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RNA sequencing (RNA-Seq) is a popular method for measuring gene expression in non-model organisms, including wild populations. While RNA-Seq can measure gene expression variation among wild-caught individuals and can yield important biological insights into organism function, sampling methods may also influence 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 and whole mRNA-Seq. We evaluated effects of dip netting versus electrofishing, and of harvesting tissue immediately versus 5 minutes after euthanasia on estimated gene expression in blood, gill, and muscle. We detected 30% more genes with whole mRNA-Seq than with 3’ RNA-Seq and found that 58% of genes were significantly differently expressed between 3’ RNA-Seq and whole mRNA-Seq. Our findings indicate that 3’ RNA-Seq and whole mRNA-Seq are robust to the technical variables related to the field sampling approaches tested here with a lack of differential gene expression among sampling methods and tissue collection time after euthanasia. However, we found that gene expression varied based on which RNA-Seq library type was used on the same set of samples. Our study suggests researchers could safely rely on different fish sampling strategies in the field and save money and analyze more individuals using 3’ RNA-Seq, but should use whole mRNA-Seq when working with a species without good genomic resources, and when maximizing the number of genes identified and detecting alternative splicing are important.

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. maculariusreference 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 previousE. maculariusreference 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 newE. maculariusreference assembly is available on NCBI at JAOPLA010000000. The genome version and its associated annotations are also available via this Figshare repositoryhttps://doi.org/10.6084/m9.figshare.20069273.

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.

Significance Beyond sugars, many types of nectar solutes play important ecological roles; however, the molecular basis for the diversity of nectar composition across species is less explored. One rare trait among flowering plants is the production of colored nectar, which may function to attract and guide prospective pollinators. Our findings indicate convergent evolution of a red-colored nectar has occurred across two distantly related plant species. Behavioral data show that the red pigment attracts diurnal geckos, the likely pollinator of one of these plants. These findings join a growing list of examples of distinct biochemical and molecular mechanisms underlying evolutionary convergence and provide a fascinating system for testing how interactions across species drive the evolution of novel pigments in an understudied context.

Pseudemys alabamensis is one of the most endangered turtle species in the United States due to its small population size and restricted geographic distribution in coastal Alabama and Mississippi. Increased urbanization and climate change impacts in the region further threaten this species. Populations of P. alabamensis are geographically isolated from one another by land and salt water, which could act as barriers to intraspecific gene flow. It is currently unknown how differentiated these isolated populations are from one another or 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 within the same geographic area as P. alabamensis and are known to hybridize with each other. These more abundant species could further 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 the mitochondrial (mtDNA) control region and nuclear microsatellite markers to assess genetic variation within and among populations of this species throughout its range and estimate admixture with co-occurring Pseudemys species. Genetic diversity of P. alabamensis was lower than expected at both markers (no variation in mtDNA and excess of homozygosity in microsatellites). We found evidence of genetic differentiation between Alabama and Mississippi populations as well as two populations (Fowl River, Alabama and Biloxi, Mississippi) with low estimated breeding sizes and signs of inbreeding. Finally, we found evidence of admixture of P. alabamensis with P. concinna/P. floridana and Pseudemys peninsularis (a species not native to Alabama or Mississippi). Our results indicate that 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 Mississippi and Alabama populations, and regular population monitoring activities such as nest surveillance and estimates of recruitment.

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.

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, while 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.

Animals are exposed to different visual stimuli that influence how they perceive and interact with their environment. Visual information such as shape and colour can help the animal detect, discriminate and make appropriate behavioural decisions for mate selection, communication, camouflage, and foraging. In all major vertebrate groups, it has been shown that certain species can discriminate and prefer certain colours and that colours may increase the response to a stimulus. However, because colour is often studied together with other potentially confounding factors, it is still unclear to what extent colour discrimination plays a crucial role in the perception of and attention towards biologically relevant and irrelevant stimuli. To address these questions in reptiles, we assessed the response of three gecko speciesCorrelophus ciliatus, Eublepharis macularius, andPhelsuma laticaudato familiar and novel 2D images in colour or grayscale. We found that while all species responded more often to the novel than to the familiar images, colour information did not influence object discrimination. We also found that the duration of interaction with images was significantly longer for the diurnal species,P. laticauda, than for the two nocturnal species, but this was independent from colouration. Finally, no differences among sexes were observed within or across species. Our results indicate that geckos discriminate between 2D images of different content independent of colouration, suggesting that colouration does not increase detectability or intensity of the response. These results are essential for uncovering which visual stimuli produce a response in animals and furthering our understanding of how animals use colouration and colour vision.

RNA sequencing (RNA-Seq) is becoming a popular method for measuring gene expression in non-model organisms, including wild populations sampled in the field. While RNA-Seq can be used to measure gene expression variation among wild-caught individuals and can yield important biological insights into organismal function, technical variables may also influence gene expression estimates. We examined the influence of multiple technical variables on estimated gene expression in a non-model fish species, the westslope cutthroat trout (Oncorhynchus clarkii lewisi), using two RNA-Seq methods: 3’ RNA-Seq and whole mRNA-Seq. We evaluated the effects of dip netting versus electrofishing, and of harvesting tissue immediately versus 5 minutes after euthanasia on estimated gene expression in blood, gill, muscle, and liver. We found higher RNA degradation in the liver compared to the other tissues. There were fewer expressed genes in blood compared to gill and muscle. We found no difference in gene expression among sampling methods or due to a delay in tissue collection. However, we detected fewer genes with 3’ RNA-Seq than with whole mRNA-Seq and found statistically significant differences in gene expression between 3’ RNA-Seq and whole mRNA-Seq. The magnitude and direction of these differences does not appear to be dependent on gene type or length. Our findings indicate that RNA-Seq is robust to the technical variables related to the field sampling techniques tested here but varies based on the tissue sampled and the RNA-Seq library used. This study advances understanding of usefulness of RNA-Seq to study gene expression variation in evolution, ecology, and conservation.

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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11357-021-00393-3.

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 and12,000 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.

The dorsal surfaces of many taxonomic groups often feature repetitive pattern elements consisting of stripes, spots, or bands. Here, we investigate how distinct categories of camouflage pattern work by relating them to ecological and behavioral traits in 439 species of gecko. We use phylogenetic comparative methods to test outstanding hypotheses based on camouflage theory and research in other taxa. We found that bands are associated with nocturnal activity, suggesting bands provide effective camouflage for motionless geckos resting in refugia during the day. A predicted association between stripes and diurnal activity was not supported, suggesting that stripes do not work via dazzle camouflage mechanisms in geckos. This, along with a lack of support for our prediction that plain patterning should be associated with open habitats, suggests that similar camouflage patterns do not work in consistent ways across taxa. We also found that plain and striped lineages frequently switched between using open or closed habitats, whereas spotted lineages rarely transitioned. This suggests that pattern categories differ in how specialized or generalized their camouflage is. This result has ramifications for theory on how camouflage compromises to background heterogeneity and how camouflage pattern might influence evolutionary trajectories.

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.

DNA damage has been linked to genomic instability and the progressive breakdown of cellular and organismal homeostasis, leading to the onset of disease and reduced longevity. Insults to DNA from endogenous sources include base deamination, base hydrolysis, base alkylation, and metabolism-induced oxidative damage that can lead to single-strand and double-strand DNA breaks. Alternatively, exposure to environmental pollutants, radiation or ultra-violet light, can also contribute to exogenously derived DNA damage. We previously validated a novel, high through-put approach to measure levels of DNA damage in cultured mammalian cells. This new CometChip Platform builds on the classical single cell gel electrophoresis or comet methodology used extensively in environmental toxicology and molecular biology. We asked whether the CometChip Platform could be used to measure DNA damage in samples derived from environmental field studies. To this end, we determined that nucleated erythrocytes from multiple species of turtle could be successfully evaluated in the CometChip Platform to quantify levels of DNA damage. In total, we compared levels of DNA damage in 40 animals from two species: the box turtle (Terrapene carolina) and the red-eared slider (Trachemys scripta elegans). Endogenous levels of DNA damage were identical between the two species, yet we did discover some sex-linked differences and changes in DNA damage accumulation. Based on these results, we confirm that the CometChip Platform allows for the measurement of DNA damage in a large number of samples quickly and accurately, and is particularly adaptable to environmental studies using field-collected samples. Environ. Mol. Mutagen. 59:322-333, 2018. © 2018 Wiley Periodicals, Inc.

Giant tortoises are among the longest-lived vertebrate animals and, as such, provide an excellent model to study traits like longevity and age-related diseases. However, genomic and molecular evolutionary information on giant tortoises is scarce. Here, we describe a global analysis of the genomes of Lonesome George—the iconic last member of Chelonoidis abingdonii—and the Aldabra giant tortoise (Aldabrachelys gigantea). Comparison of these genomes with those of related species, using both unsupervised and supervised analyses, led us to detect lineage-specific variants affecting DNA repair genes, inflammatory mediators and genes related to cancer development. Our study also hints at specific evolutionary strategies linked to increased lifespan, and expands our understanding of the genomic determinants of ageing. These new genome sequences also provide important resources to help the efforts for restoration of giant tortoise populations., We thank Banco Santander for funding a short stay of S.F.-R. and D.C.-I. at Yale University. V.Q. is supported by grants from the Principado de Asturias and Ministerio de Economía y Competitividad, including FEDER funding. L.F.K.K. is supported by an FPI fellowship associated with BFU2014-55090-P (FEDER). T.M.-B. is supported by MINECO BFU2017-86471-P (MINECO/FEDER, UE), an NIH U01 MH106874 grant, the Howard Hughes International Early Career programme, Obra Social ‘La Caixa’ and Secretaria d’Universitats i Recerca, and CERCA Programme del Departament d’Economia i Coneixement de la Generalitat de Catalunya. C.L.-O. is supported by grants from the European Research Council (DeAge; ERC Advanced Grant), Ministerio de Economía y Competitividad, Instituto de Salud Carlos III (RTICC) and Progeria Research Foundation. The Instituto Universitario de Oncología is supported by Fundación Bancaria Caja de Ahorros de Asturias. We also thank staff at the Galapagos National Park and Galapagos Conservancy for logistic and financial support.

Seagrass meadows form ecologically and economically valuable coastal habitat on every continental margin except the Antarctic, but their areal extent is declining by approximately 2–5 % per year. Seagrass wasting disease is a contributing factor in these declines, with the protist Labyrinthula identified as the etiologic agent. To help elucidate the role of Labyrinthula spp. in global seagrass declines, we surveyed roughly one fourth of all seagrass species to identify Labyrinthula diversity at the strain and/or species level, combining results from culturing methods and two common nuclear DNA markers: the ITS and 18S regions of the ribosomal RNA gene complex. After assaying a subset of the resulting isolates (of which 170 were newly sequenced), we produced a cladogenic context for putative seagrass-pathogenic versus non-pathogenic Labyrinthula while also defining host and geographic ranges. Assays also suggest that pathogenicity is consistently high (when present; and, even when comparing susceptibility of US East- versus West Coast Zostera marina hosts) while virulence is variable, that some isolate-host combinations have the potential for host cross-infection, and that several modes of transmission can be effective. Taken together, these data provide additional means for delimiting putative species of Labyrinthula, suggesting at least five seagrass-pathogenic and perhaps ten or more non-pathogenic marine “species”, yielding a working definition for ecologists and epidemiologists attempting to reconcile the sundry data related to seagrass wasting disease.

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., Author summary The observable characteristics of an organism make up its phenotype. Variation among phenotypes is due to genetic differences, environmental factors and developmental noise (effects due to inherent stochasticity) during development. We used mathematical models to investigate the contributions of variation of the developmental process due to genetic and environmental factors (treated in this work as a single unit) versus developmental noise (unavoidable variation within the developmental program) to the development of pigment patterns on gecko heads. We found that for our cohort, the proportion of phenotypic variation due to variation in the unit composed of genotypic and environmental variation is larger than that due to developmental noise. Furthermore, by allowing the parameters of the mathematical model to vary, we generated the full extent of potential phenotypic pattern variation that could occur on the head of geckos. This serves to further study the influence of the buffering mechanisms (canalization, selection, and developmental stability) limiting phenotypic variation. This approach can be applied to any regular morphological trait that results from self-organized processes such as reaction-diffusion mechanisms, including the frequently found striped and spotted patterns of animal pigmentation patterning, patterning of bones in vertebrate limbs, and body segmentation in segmented animals.

Self-righting, the capacity of an animal to self-turn after falling on its back, is a fitness-related trait. Delayed self-righting can result in loss of mating opportunities or death. Traits involved in self-righting may therefore be under selection. Galápagos giant tortoises have two main shell morphologies - saddleback and domed – that have been proposed to be adaptive. The more sloped shape on the sides of the shell and the longer extension of neck and legs of the saddlebacks could have evolved to optimize self-righting. The drier environments with more uneven surfaces where the saddleback tortoises occur increases their risk to fall on their back while walking. The ability to fast overturn could reduce the danger of dying. To test this hypothesis, we used 3D shell reconstructions of 89 Galápagos giant tortoises from three domed and two saddleback species to compare self-righting potential of the two shell morphotypes. Our results indicate that saddleback shells require higher energy input to self-right than domed ones. This suggests that several traits associated with the saddleback shell morphology could have evolved to facilitate self-righting. Studying the functional performances of fitness-related traits, as in this work, could provide important insight into the adaptive value of traits.

DNA damage has been linked to genomic instability and the progressive breakdown of cellular and organismal homeostasis, leading to the onset of disease and reduced longevity. Insults to DNA from endogenous sources include base deamination, base hydrolysis, base alkylation, and metabolism-induced oxidative damage that can lead to single-strand and double-strand DNA breaks. Alternatively, exposure to environmental pollutants, radiation or ultra-violet light, can also contribute to exogenously derived DNA damage. We previously validated a novel, high through-put approach to measure levels of DNA damage in cultured mammalian cells. This new CometChip Platform builds on the classical single cell gel electrophoresis or comet methodology used extensively in environmental toxicology and molecular biology. We asked whether the CometChip Platform could be used to measure DNA damage in samples derived from environmental field studies. To this end, we determined that nucleated erythrocytes from multiple species of turtle could be successfully evaluated in the CometChip Platform to quantify levels of DNA damage. In total, we compared levels of DNA damage in 40 animals from two species: the box turtle (Terrapene carolina) and the red-eared slider (Trachemys scripta elegans). Endogenous levels of DNA damage were identical between the two species, yet we did discover some sex-linked differences and changes in DNA damage accumulation. Based on these results, we confirm that the CometChip Platform allows for the measurement of DNA damage in a large number of samples quickly and accurately, and is particularly adaptable to environmental studies using field-collected samples. Environ. Mol. Mutagen. 59:322-333, 2018. © 2018 Wiley Periodicals, Inc.

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.

Cancer researchers have traditionally used the mouse and the rat as staple model organisms. These animals are very short-lived, reproduce rapidly, and are highly prone to cancer. They have been very useful for modeling some human cancer types and testing experimental treatments; however, these cancer-prone species offer little for understanding the mechanisms of cancer resistance. Recent technological advances have expanded research bestiary to non-standard model organisms that possess unique traits of very high value to humans such as cancer resistance and longevity. In recent years, several discoveries have been made in non-standard mammalian species providing new insights on the natural mechanisms of cancer resistance. These include mechanisms of cancer resistance in the naked mole rat, blind mole rat and elephant. In each of these species, evolution took a different path leading to novel mechanisms. Many other long-lived mammalian species display cancer resistance, including whales, grey squirrels, microbats, cows and horses. Understanding the molecular mechanisms of cancer resistance in all of these species is important and timely as ultimately, these mechanisms could be harnessed for the development of human cancer therapies.

We examined two image-based methods, photogrammetry and stereo vision, used for reconstructing the threedimensional form of biological organisms under field conditions. We also developed and tested a third ‘hybrid’ method, which combines the other two techniques. We tested these three methodologies using two different cameras to obtain digital images of museum and field sampled specimens of giant tortoises. Both the precision and repeatability of the methods were assessed statistically on the same specimens by comparing geodesic and Euclidean measurements made on the digital models with linear measurements obtained with caliper and flexible tape. We found no substantial difference between the three methods in measuring the Euclidean distance between landmarks, but spatially denser models (stereo vision and ‘hybrid’) were more accurate for geodesic distances. The use of different digital cameras did not influence the results. Image-based methods require only inexpensive instruments and appropriate software, and allow reconstruction of the three-dimensional forms (including their curved surfaces) of organisms sampled in the field. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 425–436.

The Magdalena River Turtle (Podocnemis lewyana) is a Colombian endemic species, endangered due to human exploitation and habitat destruction. To date, this species is poorly known ecologically and data on its genetic diversity are lacking. Here we report on the first genetic survey of the species across its distribution range. We obtained mitochondrial DNA sequences (488 bp) of the cytochrome b gene from 109 individuals. Samples belong to populations located at several different localities, grouped in five regions, along the four main river basins: Magdalena, Cauca, San Jorge, and Sinú drainages. We found two haplotypes, which differ in only one nucleotide substitution and which are represented with different frequencies in the five geographic regions. These results suggest that P. lewyana harbors little genetic variation and is a genetically uniform species, but more variable markers (i.e., microsatellites) should be used to unravel fine-scale phylogeographic structures in this species.

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.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12862-015-0329-x) contains supplementary material, which is available to authorized users.

Aim Amphibians are a model group for studies of the biogeographical origins of salt-intolerant taxa on oceanic islands. We used the Gulf of Guinea islands to explore the biogeographical origins of island endemism of one species of frog, and used this to gain insights into potential colonization mechanisms. Location Sao Tome and Principe, two of the four major islands in the Gulf of Guinea, West Africa, are truly oceanic and have an exceptionally high biodiversity. Methods Mitochondrial DNA is used to test the endemic status of a frog from Sao Tome and compare it with congeneric taxa from tropical Africa. Existing data on surface currents, surface salinity, atmospheric circulation and bird migration in the Gulf of Guinea are summarized to address hypotheses concerning colonization mechanisms. Results The endemic status of Ptychadena newtoni (Bocage) is supported here by mitochondrial DNA sequences, and analysis of this and other molecular data indicates that an East African species close to Ptychadena mascareniensis (Dumeril and Bibron) is its nearest relative. We refute the possibility that this population was anthropogenically introduced, in favour of a natural dispersal mechanism. Main conclusions With six endemic frogs and one caecilian, the Gulf of Guinea islands harbour a diverse amphibian fauna. Five of these species appear to have their closest relatives in East Africa. Insufficient evidence exists for transportation by storms, birds or rafts alone. However, we propose a synergy of rafting, favourable surface currents and a reduction in salinity of surface waters. Catastrophic events, or wet periods in climatic history, could allow freshwater paths to open far enough to enable continental flora and fauna to reach these and other isolated oceanic islands.

Mantella bernhardi is an endemic species of Malagasy poison frog threatened by loss and fragmentation of its natural habitat and collection for the pet trade. It is classified as threatened according to the International Union for Conservation of Nature and Natural Resources (IUCN) categories and included in Appendix II of the Convention on the International Trade of Endangered Species (CITES). A recent survey has increased the known distributional range of the species from one to eight populations across southeastern Madagascar, but little is known about its biology and genetic diversity. Here we estimate inter- and intrapopulation mitochondrial genetic variation of four populations. Populations from the northern and southern parts of the distributional range showed a high degree of divergence (maximum of 11.35% in cytochrome b) and were recovered as reciprocally monophyletic groups. Nine haplotypes were detected in the northern and 12 in the southern populations. The population from Ranomafana National Park showed the lowest number of haplotypes and nucleotide diversity, and shared its most common haplotype with the second northern population from Tolongoina. All the other detected haplotypes were unique to each of the four populations. This suggests the existence of important barriers to gene flow, pre-dating human colonization of Madagascar at about 2000 years ago, in distinct contrast to other Mantella species that show a high degree of haplotype sharing throughout their range. The continued habitat fragmentation within the distribution range of M. bernhardi prevents any connection between its populations. Our data indicate the existence of at least two different management units for conservation in this species, corresponding to the North and South of its distribution range, and highlight the existence of strong regional endemism in southeastern Madagascar.

We investigated the degree and distribution of the genetic variation, and phylogeography, of two species of Malagasy poison frogs, Mantella cowani and M. baroni. The former is critically endangered due to its restricted distribution, habitat destruction and overcollection for the pet trade. Analysis of 526 bp of mtDNA (cytochrome b) resulted in separate haplotype networks for the two species, and discovered hybridization at a single locality. The two networks confirm the status of M. baroni and M. cowani as separate evolutionary species and units for conservation. Within both mitochondrial haplotype networks, specimens from different localities shared numerous identical haplotypes, even those from the most distant sample sites of M. baroni. Most populations were characterized by high haplotype diversity and no haplotype clades exclusive to geographical regions were observed. Protection of a few large populations of these species is therefore likely to conserve much of the mtDNA genetic diversity found in the entire species. While M. baroni is widespread and occurs in many nature reserves, we recommend efficient legal protection of some M. cowani habitats to protect this species against extinction.

Malagasy poison frogs of the genus Mantella are diurnal and toxic amphibians of highly variable and largely aposematic coloration. Previous studies provided evidence for several instances of homoplastic colour evolution in this genus but were unable to sufficiently resolve relationships among major species groups or to clarify the phylogenetic position of several crucial taxa. Here, we provide cytochrome b data for 143 individuals of three species in the Mantella madagascariensis group, including four newly discovered populations. Three of these new populations are characterized by highly variable coloration and patterns but showed no conspicuous increase of haplotype diversity which would be expected under a scenario of secondary hybridization or admixture of chromatically uniform populations. Several populations of these variable forms and of M. crocea were geographically interspersed between the distribution areas of Mantella aurantiaca and Mantella milotympanum . This provides further support for the hypothesis that the largely similar uniformly orange colour of the last two species evolved in parallel. Phylogenies based on over 2000 bp of two nuclear genes ( Rag-1 and Rag-2 ) identified reliably a clade of the Mantella betsileo and Mantella laevigata groups as sister lineage to the M. madagascariensis group, but did not support species within the latter group as monophyletic. The evolutionary history of these frogs might have been characterized by fast and recurrent evolution of colour patterns, possibly triggered by strong selection pressures and mimicry effects, being too complex to be represented by simple bifurcating models of phylogenetic reconstruction.

The diurnal, brightly colored, and toxic frogs of the genus Mantella are among the most prominent representatives of the endemic anuran fauna of Madagascar. Especially three closely related species, M. aurantiaca, M. crocea, and M. milotympanum, are intensively collected for the pet trade, although basic data on their natural history and genetic diversity are still lacking. Our phylogenetic analyses based on 2.8 kbp of partial 16S rRNA, 12S rRNA, cytochrome b, and rhodopsin DNA sequences confirmed that these species belong to one of the five major clades in Mantella, the M. madagascariensis group. A haplotype network constructed using 830 bp of cytochrome b in 49 individuals from seven populations revealed that M. milotympanum and M. crocea have largely similar haplotypes sharing, confirming doubts about the species validity of M. milotympanum and indicating independent evolution of bright orange pattern in M. milotympanum and M. aurantiaca. Further, clustering of four individuals of M. aurantiaca from Andranomena with M. crocea suggests incomplete lineage sorting or introgression resulting from secondary contact of refugial populations. AMOVA confirmed significant intrapopulation nucleotide diversity (>20%). These diversity patterns and our field observations indicate relatively large population sizes. Hence, overcollecting is probably a minor problem and conservation efforts should rather focus on saving some large populations from habitat destruction through logging and forest fires.

International audience; Genetic diversity is the amount of variation observed between DNA sequences from distinct individuals of a given species. This pivotal concept of population genetics has implications for species health, domest-ication, management and conservation. Levels of genetic diversity seem to vary greatly in natural populations and species, but the determinants of this variation, and particularly the relative influences of species biology and ecology versus population history, are still largely mysterious 1,2. Here we show that the diversity of a species is predictable , and is determined in the first place by its ecological strategy. We investigated the genome-wide diversity of 76 non-model animal species by sequencing the transcriptome of two to ten individuals in each species. The distribution of genetic diversity between species revealed no detectable influence of geographic range or invasive status but was accurately predicted by key species traits related to parental investment: long-lived or low-fecundity species with brooding ability were genetically less diverse than short-lived or highly fecund ones. Our analysis demonstrates the influence of long-term life-history strategies on species response to short-term environmental perturbations, a result with immediate implications for conservation policies. Since the early studies of evolutionary genetics, there has been no understanding of how and why genetic diversity levels vary between species. This old puzzle, considered four decades ago as 'the central problem in population genetics'

Pyxis arachnoides is a Critically Endangered Malagasy tortoise for which little data about its natural history and genetic variation are available. This lack of knowledge complicates undertaking conservation measures considering Pyxis genetic diversity. Here, we describe six new microsatellites for Pyxis arachnoides from wild specimens and several localities, and the cross amplification of two Astrochelys radiata microsatellites. Of these microsatellites, two are monomorphic among the three distinct subspecies and a closely related species, while the remaining six are polymorphic. Microsatellite data are needed for this species to study the inter- and intra-subspecies genetic diversity, especially for the highly endangered subspecies P. a. brygooi, which currently occurs only in remote localities. The availability of polymorphic microsatellites will allow identifying the population of origin of individuals and will aid in justifying their management as separate evolutionary lineages for conservation purposes. The rationale is to preserve both evolutionary processes and adaptive potential., DRV was supported by a Spanish Ministry of Science and Innovation grant (CGL2009-10198).

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.

Molecular biology techniques are useful for taxonomic assignment, but they are not always accessible and can be expensive and time consuming to perform. Morphological methods to identify the origin of individuals could be valuable if they can be performed rapidly, accurately, and with minimal resources. In order to correctly assign the origin of individuals from two distinct tortoise lineages, we studied here the accuracy of shape statistics depending on the inclusion of different numbers of shape components. Misleading assignment may occur if an optimal balance between the number of shape variables and the number of sampled individuals is not respected, especially when more variables than specimens are available. Assignment of museum samples of unknown origin suggests that they mostly belong to only one of the two lineages.

Galápagos tortoises represent the only surviving lineage of giant tortoises that exhibit two different types of shell morphology. The taxonomy of Galápagos tortoises was initially based mainly on diagnostic morphological characters of the shell, but has been clarified by molecular studies indicating that most islands harbor monophyletic lineages, with the exception of Isabela and Santa Cruz. On Santa Cruz there is strong genetic differentiation between the two tortoise populations (Cerro Fatal and La Reserva) exhibiting domed shell morphology. Here we integrate nuclear microsatellite and mitochondrial data with statistical analyses of shell shape morphology to evaluate whether the genetic distinction and variability of the two domed tortoise populations is paralleled by differences in shell shape. Based on our results, morphometric analyses support the genetic distinction of the two populations and also reveal that the level of genetic variation is associated with morphological shell shape variation in both populations. The Cerro Fatal population possesses lower levels of morphological and genetic variation compared to the La Reserva population. Because the turtle shell is a complex heritable trait, our results suggest that, for the Cerro Fatal population, non-neutral loci have probably experienced a parallel decrease in variability as that observed for the genetic data.

6 páginas, 2 figuras, 2 tablas.-- et al., We are thankful to the Italian Ministero dell’Ambiente e della Tutela del Territorio (Prot.DPN-2007-0014603, DPN-2008- 0008210, DPN-2006-7546 and 9333-03/04/2007) for issuing the permits for tissue collection. Field work has been supported by a grant of the Societas Europea Herpetologica to A.vdM., Y.C., M.M., M.V., S.C. was supported by grant CGL2005-06876/BOS from the Ministerio de Educación y Ciencia, Spain. This is the ISE-M publication ISEM 2008-128 of Y.C.

Background The Recombination Activating Proteins, RAG1 and RAG2, play a crucial role in the immune response in vertebrates. Among the nuclear markers currently used for phylogenetic purposes, Rag1 has especially enjoyed enormous popularity, since it successfully contributed to elucidating the relationships among and within a large variety of vertebrate lineages. We here report on a comparative investigation of the genetic variation, base composition, presence of indels, and selection in Rag1 in different vertebrate lineages (Actinopterygii, Amphibia, Aves, Chondrichthyes, Crocodylia, Lepidosauria, Mammalia, and Testudines) through the analysis of 582 sequences obtained from Genbank. We also analyze possible differences between distinct parts of the gene with different type of protein functions. Results In the vertebrate lineages studied, Rag1 is over 3 kb long. We observed a high level of heterogeneity in base composition at the 3rd codon position in some of the studied vertebrate lineages and in some specific taxa. This result is also paralleled by taxonomic differences in the GC content at the same codon position. Moreover, positive selection occurs at some sites in Aves, Lepidosauria and Testudines. Indels, which are often used as phylogenetic characters, are more informative across vertebrates in the 5' than in the 3'-end of the gene. When the entire gene is considered, the use of indels as phylogenetic character only recovers one major vertebrate clade, the Actinopterygii. However, in numerous cases insertions or deletions are specific to a monophyletic group. Conclusions Rag1 is a phylogenetic marker of undoubted quality. Our study points to the need of carrying out a preliminary investigation on the base composition and the possible existence of sites under selection of this gene within the groups studied to avoid misleading resolution. The gene shows highly heterogeneous base composition, which affects some taxa in particular and contains sites under positive selection in some vertebrate lineages in the 5'-end. The first part of the gene (5'-end) is more variable than the second (3'-end), and less affected by a heterogeneous base composition. However, in some vertebrate lineages the 5'-end of the gene is not yet widely used for phylogenetic studies.

The taxonomy of giant Galapagos tortoises (Chelonoidis spp.) is currently based primarily on morphological characters and island of origin. Over the last decade, compelling genetic evidence has accumulated for multiple independent evolutionary lineages, spurring the need for taxonomic revision. On the island of Santa Cruz there is currently a single named species, C. porteri. Recent genetic and morphological studies have shown that, within this taxon, there are two evolutionarily and spatially distinct lineages on the western and eastern sectors of the island, known as the Reserva and Cerro Fatal populations, respectively. Analyses of DNA from natural populations and museum specimens, including the type specimen for C. porteri, confirm the genetic distinctiveness of these two lineages and support elevation of the Cerro Fatal tortoises to the rank of species. In this paper, we identify DNA characters that define this new species, and infer evolutionary relationships relative to other species of Galapagos tortoises.

Background The genus Mantella, endemic poison frogs of Madagascar with 16 described species, are known in the field of international pet trade and entered under the CITES control for the last four years. The phylogeny and phylogeography of this genus have been recently subject of study for conservation purposes. Here we report on the studies of the phylogeography of the Mantella cowani group using a fragment of 453 bp of the mitochondrial cytochrome b gene from 195 individuals from 21 localities. This group is represented by five forms: M. cowani, a critically endangered species, a vulnerable species, M. haraldmeieri, and the non-threatened M. baroni, M. aff. baroni, and M. nigricans. Results The Bayesian phylogenetic and haplotype network analyses revealed the presence of three separated haplotype clades: (1) M. baroni, M. aff. baroni, M. nigricans, and putative hybrids of M. cowani and M. baroni, (2) M. cowani and putative hybrids of M. cowani and M. baroni, and (3) M. haraldmeieri. The putative hybrids were collected from sites where M. cowani and M. baroni live in sympatry. Conclusion These results suggest (a) a probable hybridization between M. cowani and M. baroni, (b) a lack of genetic differentiation between M. baroni/M. aff. baroni and M. nigricans, (c) evidence of recent gene-flow between the northern (M. nigricans), eastern (M. baroni), and south-eastern (M. aff. baroni) forms of distinct coloration, and (d) the existence of at least three units for conservation in the Mantella cowani group.