John W. Bickham

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.

Significant deviations from Hardy-Weinberg equilibrium caused by low levels of microsatellite genotyping errors

Microsatellite genotyping from samples with varying quality can result in an uneven distribution of errors. Previous studies reporting error rates have focused on estimating the effects of both randomly distributed and locus‐specific errors. Sample‐specific errors, however, can also significantly affect results in population studies despite a large sample size. From two studies including six microsatellite markers genotyped from 272 sperm whale DNA samples, and 33 microsatellites genotyped from 213 bowhead whales, we investigated the effects of sample‐ and locus‐specific errors on calculations of Hardy–Weinberg equilibrium. The results of a jackknife analysis in these two studies identified seven individuals that were highly influential on estimates of Hardy–Weinberg equilibrium for six different markers. In each case, the influential individual was homozygous for a rare allele. Our results demonstrate that Hardy–Weinberg P values are very sensitive to homozygosity in rare alleles for single individuals, and that > 50% of these cases involved genotype errors likely due to low sample quality. This raises the possibility that even small, normal levels of laboratory errors can result in an overestimate of the degree to which markers are out of Hardy–Weinberg equilibrium and hence overestimate population structure. To avoid such bias, we recommend routine identification of influential individuals and multiple replications of those samples.

Recurrent evolution of life history ecotypes in sockeye salmon: implications for conservation and future evolution

We examine the evolutionary history and speculate about the evolutionary future of three basic life history ecotypes that contribute to the biocomplexity of sockeye salmon (Oncorhynchus nerka). The ‘recurrent evolution’ (RE) hypothesis claims that the sea/river ecotype is ancestral, a ‘straying’ form with poorly differentiated (meta)population structure, and that highly structured populations of lake‐type sockeye and kokanee have evolved repeatedly in parallel adaptive radiations between recurrent glaciations of the Pleistocene Epoch. Basic premises of this hypothesis are consistent with new, independent evidence from recent surveys of genetic variation in mitochondrial and microsatellite DNA: (1) sockeye salmon are most closely related to pink (O. gorbuscha) and chum (O. keta) salmon with sea‐type life histories; (2) the sockeye life history ecotypes exist as polyphyletic lineages within large drainages and geographic regions; (3) the sea/river ecotype exhibits less genetic differentiation among populations than the lake or kokanee ecotypes both within and among drainages; and (4) genetic diversity is typically higher in the sea/river ecotype than in the lake and kokanee ecotypes. Anthropogenic modification of estuarine habitat and intensive coastal fisheries have likely reduced and fragmented historic metapopulations of the sea/river ecotype, particularly in southern areas. In contrast, the kokanee ecotype appears to be favoured by marine fisheries and predicted changes in climate.

Effects of methylmercury exposure on glutathione metabolism, oxidative stress, and chromosomal damage in captive-reared common loon (Gavia immer) chicks

We quantified the level of dietary mercury (Hg), delivered as methylmercury chloride (CH3HgCl), associated with negative effects on organ and plasma biochemistries related to glutathione (GSH) metabolism and oxidative stress, and chromosomal damage in captive-reared common loon (Gavia immer) chicks reared from hatch to 105 days. Mercury-associated effects related to oxidative stress and altered glutathione metabolism occurred at 1.2 microg Hg/g and 0.4 microg Hg/g, an ecologically relevant dietary mercury level, but not at 0.08 microg Hg/g. Among the variables that contributed most to dissimilarities in tissue chemistries between control and treatment groups were increased levels of oxidized glutathione (GSSG), GSH peroxidase, and the ratio of GSSG to GSH in brain tissue; increased levels of hepatic GSH; and decreased levels of hepatic glucose-6-phosphate dehydrogenase (G-6-PDH). Our results also suggest that chronic exposure to environmentally relevant dietary Hg levels did not result in statistically significant somatic chromosomal damage in common loon chicks.

Assessing substitution patterns, rates and homoplasy at HVRI of Steller sea lions, Eumetopias jubatus

Despite the widely recognized incidence of homoplasy characterizing this region, the hypervariable region I (HVRI) of the mitochondrial control region is one of the most frequently used genetic markers for population genetic and phylogeographic studies. We present an evolutionary analysis of HVRI and cytochrome b sequences from a range‐wide survey of 1031 Steller sea lions, Eumetopias jubatus, to quantify homoplasy and substitution rate at HVRI. Variation in HVRI was distributed across 41 variable sites in the 238‐bp segment examined. All variants at HVR1 were found to be transitions. However, our analyses suggest that a minimum of 101 changes have actually occurred within HVRI with as many as 18 substitutions occurring at a single site. By including this hidden variation into our analyses, several instances of apparent long‐range dispersal were resolved to be homoplasies and 8.5–12% of observed HVRI haplotypes were found to have geographic distributions descriptive of convergent molecular evolution rather than identity by descent. We estimate the rate of substitution at HVRI in Steller sea lions to be ∼24 times that of cytochrome b with an absolute rate of HVRI substitution estimated at 27.45% per million years. These findings have direct implications regarding the utility of HVRI data to generate a variety of evolutionary genetic hypotheses.

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.

Systematic review of small fruit-eating bats (Artibeus) from the Guianas, and a re-evaluation of A. glaucus bogotensis

Abstract We studied molecular and morphological variation in small fruit-eating bats (Artibeus) in northern South America to establish species boundaries, evolutionary relationships, and distributional limits. Although this is a speciose genus with some of the most common bats in Neotropical forests, resolution of taxonomy and their identification has been difficult. Our molecular phylogeny based on Bayesian and parsimony analyses of cytochrome b variation includes a well supported topology of A. glaucus glaucus sister to a clade of A. gnomus and A. glaucus bogotensis indicating that A. glaucus is a paraphyletic amalgam. A re-assessment of morphology corroborates differences between A. g. bogotensis from the Andean valleys of Colombia east into the Guianas and A. g. glaucus from western Amazonia. Thus, we recognize A. bogotensis and A. glaucus as distinct and allopatrically occurring species. Based on a Kimura-2 parameter model of substitution for cytochrome b, there was 1.2% sequence divergence within A. bogotensis, and 9.5% sequence divergence between A. bogotensis and A. glaucus. Compared to A. glaucus, A. bogotensis has prominent white facial stripes, a less hirsute interfemoral membrane, less robust orbitorostral region, and also lacks a small third lower molar. Within the Guiana region, there are three species with overlapping distributions (A. bogotensis, A. cinereus, and A. gnomus), however, they are sympatric only within the interior lowland forest near savannas. All other habitats including coastal forest, lowland forest, savanna, and highland forest have only two sympatric species, one of which is relatively more abundant (> 70%).

Evolutionary history of the genus Rhogeessa (Chiroptera: Vespertilionidae) as revealed by mitochondrial DNA sequences

Abstract Evolutionary relationships among bats of the genus Rhogeessa (Chiroptera: Vespertilionidae) are poorly understood because of the morphological similarity of many of the species and the limited resolution of karyotypes and allozymes in previous studies. Previous karyotypic studies reported several populations that differ by Robertsonian centric fusions, which led to a proposed mechanism of speciation called speciation by monobrachial centric fusions. Here, we present a molecular phylogenetic analysis of 8 of the 10 currently recognized species of Rhogeessa using the mitochondrial DNA gene cytochrome b as well as new karyotypic data. The results are generally consistent with speciation by monobrachial centric fusions because karyotypically distinct populations typically comprise monophyletic maternal lineages. One exception was 2 individuals that were possible hybrids between R. tumida (2n = 34) and R. aeneus (2n = 32). We found ostensible species-level differentiation among 3 karyotypically identical (2n = 34) but geographically separated populations of R. tumida. Examination of new karyotypic data shows a population from western Ecuador to have 2n = 42 and study of molecular data shows it to be phylogenetically distinct from both the karyotypically identical R. genowaysi from Mexico and the South American R. io (2n = 30) to which it was previously allocated. We recognize this population by its available name, R. velilla. We also found an unexpectedly close relationship between Baeodon alleni and R. gracilis.

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.

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.