An exploratory spatial contaminant assessment for polar bear (Ursus maritimus) liver, fat, and muscle from northern Canada.

Since the industrial era, chemicals have been ubiquitous in worldwide ecosystems. Despite the discontinued release of highly toxic persistent organic pollutants (POPs) in the environment, the levels of some POPs are still being measured in the Canadian Arctic. These contaminants are of great concern due to their persistence, toxicity, and levels of bioaccumulation in food chains. Animals occupying top trophic positions in the Canadian Arctic, particularly polar bears, are exposed to these contaminants mainly through their diet. Our study investigated the levels of 30 metals (including total and methyl mercury) alkaline and alkaline earth metals, 15 polycyclic aromatic compounds and their alkyl congeners (PACs), 6 chlordanes (CHLs), and 20 polychlorinated biphenyls (PCBs), in 49 polar bears from the Canadian Arctic. Contaminant burden was measured in liver, muscle, and fat in bears of different sex, age, and locations. A principal component analysis did not distinguish differences between age and sex profiles for most contaminants. However, the concentrations measured and their distribution in the tissues confirm findings observed in past studies. This study highlights the importance of continual monitoring of polar bear health (e.g., newly detected PACs were measured within this study) and evaluating those impacts for the next generations of polar bears.

Cytonuclear discordance, reticulation and cryptic diversity in one of North America's most common frogs.

Complicated phylogenetic histories benefit from diverse sources of inference. Pseudacris crucifer (spring peeper) spans most of eastern North America and comprises six mtDNA lineages that form multiple contact zones. The putative Miocene or early Pliocene origins of the oldest lineages within Pseudacris crucifer imply sufficient time for species-level divergence. To understand why this species appears unified while congeners have radiated, we analyze and compare male advertisement calls, mitochondrial, and nuclear markers and speak to the complex processes that have potentially influenced its contemporary patterns. We find extensive geographic and topological mitonuclear discordance, with three nuclear lineages containing 6 more-structured mtDNA lineages, and nuclear introgression at some contact zones. Male advertisement call differentiation is incongruent with the genetic structure as only one lineage appears differentiated. Occupying the Interior Highlands of the central United States, this Western lineage also has the most concordant mitochondrial and nuclear geographic patterns. Based on our findings we suggest that the antiquity of common ancestors was not as important as the maintenance of allopatry in the divergence in P. crucifer genetic lineages. We use multiple lines of evidence to generate hypotheses of isolation, reticulation, and discordance within this species and to expand our understanding of the early stages of speciation.

Can alternative mating tactics facilitate introgression across a hybrid zone by circumventing female choice?

Reproductive barriers and divergence in species' mate recognition systems underlie major models of speciation. However, hybridization between divergent species is common, and classic mechanisms to explain permeable reproductive barriers rarely consider how an individual may attain reproductive success. Alternative mating tactics (AMTs) exist in various forms across animal taxa. Such tactics may allow poorer quality individuals to gain mating opportunities and facilitate introgression either through asymmetrical positive selection or by circumventing female choice altogether in areas of secondary contact. One such tactic is satellite behaviour in frogs, where silent males perch near advertisers in an attempt to intercept females. To test whether such satellite male tactics are context-dependent and favoured by hybrids, we genotyped and quantified the morphology of 80 male spring peeper (Pseudacris crucifer) individuals involved in caller-satellite associations from a secondary contact zone between two intraspecific mitochondrial lineages. Irrespective of population, satellite behaviour was best predicted by size but not body condition. Within the contact zone, pure individuals showed a significantly greater probability of being active callers, whereas hybrids of one lineage were more likely to adopt the satellite tactic. We suggest that satellite behaviour in P. crucifer promotes introgression, breaks down reproductive isolating barriers and contributes to asymmetrical introgression in this secondary contact zone. AMTs may thus be an underexplored but important alternative to oft-discussed causes of genetic discordance found in hybrid zones.

Contact zone dynamics during early stages of speciation in a chorus frog (Pseudacris crucifer).

Characterizing the genetic and behavioural consequences of contact between previously geographically isolated lineages provides insights into the mechanisms underlying diversification and ultimately speciation. The spring peeper (Pseudacris crucifer) is a widespread Nearctic chorus frog with six divergent mitochondrial DNA (mtDNA) lineages, many of which came into secondary contact during the Holocene. We examined genetics, morphology, advertisement calls and female preference for two lineages that began diverging in allopatry in the Pliocene and now overlap in southwestern Ontario, Canada. We found non-coincident clines in mtDNA and nuclear DNA, mirroring directionality of premating isolation barriers. We also found divergence in a range of traits between these two lineages, displacement in male call attributes and female preference for calls of their natal lineage in sympatry. Hybrids were morphologically distinct from both parental lineages, but hybrid male calls were acoustically intermediate. Female hybrids showed asymmetrical preference for Eastern male calls. These results considered together provide evidence of either unidirectional hybridization or selection against hybrids, potentially implying reproductive character displacement. Our work demonstrates the utility of integrated, multi-character approaches to understanding the processes of divergence and the nature of speciation.

Approximate Bayesian computation reveals the factors that influence genetic diversity and population structure of foxsnakes.

Contemporary geographical range and patterns of genetic diversity within species reflect complex interactions between multiple factors acting across spatial and temporal scales, and it is notoriously difficult to disentangle causation. Here, we quantify patterns of genetic diversity and genetic population structure using mitochondrial DNA sequences (101 individuals, cytochrome b) and microsatellites (816 individuals, 12 loci) and use Approximate Bayesian computation methods to test competing models of the demographic history of eastern and western foxsnakes. Our analyses indicate that post-glacial colonization and past population declines, probably caused by the infilling of deciduous forest and cooler temperatures since the mid-Holocene, largely underpin large-scale genetic patterns for foxsnakes. At finer geographical scales, our results point to more recent anthropogenic habitat loss as having accentuated genetic population structure by causing further declines and fragmentation.

Evolutionary affinity of billfishes (Xiphiidae and Istiophoridae) and flatfishes (Plueronectiformes): Independent and trans-subordinal origins of endothermy in teleost fishes.

Billfishes (Scombroidei) and tunas (Scombridae), both considered part of the suborder Scombroidei, have long been studied by biologists largely because of their remarkable physiological and anatomical muscular adaptations associated with regional endothermy and continuous swimming. These attributes, combined with analyses of other morphological and molecular data, have led to a general perception that tunas and billfishes are close relatives, though this hypothesis has been vigorously debated. Using Bayesian phylogenetic analysis of nine mitochondrial and three nuclear loci (>7000bp), we show that billfishes are only distantly related to tunas, but rather share strong evolutionary affinities with flatfishes (Pleuronectiformes) and jacks (Carangidae). This phylogenetic relationship is striking because of the marked variation in phenotype and niche across these trans-ordinal groups of fishes. Billfishes and flatfishes have each evolved radically divergent morphological and physiological features: elongated bills and extraocular heater organs in billfishes, and cranial asymmetry with complete eye migration during ontogenetic development in flatfishes. Despite this divergence, we identify synapomorphies consistent with the hypothesis of a common billfish/flatfish/jack ancestor.

Evolution of the nuclear-encoded cytochrome oxidase subunits in vertebrates.

Vertebrate mitochondrial cytochrome c oxidase (COX) possesses 10 nuclear-encoded subunits. Six subunits have paralogs in mammals, but the origins and distribution of isoforms among vertebrates have not been analyzed. We used Bayesian phylogenetic analysis to interpret the origins of each subunit, inferring the roles of gene and genome duplications. The paralogous ancestries of five genes were identical throughout the major vertebrate taxa: no paralogs of COX6c and COX7c, two paralogs of COX4 and COX6a, and three paralogs of COX7a. Two genes had an extra copy in teleosts (COX5a, COX5b), and three genes had additional copies in mammals (COX6b, COX7b, COX8). Focusing on early vertebrates, we examined structural divergence and explored transcriptional profiles across zebrafish tissues. Quantitative transcript profiles revealed dramatic differences in transcript abundance for different subunits. COX7b and COX4 transcripts were typically present at very low levels, whereas COX5a and COX8 were in vast excess in all tissues. For genes with paralogs, two general patterns emerged. For COX5a and COX8, there was ubiquitous expression of one paralog, with the other paralog in lower abundance in all tissues. COX4 and COX6a shared a distinct expression pattern, with one paralog dominant in brain and gills and the other in muscles. The isoform profiles in combination with phylogenetic analyses show that vertebrate COX isoform patterns are consistent with the hypothesis that early whole genome duplications in basal vertebrates governed the isoform repertoire in modern fish and tetrapods, though more recent lineage-specific gene/genome duplications also play a role in select subunits.

Genetic variation across species' geographical ranges: the central-marginal hypothesis and beyond.

There is growing interest in quantifying genetic population structure across the geographical ranges of species to understand why species might exhibit stable range limits and to assess the conservation value of peripheral populations. However, many assertions regarding peripheral populations rest on the long-standing but poorly tested supposition that peripheral populations exhibit low genetic diversity and greater genetic differentiation as a consequence of smaller effective population size and greater geographical isolation relative to geographically central populations. We reviewed 134 studies representing 115 species that tested for declines in within-population genetic diversity and/or increases in among-population differentiation towards range margins using nuclear molecular genetic markers. On average, 64.2% of studies detected the expected decline in diversity, 70.2% of those that tested for it showed increased differentiation and there was a positive association between these trends. In most cases, however, the difference in genetic diversity between central and peripheral population was not large. Although these results were consistent across plants and animals, strong taxonomic and biogeographical biases in the available studies call for a cautious generalization of these results. Despite the large number of studies testing these simple predictions, very few attempted to test possible mechanisms causing reduced peripheral diversity or increased differentiation. Almost no study incorporated a phylogeographical framework to evaluate historical influences on contemporary genetic patterns. Finally, there has been little effort to test whether these geographical trends in putatively neutral variation at marker loci are reflected by quantitative genetic trait variation, which is likely to influence the adaptive potential of populations across the geographical range.

Applying new inter-individual approaches to assess fine-scale population genetic diversity in a neotropical frog, Eleutherodactylus ockendeni.

We assess patterns of genetic diversity of a neotropical leaflitter frog, Eleutherodactylus ockendeni, in the upper Amazon of Ecuador without a priori delineation of biological populations and with sufficiently intensive sampling to assess inter-individual patterns. We mapped the location of each collected frog across a 5.4 x 1 km landscape at the Jatun Sacha Biological Station, genotyped 185 individuals using five species-specific DNA microsatellite loci, and sequenced a fragment of mitochondrial cytochrome b for a subset of 51 individuals. The microsatellites were characterized by high allelic diversity and homozygote excess across all loci, suggesting that when pooled the sample is not a panmictic population. We conclude that the lack of panmixia is not attributable to the influence of null alleles or biased sampling of consanguineous family groups. Multiple methods of population cluster analysis, using both Bayesian and maximum likelihood approaches, failed to identify discrete genetic clusters across the sampled area. Using multivariate spatial autocorrelation, kinship coefficients and relatedness coefficients, we identify a continuous isolation by distance population structure, with a first patch size of ca. 260 m and apparently large population sizes. Analysis of mtDNA corroborates the observation of high genetic diversity at fine scales: there are multiple haplotypes, they are non-randomly distributed and a binary haplotype correlogram shows significant spatial genetic autocorrelation. We demonstrate the utility of inter-individual genetic methods and caution against making a priori assumptions about population genetic structure based simply on arbitrary or convenient patterns of sampling.

A simple molecular technique for identifying marine host fish by sequencing blood-feeding parasites.

Gnathiid isopods are common ectoparasites of fish on the Great Barrier Reef, Australia. While screening for appropriate markers for phylogenetic studies of gnathiids, we found that primers for 12S and 16S rDNA preferentially amplified the host fish DNA instead of gnathiid DNA. This amplification occurred even when using gnathiids that were not engorged with host blood and adult gnathiids that do not feed on fish blood. This method could be used in host-parasite studies to identify hosts without having to sample parasites directly from the host (which can be costly and requires considerable skill in a marine environment). Target ribosomal DNA sequences can be amplified from total DNA extracted from parasites that are captured in funnel traps or plankton tows. Sequence data from these can be used to identify the hosts that gnathiids were feeding on before capture.

Perception and history: molecular phylogeny of a diverse group of neotropical frogs, the 30-chromosome hyla (anura: hylidae).

We used 16S rRNA, 12S rRNA, and cytochrome-b sequence to investigate the history of the "30-chromosome" Hyla, a diverse assemblage of neotropical treefrogs. Three aspects of these frogs were examined: (1) phylogenetic relationships among constituent species groups, among the species of one of these groups (Hyla leucophyllata group), and among populations of Hyla leucophyllata; (2) the apparent age of cladogenetic events; and (3) the phylogeography of H. leucophyllata. Mixed success in resolving the phylogeny is not because of a lack of character variation; levels of genetic divergence are high and suggest pre-Pleistocene diversification, even among populations. Close temporal proximity of ancient cladogenetic events might make resolution of the topology difficult using any character set. At the population level, current geographic proximity is a poor predictor of phylogenetic affinity. A long history of dispersal and colonization may complicate, or even preclude, the accurate recovery of the history of this species in the Amazon Basin. It remains to be seen whether the patterns found here will prove common among neotropical frogs.

Riverine barriers and the geographic distribution of Amazonian species.

Rivers have been suggested to have played an important role in shaping present-day patterns of ecological and genetic variation among Amazonian species and communities. Recent molecular studies have provided mixed support for the hypothesis that large lowland Amazonian rivers have functioned as significant impediments to gene flow among populations of neotropical species. To date, no study has systematically evaluated the impact that riverine barriers might have on structuring whole Amazonian communities. Our analyses of the phylogeography of frogs and small mammals indicate that a putative riverine barrier (the Juruá River) does not relate to present-day patterns of community similarity and species richness. Rather, our results imply a significant impact of the Andean orogenic axis and associated thrust-and-fold lowland dynamics in shaping patterns of biotic diversity along the Juruá. Combined results of this and other studies significantly weaken the postulated role of rivers as major drivers of Amazonian diversification.

A molecular phylogeny of warbling-finches (Poospiza): paraphyly in a neotropical Emberizid genus.

We investigated the phylogenetic relationships of 12 species within a single genus of neotropical passerine (Poospiza) using 849 bp (283 codons) of the cytochrome b mitochondrial gene. We further explored evolutionary affinities of these taxa using sequence from an additional 47 thraupine (tanagers) and 7 emberizine (sparrows and buntings) genera, members of the predominantly New World family Emberizidae. Poospiza have traditionally been considered part of the emberizine radiation. However, our analyses suggest that members of this genus are more closely related to some thraupine lineages than they are to the other neotropical emberizine genera included in our study (Atlapetes, Embernagra, Melopyrrha, Phrygilus, Saltatricula, Tiaris). Although member taxa are closely related, the genus Poospiza appears to be paraphyletic with representatives of 6 thraupine genera (Cnemoscopus, Cypsnagra, Hemispingus, Nephelornis, Pyrrhocoma, Thylpopsis) interspersed among four well-supported Poospiza clades. The majority of species within this Poospiza-thraupine clade have geographic ranges that are exclusive to, or partially overlap with, the Andes Mountains. It is probable that these mountains have played an important role in driving cladogenesis within this group. Sequence divergence (transversions only; mean 4.7+/-1.3%) within the clade suggests that much of this diversification occurred within the late Miocene and Pliocene, a period coincident with major orogenic activity in central-western South America.

A comparison of RAPD versus microsatellite DNA markers in population studies of the massasauga rattlesnake.

We compared genetic differentiation among populations of the threatened massasauga rattlesnake (Sistrurus c. catenatus) using two types of nuclear molecular markers: randomly amplified polymorphic DNA (RAPD) markers and microsatellites. Analyses of molecular variance (AMOVA) and G(ST) and F(ST) analyses indicated that levels of among-population differentiation between regional populations (>100 km) were comparable for both markers. However, microsatellites were superior in population assignment tests and at discerning fine-scale genetic differentiation between subpopulations separated by tens of kilometers. These results argue that both types of markers are suitable for defining broad-scale genetic structures in snake populations and can provide important inputs into conservation initiatives of focal taxa. However, our analyses suggest that microsatellites 3re better for detecting structure at limited spatial scales.

Ridges and rivers: a test of competing hypotheses of Amazonian diversification using a dart-poison frog (Epipedobates femoralis).

Mitochondrial DNA cytochrome b sequence data from a dart-poison frog, Epipedobates femoralis, were used to test two hypotheses of Amazonian diversification: the riverine barrier and the ridge hypotheses. Samples were derived from sites located on both banks of the Rio Juruá and on both sides of the Iquitos Arch in western Amazonia. The phylogeographic structure was inconsistent with predictions of the riverine barrier hypothesis. Haplotypes from opposite river banks did not form monophyletic clades in any of our phylogenetic analyses, nor was the topology within major clades consistent with the riverine hypothesis. Further, the greatest differentiation between paired sites on opposite banks was not at the river mouth where the strongest barrier to gene flow was predicted to occur. The results instead were consistent with the hypothesis that ancient ridges (arches), no longer evident on the landscape, have shaped the phylogeographic relationships of Amazonian taxa. Two robustly supported clades map onto opposite sides of the Iquitos Arch. The mean haplotypic divergence between the two clades, in excess of 12%, suggests that this cladogenic event dates to between five and 15 million years ago. These estimates span a period of major orogenesis in western South America and presumably the formation of these ancient ridges.

Molecular phylogenetic evidence for the evolution of specialization in anemonefishes.

Anemonefishes (genera: Amphiprion and Premnas; family Pomacentridae) are a group of 28 species of coral reef fishes that are found in obligate symbiosis with large tropical sea anemones. A phylogenetic hypothesis based on morphological analyses of this group suggests that the ancestral anemonefish was a generalist with similar morphology to other pomacentrids, and that it gave rise to other anemonefish species that were more specialized for living with particular species of host anemones. To test this hypothesis we constructed a molecular phylogeny for the anemonefishes by sequencing 1140 base pairs of the cytochrome b gene and 522 base pairs of the 16S rRNA gene for six species of anemonefishes (representatives of all subgenera and species complexes) and two other pomacentrid species. Three methods of phylogenetic analysis all strongly supported the conclusion that anemonefishes are a monophyletic group. The molecular phylogeny differs from the tree based on morphological data in that the two species of specialized anemonefishes (Premnas biaculeatus and Amphiprion ocellaris) were assigned to a basal position within the clade, and the extreme host generalist (Amphiprion clarkii) to a more derived position. Thus, the initial anemonefish ancestors were probably host specialists and subsequent speciation events led to a combination of generalist and specialist groups. Further phylogenetic studies of additional anemonefish species are required to substantiate this hypothesis.

VOCAL DIALECTS AND THE STRUCTURE OF GEOGRAPHIC VARIATION IN MORPHOLOGICAL AND ALLOZYMIC CHARACTERS IN THE RUFOUS-COLLARED SPARROW, ZONOTRICHIA CAPENSIS.

The geographical patterns of variation shown at 20 allozyme and non-enzymatic protein-coding loci, in 8 external, and in 12 skeletal morphological characters in the rufous-collared sparrow, Zonotrichia capensis, were analyzed in order to test the local (genetic) adaptation hypothesis regarding the origin and maintenance of vocal dialects in birds. Approximately 20 males were collected from each of four sites within each of six different dialect zones. There was significant variability in both external and skeletal morphology among all 24 sites and among dialect groups. Average Wright's corrected fixation coefficient (FST ) was 0.118, indicating significant genetic differentiation among all sites, regardless of dialect. Hierarchical F statistics indicated that only 50% of among site variability was due to a dialect effect. Puna dialect sites were highly differentiated from all other sites with respect to both morphology (external and skeletal measures) and allozyme frequencies. Heterogeneity at the PGM-1 locus among puna scrub sites was the major cause of the high average FST across all sites, and within the puna scrub dialect. Average genetic differentiation among non-puna sites (FST = 0.018) was similar to differentiation among sites within each of the five non-puna dialect groups (mean FST = 0.0132 ± 0.0069). Hierarchical F statistics indicated that none of the among-site differentiation in this subset of samples was due to a dialect effect. These observations are not consistent with the local adaptation hypothesis. All significant genetic heterogeneity occurred among sites in mountainous habitats, and we suggest that topography and patchiness of habitat may have been major factors involved in population differentiation, rather than vocal dialects.

A survey of tissue-specific isozyme expressions during chicken ontogeny.

The ontogenetic trends in the expression of 25 isozymes in liver, gizzard, heart, and pectoralis muscle of White Leghorn chickens were examined using starch gel electrophoresis. Little change in expression during development was evident in liver S-AAT-A, GPI-A, S-ICDH-A, S-MDH-A and M-MDH-A, in gizzard S-ACON-A, ADH-A, GPI-A, HK-1, HK-3, ME-A PEP-1, and PGM-A, in heart ADH-A, HK-1, HK-3, ME-A, PEP-2, PGM-A, and LDH-A, in pectoralis M-ACON-A, S-ACON-A, ADH-A, HK-1, HK-3, ME-A, PEP-2, and PGM-A, and in liver, gizzard, and heart M-ACON-A, ALD-A, CK-A, G3PDH-A, HK-1, and PGDH-A. Increasing levels of activity were demonstrated in liver ADH-A, ME-A, and PEP-2, in heart M-MDH-A, S-ICDH-A, M-ICDH, and M-AAT-A, and in pectoralis LDH-A, LDH-B, G3PDH-3, ALD-A, CK-A, HK-2, and PGM-B. There was a decrease in the activity of HK-1 in liver and in PEP-1 and PGDH-A in pectoralis muscle throughout development. While CK-C is active in the embryonic pectoralis, CK-A is restricted to later developmental stages. Isozyme expressions in regions of the pectoralis containing fast and slow muscle fibers in 7-month-posthatch individuals were noted and found to be identical. The results underscore the need to use similar developmental stages and tissue samples in comparative electrophoretic studies of birds.