In order to evaluate the endangered status of P. alabamensis and the level of hybridization with other species, we used mitochondrial and nuclear microsatellite markers to assess genetic variation within and among populations of P. alabamensis throughout its range and estimate admixture with co-occurring Pseudemys species. In P. alabamensis, we found no variation in mitochondrial DNA and an excess of homozygosity in microsatellite data. Our results show genetic differentiation between Alabama and Mississippi populations of P. alabamensis, and low estimated breeding sizes and signs of inbreeding for two populations (Fowl River, Alabama and Biloxi, Mississippi). We also found evidence of admixture between P. alabamensis and P. concinna/P. floridana. Based on our results, P. alabamensis is highly endangered throughout its range and threatened by both low population sizes and hybridization. In order to improve the species' chances of survival, focus should be placed on habitat preservation, maintenance of genetic diversity within both the Mississippi and Alabama populations, and routine population-monitoring activities such as nest surveillance and estimates of recruitment.

Capturing and analyzing pattern diversity: an example using the melanistic spotted patterns of leopard geckos.

Animal color patterns are widely studied in ecology, evolution, and through mathematical modeling. Patterns may vary among distinct body parts such as the head, trunk or tail. As large amounts of photographic data is becoming more easily available, there is a growing need for general quantitative methods for capturing and analyzing the full complexity and details of pattern variation. Detailed information on variation in color pattern elements is necessary to understand how patterns are produced and established during development, and which evolutionary forces may constrain such a variation. Here, we develop an approach to capture and analyze variation in melanistic color pattern elements in leopard geckos. We use this data to study the variation among different body parts of leopard geckos and to draw inferences about their development. We compare patterns using 14 different indices such as the ratio of melanistic versus total area, the ellipticity of spots, and the size of spots and use these to define a composite distance between two patterns. Pattern presence/absence among the different body parts indicates a clear pathway of pattern establishment from the head to the back legs. Together with weak within-individual correlation between leg patterns and main body patterns, this suggests that pattern establishment in the head and tail may be independent from the rest of the body. We found that patterns vary greatest in size and density of the spots among body parts and individuals, but little in their average shapes. We also found a correlation between the melanistic patterns of the two front legs, as well as the two back legs, and also between the head, tail and trunk, especially for the density and size of the spots, but not their shape or inter-spot distance. Our data collection and analysis approach can be applied to other organisms to study variation in color patterns between body parts and to address questions on pattern formation and establishment in animals.

Concurrent Evolution of Antiaging Gene Duplications and Cellular Phenotypes in Long-Lived Turtles.

There are many costs associated with increased body size and longevity in animals, including the accumulation of genotoxic and cytotoxic damage that comes with having more cells and living longer. Yet, some species have overcome these barriers and have evolved remarkably large body sizes and long lifespans, sometimes within a narrow window of evolutionary time. Here, we demonstrate through phylogenetic comparative analysis that multiple turtle lineages, including Galapagos giant tortoises, concurrently evolved large bodies, long lifespans, and reduced cancer risk. We also show through comparative genomic analysis that Galapagos giant tortoises have gene duplications related to longevity and tumor suppression. To examine the molecular basis underlying increased body size and lifespan in turtles, we treated cell lines from multiple species, including Galapagos giant tortoises, with drugs that induce different types of cytotoxic stress. Our results indicate that turtle cells, in general, are resistant to oxidative stress related to aging, whereas Galapagos giant tortoise cells, specifically, are sensitive to endoplasmic reticulum stress, which may give this species an ability to mitigate the effects of cellular stress associated with increased body size and longevity.

Elevated metallothionein expression in long-lived species mediates the influence of cadmium accumulation on aging.

Cadmium (Cd) accumulates with aging and is elevated in long-lived species. Metallothioneins (MTs), small cysteine-rich proteins involved in metal homeostasis and Cd detoxification, are known to be related to longevity. However, the relationship between Cd accumulation, the role of MTs, and aging is currently unclear. Specifically, we do not know if long-lived species evolved an efficient metal stress response by upregulating their MT levels to reduce the toxic effects of environmental pollutants, such as Cd, that accumulate over their longer life span. It is also unknown if the number of MT genes, their expression, or both protect the organisms from potentially damaging effects during aging. To address these questions, we reanalyzed several cross-species studies and obtained data on MT expression and Cd accumulation in long-lived mouse models. We confirmed a relationship between species maximum life span in captive mammals and their Cd content in liver and kidney. We found that although the number of MT genes does not affect longevity, gene expression and protein amount of specific MT paralogs are strongly related to life span in mammals. MT expression rather than gene number may influence the high Cd levels and longevity of some species. In support of this, we found that overexpression of MT-1 accelerated Cd accumulation in mice and that tissue Cd was higher in long-lived mouse strains with high MT expression. We conclude that long-lived species have evolved a more efficient stress response by upregulating the expression of MT genes in presence of Cd, which contributes to elevated tissue Cd levels.