Molecular insights into the historic demography of bowhead whales: understanding the evolutionary basis of contemporary management practices.

Patterns of genetic variation observed within species reflect evolutionary histories that include signatures of past demography. Understanding the demographic component of species' history is fundamental to informed management because changes in effective population size affect response to environmental change and evolvability, the strength of genetic drift, and maintenance of genetic variability. Species experiencing anthropogenic population reductions provide valuable case studies for understanding the genetic response to demographic change because historic changes in the census size are often well documented. A classic example is the bowhead whale, Balaena mysticetus, which experienced dramatic population depletion due to commercial whaling in the late 19th and early 20th centuries. Consequently, we analyzed a large multi-marker dataset of bowhead whales using a variety of analytical methods, including extended Bayesian skyline analysis and approximate Bayesian computation, to characterize genetic signatures of both ancient and contemporary demographic histories. No genetic signature of recent population depletion was recovered through any analysis incorporating realistic mutation assumptions, probably due to the combined influences of long generation time, short bottleneck duration, and the magnitude of population depletion. In contrast, a robust signal of population expansion was detected around 70,000 years ago, followed by a population decline around 15,000 years ago. The timing of these events coincides to a historic glacial period and the onset of warming at the end of the last glacial maximum, respectively. By implication, climate driven long-term variation in Arctic Ocean productivity, rather than recent anthropogenic disturbance, appears to have been the primary driver of historic bowhead whale demography.

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 approximately 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.

Contrasting patterns of genetic diversity at three different genetic markers in a marine mammal metapopulation.

Many studies use genetic markers to explore population structure and variability within species. However, only a minority use more than one type of marker and, despite increasing evidence of a link between heterozygosity and individual fitness, few ask whether diversity correlates with population trajectory. To address these issues, we analysed data from the Steller's sea lion, Eumetiopias jubatus, where three stocks are distributed over a vast geographical range and where both genetic samples and detailed demographic data have been collected from many diverse breeding colonies. To previously published mitochondrial DNA (mtDNA) and microsatellite data sets, we have added new data for amplified fragment length polymorphism (AFLP) markers, comprising 238 loci scored in 285 sea lions sampled from 23 natal rookeries. Genotypic diversity was low relative to most vertebrates, with only 37 loci (15.5%) being polymorphic. Moreover, contrasting geographical patterns of genetic diversity were found at the three markers, with Nei's gene diversity tending to be higher for AFLPs and microsatellites in rookeries of the western and Asian stocks, while the highest mtDNA values were found in the eastern stock. Overall, and despite strongly contrasting demographic histories, after applying phylogenetic correction we found little correlation between genetic diversity and either colony size or demography. In contrast, we were able to show a highly significant positive relationship between AFLP diversity and current population size across a range of pinniped species, even though equivalent analyses did not reveal significant trends for either microsatellites or mtDNA.

Potential effects of environmental contaminants on P450 aromatase activity and DNA damage in swallows from the Rio Grande and Somerville, Texas.

Cliff swallows (Petrochelidon pyrrhonota) and cave swallows (P. fulva) were sampled during the breeding season at several locations in the Rio Grande, Texas, to evaluate the potential effects of environmental contaminants on P450 aromatase activity in brain and gonads and DNA damage in blood cells. The tritiated water-release aromatase assay was used to measure aromatase activity and flow cytometry was used to measure DNA damage in nucleated blood cells. There were no significant differences in brain and gonadal aromatase activities or in estimates of DNA damage (HPCV values) among cave swallow colonies from the Lower Rio Grande Valley (LRGV) and Somerville. However, both brain and gonadal aromatase activities were significantly higher (P < 0.05) in male cliff swallows from Laredo than in those from Somerville. Also, DNA damage estimates were significantly higher (P < 0.05) in cliff swallows (males and females combined) from Laredo than in those from Somerville. Contaminants of current high use in the LRGV, such as atrazine, and some of the highly persistent organochlorines, such as toxaphene and DDE, could be potentially associated with modulation of aromatase activity in avian tissues. Previous studies have indicated possible DNA damage in cliff swallows. We did not observe any differences in aromatase activity or DNA damage in cave swallows that could be associated with contaminant exposure. Also, the differences in aromatase activity and DNA damage between male cliff swallows from Laredo and Somerville could not be explained by contaminants measured at each site in previous studies. Our study provides baseline information on brain and gonadal aromatase activity in swallows that could be useful in future studies.

Diploid-triploid mosaicism and tissue ploidy diversity within Platemys platycephala from Suriname.

The twist-necked turtle, Platemys platycephala, is 1 of only 2 known species to possess sexual reproduction and diploid-triploid mosaicism. Previous studies have shown that mosaics occur in Suriname and French Guiana but only diploids are known from Bolivia and Brazil. In this paper, the frequency of ploidy mosaicism was studied in a large sample of P. platycephala from Suriname to more fully explore the diversity of ploidy levels within and among individuals. Flow-cytometric analysis of blood revealed a wide diversity of conditions including diploids, diploid-triploid mosaics, triploids, and triploid-tetraploid mosaics. The largest frequency class was 100% diploid, and the second largest was 100% triploid. However, mosaic individuals were observed from the entire spectrum of mixtures ranging from nearly all-diploid to nearly all-triploid and 2 individuals were triploid-tetraploid mosaics. It appears likely that diploids, triploids and mosaics do not represent distinct biotypes, but simply different conditions within a spectrum of possible ploidy mixtures. Studies of multiple tissues from 5 individuals showed blood alone is a good indicator of ploidy, but subtle differences were found among tissues for some individuals, and some individuals that were all-diploid or all-triploid in blood were found to be mosaic in other tissues. Triploidy was statistically associated with males, and we hypothesize that genome size plays a role in sex determination in this species.

Detecting genetic structure in migrating bowhead whales off the coast of Barrow, Alaska.

We develop a general framework for analysing and testing genetic structure within a migratory assemblage that is based on measures of genetic differences between individuals. We demonstrate this method using microsatellite DNA data from the Bering-Chukchi-Beaufort stock of bowhead whales (Balaena mysticetus), sampled via Inuit hunting during the spring and autumn migration off Barrow, Alaska. This study includes a number of covariates such as whale ages and the time separation between captures. Applying the method to a sample of 117 bowhead whales, we use permutation methods to test for temporal trends in genetic differences that can be ascribed to age-related effects or to timing of catches during the seasons. The results reveal a pattern with elevated genetic differences among whales caught about a week apart, and are statistically significant for the autumn migration. In contrast, we find no effects of time of birth or age-difference on genetic differences. We discuss possible explanations for the results, including population substructuring, demographic consequences of historical overexploitation, and social structuring during migration.

Deep genetic subdivision within a continuously distributed and highly vagile marine mammal, the Steller's sea lion (Eumetopias jubatus).

The Steller's sea lion Eumetopias jubatus is an endangered marine mammal that has experienced dramatic population declines over much of its range during the past five decades. Studies using mitochondrial DNA (mtDNA) have shown that an apparently continuous population includes a strong division, yielding two discrete stocks, western and eastern. Based on a weaker split within the western stock, a third Asian stock has also been defined. While these findings indicate strong female philopatry, a recent study using nuclear microsatellite markers found little evidence of any genetic structure, implying extensive paternal gene flow. However, this result was at odds with mark-recapture data, and both sample sizes and genetic resolution were limited. To address these concerns, we increased analytical power by genotyping over 700 individuals from across the species' range at 13 highly polymorphic microsatellite loci. We found a clear phylogenetic break between populations of the eastern stock and those of the western and Asian stocks. However, our data provide little support for the classification of a separate Asian stock. Our findings show that mtDNA structuring is not due simply to female philopatry, but instead reflects a genuine discontinuity within the range, with implications for both the phylogeography and conservation of this important marine mammal.

Glacial refugia and the phylogeography of Steller's sea lion (Eumatopias jubatus) in the North Pacific.

Mitochondrial DNA sequence data were used to examine the phylogeographic history of Steller's sea lions (Eumetopias jubatus) in relation to the presence of Plio-Pleistocene insular refugia. Cytochrome b and control region sequences from 336 Steller's sea lions reveal phylogenetic lineages associated with continental refugia south of the ice sheets in North America and Eurasia. Phylogenetic analysis suggests the genetic structure of E. jubatus is the result of Pleistocene glacial geology, which caused the elimination and subsequent reappearance of suitable rookery habitat during glacial and interglacial periods. The cyclic nature of geological change produced a series of independent population expansions, contractions and isolations that had analogous results on Steller's sea lions and other marine and terrestrial species. Our data show evidence of four glacial refugia in which populations of Steller's sea lions diverged. These events occurred from approximately 60,000 to 180,000 years BP and thus preceded the last glacial maximum.

Gradual and quantum genome size shifts in the hystricognath rodents.

We assessed genome size variation by flow cytometry within and among 31 species of nine families of African and South American hystricognath rodents. Interspecific variation was extensive and genome size was relatively high among the South American radiation whereas only moderate variation and smaller estimates of genome size were observed in the African counterparts. The largest genome size, indicating tetraploidy was recorded in the South American octodontid, Tympanoctomys barrerae (16.8 pg DNA). This quantum shift in DNA content represents a novel mechanism of genome evolution in mammals. As expected in polyploid organisms, varying nucleotypic effects were observed in the dimensions of the sperm cells and lymphocytes of T. barrerae. The role of control mechanisms that influence cell dimensions in polyploid organisms is discussed.

Phylogenetic relationships within the eared seals (Otariidae: Carnivora): implications for the historical biogeography of the family.

Phylogenetic relationships within the family Otariidae were investigated using two regions of the mitochondrial genome. A 360-bp region of the cytochrome b gene was employed for the primary phylogenetic analysis, while a 356-bp segment of the control region was used to enhance resolution of the terminal nodes. Traditional classification of the family into the subfamilies Arctocephalinae (fur seals) and Otariinae (sea lions) is not supported, with the fur seal Callorhinus ursinus having a basal relationship relative to the rest of the family. This is consistent with the fossil record which suggests that this genus diverged from the line leading to the remaining fur seals and sea lions about 6 million years ago (mya). There is also little evidence to support or refute the monophyly of sea lions. Four sea lion clades and five fur seal clades were observed, but relationships among these clades are unclear. Similar genetic divergences between the sea lion clades (D(a) = 0.054-0.078), as well as between the major Arctocephalus fur seal clades (D(a) = 0.040-0.069) suggest that these groups underwent periods of rapid radiation at about the time they diverged from each other. Rapid radiations of this type make the resolution of relationships between the resulting species difficult and indicate the requirement for additional molecular data from both nuclear and mitochondrial genes. The phylogenetic relationships within the family and the genetic distances among some taxa highlight inconsistencies in the current taxonomic classification of the family.

Cytochrome b sequences in black-crowned night-herons (Nycticorax nycticorax) from heronries exposed to genotoxic contaminants.

DNA sequence analysis of a 215 base-pair region of the mitochondrial cytochrome b gene was used to examine genetic variation and search for evidence of an increased mutation rate in black-crowned night-herons. We examined five populations exposed to environmental contamination (primarily PAHs and PCBs) and one reference population from the eastern U.S. There was no evidence of a high mutation rate even within populations previously shown to exhibit increased variation in DNA content among somatic cells as a result of petroleum exposure. Three haplotypes were observed among 99 individuals. The low level of variability could be evidence for a genetic bottleneck, or that cytochrome b is too conservative for use in population genetic studies of this species. With the exception of one population from Louisiana, pair-wise Phist estimates were very low, indicative of little population structure and potentially high rates of effective migration among populations.

Integration of genotoxicity and population genetic analyses in kangaroo rats (Dipodomys merriami) exposed to radionuclide contamination at the Nevada Test Site, USA.

We examined effects of radionuclide exposure at two atomic blast sites on kangaroo rats (Dipodomys merriami) at the Nevada Test Site, Nevada, USA, using genotoxicity and population genetic analyses. We assessed chromosome damage by micronucleus and flow cytometric assays and genetic variation by randomly amplified polymorphic DNA (RAPD) and mitochondrial DNA (mtDNA) analyses. The RAPD analysis showed no population structure, but mtDNA exhibited differentiation among and within populations. Genotoxicity effects were not observed when all individuals were analyzed. However, individuals with mtDNA haplotypes unique to the contaminated sites had greater chromosomal damage than contaminated-site individuals with haplotypes shared with reference sites. When interpopulation comparisons used individuals with unique haplotypes, one contaminated site had greater levels of chromosome damage than one or both of the reference sites. We hypothesize that shared-haplotype individuals are potential migrants and that unique-haplotye individuals are potential long-term residents. A parsimony approach was used to estimate the minimum number of migration events necessary to explain the haplotype distributions on a phylogenetic tree. The observed predominance of migration events into the contaminated sites supported our migration hypothesis. We conclude the atomic blast sites are ecological sinks and that immigration masks the genotoxic effects of radiation on the resident populations.

Effects of chemical contaminants on genetic diversity in natural populations: implications for biomonitoring and ecotoxicology.

The conservation of genetic diversity has emerged as one of the central issues in conservation biology. Although researchers in the areas of evolutionary biology, population management, and conservation biology routinely investigate genetic variability in natural populations, only a handful of studies have addressed the effects of chemical contamination on population genetics. Chemical contamination can cause population reduction by the effects of somatic and heritable mutations, as well as non-genetic modes of toxicity. Stochastic processes in small populations, increased mutation load, and the phenomenon of mutational meltdown are compounding factors that cause reduced fitness and accelerate the process of population extirpation. Although the original damage caused by chemical contaminants is at the molecular level, there are emergent effects at the level of populations, such as the loss of genetic diversity, that are not predictable based solely on knowledge of the mechanism of toxicity of the chemical contaminants. Therefore, the study of evolutionary toxicology, which encompasses the population-genetic effects of environmental contaminants, should be an important focus of ecotoxicology. This paper reviews the issues surrounding the genetic effects of pollution, summarizes the technical approaches that can be used to address these issues, and provides examples of studies that have addressed some of them.

Mixed-function oxygenases, oxidative stress, and chromosomal damage measured in lesser scaup wintering on the Indiana Harbor Canal.

During the winter of 1993-1994, male lesser scaup (Aythya affinis) were collected on the heavily polluted Indiana Harbor Canal, East Chicago, Indiana, and examined for several bioindicators of chemical exposure. Livers were analyzed for activities of three cytochrome P450-associated monooxygenases and four measures of oxidative stress. Blood and spleen were analyzed by flow cytometry for chromosomal damage. In a concurrent study, scaup tissues were analyzed for organic and inorganic contaminants. Ethoxyresorufin-O-dealkylase (EROD) activity in livers of scaup collected in January 1994 was significantly higher than in livers of scaup collected in March 1994 or in livers of reference birds. Three hepatic monooxygenase activities were each significantly correlated with polycyclic aromatic hydrocarbon (PAH) concentrations in scaup carcasses. Thiobarbituric acid (TBA) activity in scaup livers was positively correlated with iron, boron, and lead concentrations in livers and polychlorinated biphenyl concentrations in carcasses. TBA activity was negatively correlated with protein-bound thiol activity and mercury concentrations in livers. The coefficient of variation of DNA content in scaup blood cells was correlated with PAH concentrations in scaup carcasses. This is the first field study with birds to demonstrate a correlation between liver monooxygenase activity and carcass PAH concentrations and to show a direct correlation between PAH concentrations in tissues and somatic chromosomal damage in blood.

Biogeographic implications of cytochrome b sequences and allozymes in sockeye (Oncorhynchus nerka).

Nucleotide sequence and restriction site analyses of the cytochrome b gene of mitochondrial DNA revealed three relatively common haplotypes among sockeye salmon (N = 80) from eight populations representing four major drainages from Kamchatka (Russia), Alaska, and British Columbia. Macrogeographic variation in mtDNA was compared to that of three variable allozyme loci assayed for a much larger number of fish (N = 779). Sockeye from the Fraser River drainage of British Columbia were distinct from those from the other three drainages in both analyses. The GT haplotype was found in all four populations, but the GC haplotype was absent from the Fraser River (southern) drainage and present in the three northern drainages. The AC haplotype was common in the Fraser River drainage (frequency = 0.4), rare in the Skeena River drainage of British Columbia (0.1), and absent from the Alaskan and Russian samples. Cluster analysis (UPGMA) of allozyme allele frequencies showed a similar pattern of divergence with the populations from the Fraser River drainage being most divergent and clustering at an identity value of 0.93 with the rest of the populations. Five of the remaining six populations clustered at or above an identity value of 0.99. These data, combined with allozyme data for sockeye and mitochondrial DNA data for chinook salmon and the Steller sea lion, indicate the presence of a phylogeographic break between northern and southern populations ostensibly derived from glacial refugia in Beringia and the Columbia River, respectively.