The inference of gray whale (Eschrichtius robustus) historical population attributes from whole-genome sequences.

BACKGROUND: Commercial whaling caused extensive demographic declines in many great whale species, including gray whales that were extirpated from the Atlantic Ocean and dramatically reduced in the Pacific Ocean. The Eastern Pacific gray whale has recovered since the 1982 ban on commercial whaling, but the Western Pacific gray whale-once considered possibly extinct-consists of only about 200 individuals and is considered critically endangered by some international authorities. Herein, we use whole-genome sequencing to investigate the demographic history of gray whales from the Pacific and use environmental niche modelling to make predictions about future gene flow. RESULTS: Our sequencing efforts and habitat niche modelling indicate that: i) western gray whale effective population sizes have declined since the last glacial maximum; ii) contemporary gray whale genomes, both eastern and western, harbor less autosomal nucleotide diversity than most other marine mammals and megafauna; iii) the extent of inbreeding, as measured by autozygosity, is greater in the Western Pacific than in the Eastern Pacific populations; and iv) future climate change is expected to open new migratory routes for gray whales. CONCLUSION: Our results indicate that gray whale genomes contain low nucleotide diversity and have been subject to both historical and recent inbreeding. Population sizes over the last million years likely peaked about 25,000 years before present and have declined since then. Our niche modelling suggests that novel migratory routes may develop within the next century and if so this could help retain overall genetic diversity, which is essential for adaption and successful recovery in light of global environmental change and past exploitation.

Genetic data reveal mixed-stock aggregations of gray whales in the North Pacific Ocean.

Gray whales (Eschrichtius robustus) in the Western Pacific are critically endangered, whereas in the Eastern Pacific, they are relatively common. Holocene environmental changes and commercial whaling reduced their numbers, but gray whales in the Eastern Pacific now outnumber their Western counterparts by more than 100-fold. Herein, we investigate the genetic diversity and population structure within the species using a panel of genic single nucleotide polymorphisms. Results indicate the gray whale gene pool is differentiated into two substocks containing similar levels of genetic diversity, and that both our Eastern and Western geographical samples represent mixed-stock aggregations. Ongoing or future gene flow between the stocks may conserve genetic diversity overall, but admixture has implications for conservation of the critically endangered Western gray whale.

Patterns of genome size diversity in bats (order Chiroptera).

Despite being a group of particular interest in considering relationships between genome size and metabolic parameters, bats have not been well studied from this perspective. This study presents new estimates for 121 "microbat" species from 12 families and complements a previous study on members of the family Pteropodidae ("megabats"). The results confirm that diversity in genome size in bats is very limited even compared with other mammals, varying approximately 2-fold from 1.63 pg in Lophostoma carrikeri to 3.17 pg in Rhinopoma hardwickii and averaging only 2.35 pg ± 0.02 SE (versus 3.5 pg overall for mammals). However, contrary to some other vertebrate groups, and perhaps owing to the narrow range observed, genome size correlations were not apparent with any chromosomal, physiological, flight-related, developmental, or ecological characteristics within the order Chiroptera. Genome size is positively correlated with measures of body size in bats, though the strength of the relationships differs between pteropodids ("megabats") and nonpteropodids ("microbats").

DNA damage in cichlids from an oil production facility in Guatemala.

This study focused on several wetlands in Laguna del Tigre National Park (Guatemala) as part of Conservation International's Rapid Assessment Program. Sediment and water samples were collected from a laguna near Xan field, Guatemala's largest oil facility, and three other sites for determination of levels of polycyclic aromatic hydrocarbons (PAHs). Cichlid fish (Thorichthys meeki and Vieja synspila) were collected for determination of DNA strand breakage (by gel electrophoresis), chromosomal breakage (flow cytometry), and fin erosion. For T. meeki from Xan field, chromosomal breakage and strand breakage was greater than in at least two of the three reference sites. For V. synspila, chromosomal breakage and strand breakage were greater in Xan than one of the two reference sites. Fin erosion was observed only at the Xan laguna. Genetic biomarker effects and fin erosion, along with patterns of aqueous PAH concentrations, indicate that fish are affected by anthropogenic contaminants. PAHs were elevated at some reference sites, but environmental forensic analysis suggested a pyrogenic or diagenic origin. It is possible that oil field brines injected into the ground water caused fin erosion and genotoxicity in fish at Xan field, and it is also possible that pyrogenic PAHs influence levels of DNA damage in reference sites. These analyses represent one of the first efforts to examine genotoxicity in native Mesoamerican cichlids.

The four cornerstones of Evolutionary Toxicology.

Evolutionary Toxicology is the study of the effects of chemical pollutants on the genetics of natural populations. Research in Evolutionary Toxicology uses experimental designs familiar to the ecotoxicologist with matched reference and contaminated sites and the selection of sentinel species. It uses the methods of molecular genetics and population genetics, and is based on the theories and concepts of evolutionary biology and conservation genetics. Although it is a relatively young field, interest is rapidly growing among ecotoxicologists and more and more field studies and even controlled laboratory experiments are appearing in the literature. A number of population genetic impacts have been observed in organisms exposed to pollutants which I refer to here as the four cornerstones of Evolutionary Toxicology. These include (1) genome-wide changes in genetic diversity, (2) changes in allelic or genotypic frequencies caused by contaminant-induced selection acting at survivorship loci, (3) changes in dispersal patterns or gene flow which alter the genetic relationships among populations, and (4) changes in allelic or genotypic frequencies caused by increased mutation rates. It is concluded that population genetic impacts of pollution exposure are emergent effects that are not necessarily predictable from the mode of toxicity of the pollutant. Thus, to attribute an effect to a particular contaminant requires a careful experimental design which includes selection of appropriate reference sites, detailed chemistry analyses of environmental samples and tissues, and the use of appropriate biomarkers to establish exposure and effect. This paper describes the field of Evolutionary Toxicology and discusses relevant field studies and their findings.

Evolutionary toxicology: contaminant-induced genetic mutations in mosquitofish from Sumgayit, Azerbaijan.

This study builds on a long-term program that has shown Sumgayit, Azerbaijan to contain wetlands with high levels of a diversity of chemical contaminants. Previous contaminant and biomarker studies of turtles and frogs showed a correlation between somatic chromosomal damage and chemical contaminants at Sumgayit. The objective of this study was to determine if a recently arrived species (mosquitofish) has genetic impacts similar to native species (marsh frogs) thus confirming the pattern is not the result of historical events such as glacial cycles, but is associated with recent chemical contamination. Nucleotide sequences of the mtDNA control region of invasive mosquitofish (Gambusia holbrooki) from Sumgayit were compared to mosquitofish from pristine sites in Europe and Azerbaijan and to native North American populations. Persistent heteroplasmy for a hyper-mutable simple sequence repeat and low haplotype and nucleotide diversities were observed in all invasive populations. However, Sumgayit possessed four de novo haplotypes and heteroplasmic conditions. All of the observed variable nucleotide positions were within or adjacent to a cytosine mononucleotide repeat. This repeat was within a conserved secondary structure; the region likely undergoes expansion and contraction at a rate sufficient to prevent fixation of the common 1/3 heteroplasmy. Whereas the 1/3 heteroplasmy appeared coincident with the establishment of mosquitofish in Europe, other forms of heteroplasmy resulted from contaminant-induced de novo mutations in Sumgayit. We conclude that Sumgayit is a mutational hotspot caused by legacy contaminants from chemical factories from the era of the Soviet Union.

Genotoxicity in Atlantic killifish (Fundulus heteroclitus) from a PAH-contaminated Superfund site on the Elizabeth River, Virginia.

The Atlantic Wood Industries Superfund site (AWI) on the Elizabeth River in Portsmouth, VA is heavily contaminated with polycyclic aromatic hydrocarbons (PAHs) from a wood treatment facility. Atlantic killifish, or mummichog (Fundulus heteroclitus), at this Superfund site are exposed to very high concentrations of several carcinogens. In this study, we measured PAH concentrations in both fish tissues and sediments. Concurrently, we assessed different aspects of genotoxicity in the killifish exposed in situ. Both sediment and tissue PAH levels were significantly higher in AWI samples, relative to a reference site, but the chemistry profile was different between sediments and tissues. Killifish at AWI exhibited higher levels of DNA damage compared to reference fish, as measured via the flow cytometric method (FCM), and the damage was consistent with sediment PAH concentrations. Covalent binding of benzo[a]pyrene (BaP) metabolites to DNA, as measured via LC-MS/MS adduct detection methods, were also elevated and could be partially responsible for the DNA damage. Using similar LC-MS/MS methods, we found no evidence that oxidative DNA adducts had a role in observed genotoxicity.

The effects of silver nanoparticles on fathead minnow (Pimephales promelas) embryos.

Nanoparticles are being used in many commercial applications. We describe the toxicity of two commercial silver (Ag) nanoparticle (NP) products, NanoAmor and Sigma on Pimephales promelas embryos. Embryos were exposed to varying concentrations of either sonicated or stirred NP solutions for 96 h. LC(50) values for NanoAmor and Sigma Ag NPs were 9.4 and 10.6 mg/L for stirred and 1.25 and 1.36 mg/L for sonicated NPs, respectively. Uptake of Ag NPs into the embryos was observed after 24 h using Transmission Electron Microscopy and Ag NPs induced a concentration-dependent increase in larval abnormalities, mostly edema. Dissolved Ag released from Ag NPs was measured using Inductively Coupled-Mass Spectrometry and the effects tested were found to be three times less toxic when compared to Ag nitrate (AgNO(3)). The percentage of dissolved Ag released was inversely proportional to the concentration of Ag NPs with the lowest (0.625 mg/L) and highest (20 mg/L) concentrations tested releasing 3.7 and 0.45% dissolved Ag, respectively and percent release was similar regardless if concentrations were stirred or sonicated. Thus increased toxicity after sonication cannot be solely explained by dissolved Ag. We conclude that both dissolved and particulate forms of Ag elicited toxicity to fish embryos.

Bioenergy sustainability in China: potential and impacts.

The sustainability implications of bioenergy development strategies are large and complex. Unlike conventional agriculture, bioenergy production provides an opportunity to design systems for improving eco-environmental services. Different places have different goals and solutions for bioenergy development, but they all should adhere to the sustainability requirements of the environment, economy, and society. This article serves as a brief overview of China's bioenergy development and as an introduction to this special issue on the impacts of bioenergy development in China. The eleven articles in this special issue present a range of perspectives and scenario analyses on bioenergy production and its impacts as well as potential barriers to its development. Five general themes are covered: status and goals, biomass resources, energy plants, environmental impacts, and economic and social impacts. The potential for bioenergy production in China is huge, particularly in the central north and northwest. China plans to develop a bioenergy capacity of 30GW by 2020. However, realization of this goal will require breakthroughs in bioenergy landscape design, energy plant biotechnology, legislation, incentive policy, and conversion facilities. Our analyses suggest that (1) the linkage between bioenergy, environment, and economy are often circular rather than linear in nature; (2) sustainability is a core concept in bioenergy design and the ultimate goal of bioenergy development; and (3) each bioenergy development scheme must be region-specific and designed to solve local environmental and agricultural problems.

Determinants of genetic variation across eco-evolutionary scales in pinnipeds.

The effective size of a population (Ne), which determines its level of neutral variability, is a key evolutionary parameter. Ne can substantially depart from census sizes of present-day breeding populations (NC) as a result of past demographic changes, variation in life-history traits and selection at linked sites. Using genome-wide data we estimated the long-term coalescent Ne for 17 pinniped species represented by 36 population samples (total n = 458 individuals). Ne estimates ranged from 8,936 to 91,178, were highly consistent within (sub)species and showed a strong positive correlation with NC ([Formula: see text] = 0.59; P = 0.0002). Ne/NC ratios were low (mean, 0.31; median, 0.13) and co-varied strongly with demographic history and, to a lesser degree, with species' ecological and life-history variables such as breeding habitat. Residual variation in Ne/NC, after controlling for past demographic fluctuations, contained information about recent population size changes during the Anthropocene. Specifically, species of conservation concern typically had positive residuals indicative of a smaller contemporary NC than would be expected from their long-term Ne. This study highlights the value of comparative population genomic analyses for gauging the evolutionary processes governing genetic variation in natural populations, and provides a framework for identifying populations deserving closer conservation attention.

Speciation by monobrachial centric fusions: a test of the model using nuclear DNA sequences from the bat genus Rhogeessa.

Members of Rhogeessa are hypothesized to have undergone speciation via chromosomal rearrangements in a model termed speciation by monobrachial centric fusions. Recently, mitochondrial cytochrome-b sequence data tentatively supported this hypothesis but could not explicitly test the model's expectations regarding interbreeding among karyotypic forms. These data showed potential evidence for hybridization or incomplete lineage sorting between the karyotypically distinct R. tumida and R. aeneus and identified multiple lineages of karyotypically identical R. tumida. Here, we present a more comprehensive test of speciation by monobrachial centric fusions in Rhogeessa. Our analysis is based on sequence data from two nuclear loci: paternally inherited ZFY and autosomal MPI genes. These data provide results consistent either with incomplete lineage sorting or ancient hybridization to explain alleles shared at low frequency between R. aeneus and R. tumida. Recent and ongoing hybridization between any species can be ruled out. These data confirm the presence of multiple lineages of the 2n=34 karyotypic form ("R. tumida") that are not each other's closest relatives. These results are generally consistent with speciation by monobrachial centric fusions, although additional modes of speciation have also occurred in Rhogeessa. Phylogeographic analyses indicate habitat differences may be responsible for isolation and divergence between different lineages currently referred to as R. tumida.

Wildlife toxicology: biomarkers of genotoxic exposures at a hazardous waste site.

A large number of hazardous waste sites in the United States have undergone the initial stages of remediation or containment. At many of the remaining sites, the potential for exposure to ecological receptors is a primary concern. This manuscript reports on studies to investigate the impact on ecological receptors exposed to complex mixtures at a former creosote facility. Currently there are isolated areas on-site that were not addressed in the initial removal action that appear to be releasing polycyclic aromatic hydrocarbons (PAHs) to the surrounding environment. The U.S. EPA collected environmental samples and performed ex situ sediment bioassays to measure chronic toxicity; whereas, this study describes an in situ study to measure biomarkers of effect in two ecological receptors. Mosquitofish (Gambusia affinis) and cricket frogs (Acris crepitans) were collected from a small intermittent creek adjacent to the site, and reference stations. A weight-of-evidence ecological risk assessment was completed for the amphibian and fish communities. The ecological risk assessment was developed using analysis of media chemistry, body burden of specific PAHs, bioassay results, community surveys, and cellular genome size variation as a biomarker of genotoxicity. Flow cytometric estimates of chromosomal damage were significantly elevated for both mosquitofish and cricket frogs inhabiting the contaminated site, relative to at least one reference site. Surface water screening values for fish and amphibians exceeded screening values for PAHs by more than one order of magnitude in the on-site creek, and sediment PAH concentrations were extremely high (up to 1,549 microg/dry g). Tissue concentrations of PAHs were below screening values. Media chemistry, bioassay and genotoxicity data all support the same conclusion that on-site PAHs continue to impact aquatic receptors. The genotoxicity findings are consistent with and contribute to results of the weight-of-evidence ecological risk assessment. The results support continuing efforts to incorporate biomarkers as valuable lines of evidence within ecological risk assessment.

In situ biomonitoring of PAH-contaminated sediments using juvenile coho salmon (Oncorhynchus kisutch).

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous marine and freshwater sediment contaminants. Extensive data exist to confirm that PAHs are toxic to aquatic receptors. However, limited information is available regarding the bioavailability and genotoxicity of sediment PAHs to aquatic organisms. This study investigated an integrated biomonitoring approach using chemical analyses and biomarkers to characterize the bioavailability and genotoxicity of a complex PAH mixture in freshwater lake sediments associated with a former manufactured gas plant (MGP). Sediment PAH genotoxicity was assessed by flow cytometry (FCM), DNA adduct (32)P-postlabeling, and erythrocyte micronuclei in juvenile coho salmon (Oncorhynchus kisutch) caged in the water column. Significant PAH-induced genotoxicity was observed with FCM and (32)P-postlabeling, but not with erythrocyte micronuclei. Chromosome damage in peripheral blood and hepatic DNA adducts correlated with sediment, but not water column PAH concentrations. Total hepatic DNA adducts in salmon caged nearest the former MGP facility was 39+/-6.5 (RALx10(9)), while salmon caged in a reference lake had 28+/-2.3 total hepatic DNA adducts per 10(9) nucleotides. These results indicate that in situ biomonitoring using biomarkers and caged fish can be a sensitive indicator of genotoxic PAHs in sediments.

Evolutionary toxicology in an omics world.

Evolutionary toxicology is a young field that has grown rapidly in the past two decades. The potential of this field comes from the ability to link chemical contamination to multigenerational and population-wide effects in various species. The advancements and rapidly decreasing costs of -omic tools are improving the power and resolution of evolutionary toxicology studies. In this manuscript, we aim to address the trajectories and perspectives for conducting evolutionary toxicology studies with -omic approaches. We discuss the complementarity of using multiple -omic tools (genomics, eDNA, transcriptomics, proteomics, and metabolomics) for utility in understanding the toxicological relevance of adaptive responses in populations. In addition, we discuss phenotypic plasticity and its relevance to transcriptomic studies in toxicology. As evolutionary toxicology grows and expands its capacity to link toxicology with population-wide end points, we emphasize the applications of such studies in answering questions about ecological and population health, as well as future applicability to regulation. Thus, we aim to emphasize the enormous potential for evolutionary toxicology in an -omics world and give perspectives on the directions of future investigations.

Nuclear and mtDNA phylogenetic analyses clarify the evolutionary history of two species of native Hawaiian bats and the taxonomy of Lasiurini (Mammalia: Chiroptera).

Previous studies on genetics of hoary bats produced differing conclusions on the timing of their colonization of the Hawaiian Islands and whether or not North American (Aeorestes cinereus) and Hawaiian (A. semotus) hoary bats are distinct species. One study, using mtDNA COI and nuclear Rag2 and CMA1, concluded that hoary bats colonized the Hawaiian Islands no more than 10,000 years ago based on indications of population expansion at that time using Extended Bayesian Skyline Plots. The other study, using 3 mtDNA and 1 Y-chromosome locus, concluded that the Hawaiian Islands were colonized about 1 million years ago. To address the marked inconsistencies between those studies, we examined DNA sequences from 4 mitochondrial and 2 nuclear loci in lasiurine bats to investigate the timing of colonization of the Hawaiian Islands by hoary bats, test the hypothesis that Hawaiian and North American hoary bats belong to different species, and further investigate the generic level taxonomy within the tribe. Phylogenetic analysis and dating of the nodes of mtDNA haplotypes and of nuclear CMA1 alleles show that A. semotus invaded the Hawaiian Islands approximately 1.35 Ma and that multiple arrivals of A. cinereus occurred much more recently. Extended Bayesian Skyline plots show population expansion at about 20,000 years ago in the Hawaiian Islands, which we conclude does not represent the timing of colonization of the Hawaiian Islands given the high degree of genetic differentiation among A. cinereus and A. semotus (4.2% divergence at mtDNA Cytb) and the high degree of genetic diversity within A. semotus. Rather, population expansion 20,000 years ago could have resulted from colonization of additional islands, expansion after a bottleneck, or other factors. New genetic data also support the recognition of A. semotus and A. cinereus as distinct species, a finding consistent with previous morphological and behavioral studies. The phylogenetic analysis of CMA1 alleles shows the presence of 2 clades that are primarily associated with A. semotus mtDNA haplotypes, and are unique to the Hawaiian Islands. There is evidence for low levels of hybridization between A. semotus and A. cinereus on the Hawaiian Islands, but it is not extensive (<15% of individuals are of hybrid origin), and clearly each species is able to maintain its own genetic distinctiveness. Both mtDNA and nuclear DNA sequences show deep divergence between the 3 groups (genera) of lasiurine bats that correspond to the previously recognized morphological differences between them. We show that the Tribe Lasiurini contains the genera Aeorestes (hoary bats), Lasiurus (red bats), and Dasypterus (yellow bats).

Characterization of the Gray Whale Eschrichtius robustus Genome and a Genotyping Array Based on Single-Nucleotide Polymorphisms in Candidate Genes.

Genetic and genomic approaches have much to offer in terms of ecology, evolution, and conservation. To better understand the biology of the gray whale Eschrichtius robustus (Lilljeborg, 1861), we sequenced the genome and produced an assembly that contains ∼95% of the genes known to be highly conserved among eukaryotes. From this assembly, we annotated 22,711 genes and identified 2,057,254 single-nucleotide polymorphisms (SNPs). Using this assembly, we generated a curated list of candidate genes potentially subject to strong natural selection, including genes associated with osmoregulation, oxygen binding and delivery, and other aspects of marine life. From these candidate genes, we queried 92 autosomal protein-coding markers with a panel of 96 SNPs that also included 2 sexing and 2 mitochondrial markers. Genotyping error rates, calculated across loci and across 69 intentional replicate samples, were low (0.021%), and observed heterozygosity was 0.33 averaged over all autosomal markers. This level of variability provides substantial discriminatory power across loci (mean probability of identity of 1.6 × 10-25 and mean probability of exclusion >0.999 with neither parent known), indicating that these markers provide a powerful means to assess parentage and relatedness in gray whales. We found 29 unique multilocus genotypes represented among our 36 biopsies (indicating that we inadvertently sampled 7 whales twice). In total, we compiled an individual data set of 28 western gray whales (WGSs) and 1 presumptive eastern gray whale (EGW). The lone EGW we sampled was no more or less related to the WGWs than expected by chance alone. The gray whale genomes reported here will enable comparative studies of natural selection in cetaceans, and the SNP markers should be highly informative for future studies of gray whale evolution, population structure, demography, and relatedness.

Evolutionary toxicology: population-level effects of chronic contaminant exposure on the marsh frogs (Rana ridibunda) of Azerbaijan.

We used molecular methods and population genetic analyses to study the effects of chronic contaminant exposure in marsh frogs from Sumgayit, Azerbaijan. Marsh frogs inhabiting wetlands in Sumgayit are exposed to complex mixtures of chemical contaminants, including petroleum products, pesticides, heavy metals, and many other industrial chemicals. Previous results documented elevated estimates of genetic damage in marsh frogs from the two most heavily contaminated sites. Based on mitochondrial DNA (mtDNA) control region sequence data, the Sumgayit region has reduced levels of genetic diversity, likely due to environmental degradation. The Sumgayit region also acts as an ecological sink, with levels of gene flow into the region exceeding gene flow out of the region. Additionally, localized mtDNA heteroplasmy and diversity patterns suggest that one of the most severely contaminated sites in Sumgayit is acting as a source of new mutations resulting from an increased mutation rate. This study provides an integrated method for assessing the cumulative population impacts of chronic contaminant exposure by studying both population genetic and evolutionary effects.

Biomarkers of exposure and effects of environmental contaminants on swallows nesting along the Rio Grande, Texas, USA.

We collected adult cave swallows (Petrochelidon fulva) and cliff swallows (P. pyrrhonota) during the breeding seasons in 1999 and 2000 from eight locations along the Rio Grande from Brownsville to El Paso (unless otherwise specified, all locations are Texas, USA) and an out-of-basin reference location. Body mass, spleen mass, hepatosomatic index (HSI), gonadosomatic index (GSI), thyroxine (T4) in plasma, DNA damage measured as the half-peak coefficient of variation of DNA content (HPCV) in blood cells, as well as acetylcholinesterase and butyrylcholinesterase in brain were compared with concentrations of organochlorines, metals, and metalloids in carcasses to determine potential effects of contaminants on swallows during the breeding season. Concentrations of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE) were significantly greater in swallows from El Paso than in those from most locations, except for Pharr and Llano Grande. All swallows from these three locations had p,p'-DDE concentrations of 3 microg/g wet weight or greater. Swallows from El Paso either had or shared the highest concentrations of p,p'-DDE, polychlorinated biphenyls, and 13 inorganic elements. Swallows from El Paso exhibited greater spleen mass and HPCV values as well as lower T4 values compared with those from other locations. Thyroxine was a potential biomarker of contaminant exposure in swallows of the Rio Grande, because it was negatively correlated with p,p'-DDE and Se. Spleen mass was positively correlated with selenium and HSI and negatively correlated with body mass, GSI, Mn, and Ni. Overall, the present study suggests that insectivorous birds living in areas of high agricultural and industrial activity along the Rio Grande bioaccumulate environmental contaminants. These contaminants, particularly p,p'-DDE, may be among multiple factors that impact endocrine and hematopoietic function in Rio Grande swallows.