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.

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.

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.

Priority screening of contaminants of emerging concern (CECs) in surface water: Comparing cell-based bioassays and exposure-activity ratios (EARs).

In this study, we compared a wide range of cell-based bioassays to the use of chemical analysis followed by exposure-activity ratio (EAR) and Toxicological Prioritization index (ToxPi) for prioritizing chemicals, sites, and hazard concerns in water samples. Surface water samples were collected from nine sites in three Central Pennsylvania streams and analyzed for a forty-six contaminants of emerging concern (CECs), including pesticides, personal care products, and pharmaceuticals. Cell-based reporter assays evaluated human and zebrafish molecular initiating events (MIEs) in endocrine and metabolic disruption, altered lipid metabolism, and oxidative stress. Bioassays showed that 12 out of 40 assays had at least one site with activity over the effect-based trigger (EBT) values. The receptors that exhibited the highest number of samples above the EBT that would be expected to cause toxicity were Aryl hydrocarbon receptor (AhR, human and zebrafish), Pregnane X Receptor (PXR), Estrogen Receptor-beta (ERB), and Androgen Receptor (AR). Characterizing the collection sites by their bioactivity aligned closely with the stream in which samples were collected. The sum of all EARs for each chemical indicated that the pharmaceutical Carbamazepine and the pesticides Carbaryl and Atrazine posed the greatest concern. However, predicted activity and site prioritization based on individual chemical analysis and calculated EAR were different than those measured by bioassay, indicating that biologically active chemicals are present in the samples that were not included in the targeted analytes. Taken together, these data show that chemical analysis and EAR analysis are beneficial for prioritization of chemicals, whereas mechanism-based bioassays are more inclusive of known as well as unknown chemical contaminants and thus of more use for overall water quality analysis and site prioritization.

Chemical prioritization of pharmaceuticals and personal care products in an urban tributary of the Potomac River.

Pharmaceuticals and personal care products (PPCPs) are incredibly diverse in terms of chemical structures, physicochemical properties, and modes of action, making their environmental impacts challenging to assess. New chemical prioritization methodologies have emerged that compare contaminant monitoring concentrations to multiple toxicity data sources, including whole organism and high-throughput data, to develop a list of "high priority" chemicals requiring further study. We applied such an approach to assess PPCPs in Hunting Creek, an urban tributary of the Potomac River near Washington, DC, which has experienced extensive human population growth. We estimated potential risks of 99 PPCPs from surface water and sediment collected upstream and downstream of a major wastewater treatment plant (WWTP), nearby combined sewer overflows (CSO), and in the adjacent Potomac River. The greatest potential risks to the aquatic ecosystem occurred near WWTP and CSO outfalls, but risk levels rapidly dropped below thresholds of concern - established by previous chemical prioritization studies - in the Potomac mainstem. These results suggest that urban tributaries, rather than larger rivers, are important to monitor because their lower or intermittent flow may not adequately dilute contaminants of concern. Common psychotropics, such as fluoxetine and venlafaxine, presented the highest potential risks, with toxicity quotients often > 10 in surface water and > 1000 in sediment, indicating the need for further field studies. Several ubiquitous chemicals such as caffeine and carbamazepine also exceeded thresholds of concern throughout our study area and point to specific neurotoxic and endocrine modes of action that warrant further investigation. Since many "high priority" chemicals in our analysis have also triggered concerns in other areas around the world, better coordination is needed among environmental monitoring programs to improve global chemical prioritization efforts.

Critical review of the phytohemagglutinin assay for assessing amphibian immunity.

Infectious diseases are a major driver of the global amphibian decline. In addition, many factors, including genetics, stress, pollution, and climate change can influence the response to pathogens. Therefore, it is important to be able to evaluate amphibian immunity in the laboratory and in the field. The phytohemagglutinin (PHA) assay is an inexpensive and relatively non-invasive tool that has been used extensively to assess immunocompetence, especially in birds, and more recently in amphibians. However, there is substantial variation in experimental methodology among amphibian PHA studies in terms of species and life stages, PHA doses and injection sites, and use of experimental controls. Here, we compile and compare all known PHA studies in amphibians to identify knowledge gaps and develop best practices for future work. We found that research has only been conducted on a limited number of species, which may not reflect the diversity of amphibians. There is also a lack of validation studies in most species, so that doses and timing of PHA injection and subsequent swelling measurements may not effectively evaluate immunocompetence. Based on these and other findings, we put forward a set of recommendations to make future PHA studies more consistent and improve the ability to utilize this assay in wild populations, where immune surveillance is greatly needed.

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.

Variation in copper sensitivity between laboratory and wild strains of Caenorhabditis elegans.

Ecological risk assessments of chemicals are frequently based on laboratory toxicity data from a small number of model species that may be reared in labs for years or decades. These populations can undergo many processes in the lab including artificial selection, founder effect, and genetic drift, and may not adequately represent their wild counterparts, potentially undermining the goal of protecting natural populations. Here we measure variation in lethality to copper chloride among strains of an emerging model species in toxicology, Caenorhabditis elegans. We tested four wild strains from Chile, Germany, Kenya, and Madeira (Portugal) against several versions of the standard laboratory N2 strain from Bristol, UK used in molecular biology. The four wild strains were more sensitive than any of the N2 strains tested with copper. We also found that the standard N2 strain cultured in the laboratory for >1 year was less sensitive than a recently cultured N2 strain as well as a cataloged ancestral version of the N2 strain. These results suggest that toxicologists should be cognizant of performing toxicity testing with long-held animal cultures, and should perhaps use multiple strains as well as renew cultures periodically in the laboratory. This study also shows that multi-strain toxicity testing with nematodes is highly achievable and useful for understanding variation in intra- and interspecific chemical sensitivity.

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.

Historical DNA analysis reveals living descendants of an extinct species of Galápagos tortoise.

Giant tortoises, a prominent symbol of the Galápagos archipelago, illustrate the influence of geological history and natural selection on the diversification of organisms. Because of heavy human exploitation, 4 of the 15 known species (Geochelone spp.) have disappeared. Charles Darwin himself detailed the intense harvesting of one species, G. elephantopus, which once was endemic to the island of Floreana. This species was believed to have been exterminated within 15 years of Darwin's historic visit to the Galápagos in 1835. The application of modern DNA techniques to museum specimens combined with long-term study of a system creates new opportunities for identifying the living remnants of extinct taxa in the wild. Here, we use mitochondrial DNA and microsatellite data obtained from museum specimens to show that the population on Floreana was evolutionarily distinct from all other Galápagos tortoise populations. It was demonstrated that some living individuals on the nearby island of Isabela are genetically distinct from the rest of the island's inhabitants. Surprisingly, we found that these "non-native" tortoises from Isabela are of recent Floreana ancestry and closely match the genetic data provided by the museum specimens. Thus, we show that the genetic line of G. elephantopus has not been completely extinguished and still exists in an intermixed population on Isabela. With enough individuals to commence a serious captive breeding program, this finding may help reestablish a species that was thought to have gone extinct more than a century ago and illustrates the power of long-term genetic analysis and the critical role of museum specimens in conservation biology.

Intra- and interspecific toxicity testing methods and data for nematodes exposed to metals.

Twenty-four hour median lethal concentration (LC50) toxicity tests were performed with five species of nematodes (Caenorhabditis elegans, Caenorhabditis briggsae, Pristionchus pacificus, Oscheius tipulae, and Oscheius myriophila) in response to copper chloride and zinc chloride. In addition, lethality tests were also performed with seven strains of C. elegans (N2 > 1 year in culture, N2 newly acquired, N2 ancestral, ED3053, JU258, JU1171, and MY1) exposed to copper chloride. Nominal chemical concentrations were validated and analyzed according to U.S. Environmental Protection Agency method 6010 using inductively coupled plasma-atomic emission spectroscopy (ICP-AES). This paper combines the datasets previously published separately by Heaton et al. (2020, 2022). The goal is to catalog all raw and analyzed toxicity data collected from both studies in a single consistent information source for use by the scientific community.

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.

Genetic differentiation between marine iguanas from different breeding sites on the island of Santa Fe (Galapagos Archipelago).

We studied patterns of genetic diversity within and among 5 populations (318 individuals) of Galápagos marine iguanas (Amblyrhynchus cristatus) from the island Santa Fé. Populations were separated by distances of 0.2 to 9.9 km. We sequenced 1182 base pairs of the mitochondrial control region and screened 13 microsatellite loci for variability. We also added data from 5 populations (397 individuals) sampled on 4 neighboring islands (Santa Cruz, Floreana, Espanola, and San Cristobal). The 5 Santa Fé populations, revealed as genetically distinct from populations on other islands, present relatively low levels of genetic diversity, which are similar for both microsatellite (average observed heterozygosity from 0.7686 to 0.7773) and mitochondrial DNA (mtDNA) markers (haplotypic and nucleotide diversity from 0.587 to 0.728 and from 0.00079 to 0.00293, respectively), and comparable with those observed in similar-sized sampling sites on other islands. There was frequency-based evidence of genetic structure between northern and southern sites on Santa Fé (F(st) of 0.0027-0.0115 for microsatellite and 0.0447-0.2391 for mtDNA), but the 4 southern sites showed little differentiation. Most of the intra-island genetic variation was allocated within rather than between sites. There was no evidence of sex-biased dispersal or population substructuring due to lek-mating behavior, suggesting that these 2 observed behaviors are not strong enough to leave an evolutionary signal on genetic patterns in this species.

Interspecific Variation in Nematode Responses to Metals.

Performing toxicity testing on multiple species with differing degrees of evolutionary relatedness can provide important information on how chemical sensitivity varies among species and can help pinpoint the biological drivers of species sensitivity. Such knowledge could ultimately be used to design better multispecies predictive ecological risk assessment models and identify particularly sensitive species. However, laboratory toxicity tests involving multiple species can also be resource intensive, especially when each species has unique husbandry conditions. We performed lethality tests with 2 metals, copper chloride and zinc chloride, on 5 different nematode species, which are nested in their degree of evolutionary relatedness: Caenorhabditis briggsae, Caenorhabditis elegans, Oscheius myriophila, Oscheius tipulae, and Pristionchus pacificus. All species were successfully cultured and tested concurrently with limited resources, demonstrating that inexpensive, multispecies nematode toxicity testing systems are achievable. The results indicate that P. pacificus is the most sensitive to both metals. Conversely, C. elegans is the least sensitive species to copper, but the second most sensitive to zinc, indicating that species relationships do not necessarily predict species sensitivity. Toxicity testing with additional nematode species and types of chemicals is feasible and will help form more generalizable conclusions about relative species sensitivity. Environ Toxicol Chem 2020;39:1006-1016. © 2020 SETAC.

Progressive colonization and restricted gene flow shape island-dependent population structure in Galápagos marine iguanas (Amblyrhynchus cristatus).

BACKGROUND: Marine iguanas (Amblyrhynchus cristatus) inhabit the coastlines of large and small islands throughout the Galápagos archipelago, providing a rich system to study the spatial and temporal factors influencing the phylogeographic distribution and population structure of a species. Here, we analyze the microevolution of marine iguanas using the complete mitochondrial control region (CR) as well as 13 microsatellite loci representing more than 1200 individuals from 13 islands. RESULTS: CR data show that marine iguanas occupy three general clades: one that is widely distributed across the northern archipelago, and likely spread from east to west by way of the South Equatorial current, a second that is found mostly on the older eastern and central islands, and a third that is limited to the younger northern and western islands. Generally, the CR haplotype distribution pattern supports the colonization of the archipelago from the older, eastern islands to the younger, western islands. However, there are also signatures of recurrent, historical gene flow between islands after population establishment. Bayesian cluster analysis of microsatellite genotypes indicates the existence of twenty distinct genetic clusters generally following a one-cluster-per-island pattern. However, two well-differentiated clusters were found on the easternmost island of San Cristóbal, while nine distinct and highly intermixed clusters were found on youngest, westernmost islands of Isabela and Fernandina. High mtDNA and microsatellite genetic diversity were observed for populations on Isabela and Fernandina that may be the result of a recent population expansion and founder events from multiple sources. CONCLUSIONS: While a past genetic study based on pure FST analysis suggested that marine iguana populations display high levels of nuclear (but not mitochondrial) gene flow due to male-biased dispersal, the results of our sex-biased dispersal tests and the finding of strong genetic differentiation between islands do not support this view. Therefore, our study is a nice example of how recently developed analytical tools such as Bayesian clustering analysis and DNA sequence-based demographic analyses can overcome potential biases introduced by simply relying on FST estimates from markers with different inheritance patterns.

Evaluating the role of fish as surrogates for amphibians in pesticide ecological risk assessment.

Ecological risk of chemicals to aquatic-phase amphibians has historically been evaluated by comparing estimated environmental concentrations in surface water to surrogate toxicity data from fish species. Despite their obvious similarities, there are biological disparities among fish and amphibians that could affect their exposure and response to chemicals. Given the alarming decline in amphibians, in which anthropogenic pollutants play at least some role, investigating the risk of chemicals to amphibians is becoming increasingly important. Here, we evaluate relative sensitivity of fish and larval aquatic-phase amphibians to 45 different pesticides using existing data from three standardized toxicity test designs: (1) amphibian metamorphosis assay (AMA) with the African clawed frog (Xenopus laevis); (2) fish short-term reproduction assay (FSTRA) with the fathead minnow (Pimephales promelas); (3) fish early life stage test (ELS) with fathead minnows or rainbow trout (Oncorhynchus mykiss). The advantage of this dataset over previous work is that the underlying studies are consistent in exposure method, study duration, test species, endpoints measured, and number of concentrations tested. We found very strong positive relationships between fish and frog lowest adverse effect concentrations (LOAEC) for survival [Spearman's rank correlation (rs) = 0.88], body weight (rs = 0.86), and length (rs = 0.89) with only one out of 45 chemicals (propiconazole) exhibiting 100-folder greater sensitivity in frogs relative to fish. While our results suggest comparable toxicity for pesticides between fish and aquatic-phase amphibians under these test conditions, further research with a greater diversity of amphibians and exposure scenarios will help determine the relevance of these results across species and life stages.

Sialic acid on avian erythrocytes.

Understanding variation in physiological traits across taxa is a central question in evolutionary biology that has wide-ranging implications in biomedicine, disease ecology, and environmental protection. Sialic acid (Sia), and in particular, 5-N-acetylneuraminic acid (Neu5Ac), is chemically bound to galactose and the underlying glycan via α2-3 or α2-6 glycosidic linkage (i.e., Siaα2-3Galactose or Siaα2-6Galactose), conferring two different cell surface structures that affects cell to cell communication and interactions with foreign agents including microparasites and toxins. As an initial step towards understanding variation of Sia across the class Aves, we collected red blood cells (RBCs or erythrocytes) and measured Sia quantity in 76 species and 340 individuals using HPLC-MS/MS and glycosidic linkage type in 24 species and 105 individuals using hemagglutination assay. Although Sia quantity did not, α2-6 glycosidic linkage did exhibit a discernable phylogenetic pattern as evaluated by a phylogenetic signal (λ) value of 0.7. Sia quantity appeared to be higher in after hatch year birds than hatch year birds (P < 0.05); moreover, ~80% of the measured Sia across all individuals or species was expressed by ~20% of the individuals or species. Lastly, as expected, we detected a minimal presence of 5-N-glycolylneuraminic acid in the avian RBCs tested. These data provide novel insights and a large baseline dataset for further study on the variability of Sia in the class Aves which might be useful for understanding Sia dependent processes in birds.

Giant tortoise genomes provide insights into longevity and age-related disease.

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.