Incorporating non-equilibrium dynamics into demographic history inferences of a migratory marine species.

Understanding how dispersal and gene flow link geographically separated the populations over evolutionary history is challenging, particularly in migratory marine species. In southern right whales (SRWs, Eubalaena australis), patterns of genetic diversity are likely influenced by the glacial climate cycle and recent history of whaling. Here we use a dataset of mitochondrial DNA (mtDNA) sequences (n = 1327) and nuclear markers (17 microsatellite loci, n = 222) from major wintering grounds to investigate circumpolar population structure, historical demography and effective population size. Analyses of nuclear genetic variation identify two population clusters that correspond to the South Atlantic and Indo-Pacific ocean basins that have similar effective breeder estimates. In contrast, all wintering grounds show significant differentiation for mtDNA, but no sex-biased dispersal was detected using the microsatellite genotypes. An approximate Bayesian computation (ABC) approach with microsatellite markers compared the scenarios with gene flow through time, or isolation and secondary contact between ocean basins, while modelling declines in abundance linked to whaling. Secondary-contact scenarios yield the highest posterior probabilities, implying that populations in different ocean basins were largely isolated and came into secondary contact within the last 25,000 years, but the role of whaling in changes in genetic diversity and gene flow over recent generations could not be resolved. We hypothesise that these findings are driven by factors that promote isolation, such as female philopatry, and factors that could promote dispersal, such as oceanographic changes. These findings highlight the application of ABC approaches to infer the connectivity in mobile species with complex population histories and, currently, low levels of differentiation.

Detecting dyads of related individuals in large collections of DNA-profiles by controlling the false discovery rate.

The search for pairs (dyads) of related individuals in large databases of DNA-profiles has become an increasingly important inference tool in ecology. However, the many, partly dependent, pairwise comparisons introduce statistical issues. We show that the false discovery rate (FDR) procedure is well suited to control for the proportion of false positives, i.e. dyads consisting of unrelated individuals, which under normal circumstances would have been labelled as related individuals. We verify the behaviour of the standard FDR procedure by simulation, demonstrating that the FDR procedure works satisfactory in spite of the many dependent pairwise comparisons involved in an exhaustive database screening. A computer program that implements this method is available online. In addition, we propose to implement a second stage in the procedure, in which additional independent genetic markers are used to identify the false positives. We demonstrate the application of the approach in an analysis of a DNA database consisting of 3300 individual minke whales (Balaenoptera acutorostrata) each typed at ten microsatellite loci. Applying the standard procedure with an FDR of 50% led to the identification of 74 putative dyads of 1st- or 2nd-order relatives. However, introducing the second step, which involved additional genotypes at 15 microsatellite loci, revealed that only 21 of the putative dyads can be claimed with high certainty to be true dyads.

Statistical approaches to paternity analysis in natural populations and applications to the North Atlantic humpback whale.

We present a new method for paternity analysis in natural populations that is based on genotypic data that can take the sampling fraction of putative parents into account. The method allows paternity assignment to be performed in a decision theoretic framework. Simulations are performed to evaluate the utility and robustness of the method and to assess how many loci are necessary for reliable paternity inference. In addition we present a method for testing hypotheses regarding relative reproductive success of different ecologically or behaviorally defined groups as well as a new method for estimating the current population size of males from genotypic data. This method is an extension of the fractional paternity method to the case where only a proportion of all putative fathers have been sampled. It can also be applied to provide abundance estimates of the number of breeding males from genetic data. Throughout, the methods were applied to genotypic data collected from North Atlantic humpback whales (Megaptera novaeangliae) to test if the males that appear dominant during the mating season have a higher reproductive success than the subdominant males.

Single-locus tests of microsatellite evolution: multi-step mutations and constraints on allele size.

We evaluate some common simulation procedures as well as a recently developed likelihood method used for testing hypotheses regarding microsatellite evolution. Results from simulated data revealed that the tests for the detection of multi-step mutations in general have some power, whereas tests for the presence of constraints on the repeat number have only very limited power. The tests were applied to population data obtained from nine different baleen whale populations. High agreement was found between results obtained using the simulation-based approach and results obtained using a likelihood ratio test. In four of the nine population samples the tests rejected the one-step mutation model. In two instances the significant deviation was due to excess of heterozygosity and in two instances to a reduced level of heterozygosity relative to the expectations under the stepwise mutation model. The former significant deviation was consistent with occasional multi-step mutations, whereas the latter may indicate the presence of constraints on the number of repeats.

Multiple levels of single-strand slippage at cetacean tri- and tetranucleotide repeat microsatellite loci.

Between three and six tri- and tetranucleotide repeat microsatellite loci were analyzed in 3720 samples collected from four different species of baleen whales. Ten of the 18 species/locus combinations had imperfect allele arrays, i.e., some alleles differed in length by other than simple integer multiples of the basic repeat length. The estimate of the average number of alleles and heterozygosity was higher at loci with imperfect allele arrays relative to those with perfect allele arrays. Nucleotide sequences of 23 different alleles at one tetranucleotide repeat microsatellite locus in fin whales, Balaenoptera physalus, and humpback whales, Megaptera novaeangliae, revealed sequence changes including perfect repeats only, multiple repeats, and partial repeats. The relative rate of the latter two categories of mutation was estimated at 0.024 of the mutation rate involving perfect repeats only. It is hypothesized that single-strand slippage of partial repeats may provide a mechanism for counteracting the continuous expansion of microsatellite loci, which is the logical consequence of recent reports demonstrating directional mutations. Partial-repeat mutations introduce imperfections in the repeat array, which subsequently could reduce the rate of single-strand slippage. Limited computer simulations confirmed this predicted effect of partial-repeat mutations.

Population genetic structure of North Atlantic, Mediterranean Sea and Sea of Cortez fin whales, Balaenoptera physalus (Linnaeus 1758): analysis of mitochondrial and nuclear loci.

Samples were collected from 407 fin whales, Balaenoptera physalus, at four North Atlantic and one Mediterranean Sea summer feeding area as well as the Sea of Cortez in the Pacific Ocean. For each sample, the sex, the sequence of the first 288 nucleotides of the mitochondrial (mt) control region and the genotype at six microsatellite loci were determined. A significant degree of divergence was detected at all nuclear and mt loci between North Atlantic/Mediterranean Sea and the Sea of Cortez. However, the divergence time estimated from the mt sequences was substantially lower than the time elapsed since the rise of the Panama Isthmus, suggesting occasional gene flow between the North Pacific and North Atlantic ocean after the separation of the two oceans. Within the North Atlantic and Mediterranean Sea, significant levels of heterogeneity were observed in the mtDNA between the Mediterranean Sea, the eastern (Spain) and the western (the Gulf of Maine and the Gulf of St Lawrence) North Atlantic. Samples collected off West Greenland and Iceland could not be unequivocally assigned to either of the two areas. The homogeneity tests performed using the nuclear data revealed significant levels of divergence only between the Mediterranean Sea and the Gulf of St Lawrence or West Greenland. In conclusion, our results suggest the existence of several recently diverged populations in the North Atlantic and Mediterranean Sea, possibly with some limited gene flow between adjacent populations, a population structure which is consistent with earlier population models proposed by Kellogg, Ingebrigtsen, and Sergeant.

Genetic tagging of humpback whales.

The ability to recognize individual animals has substantially increased our knowledge of the biology and behaviour of many taxa. However, not all species lend themselves to this approach, either because of insufficient phenotypic variation or because tag attachment is not feasible. The use of genetic markers ('tags') represents a viable alternative to traditional methods of individual recognition, as they are permanent and exist in all individuals. We tested the use of genetic markers as the primary means of identifying individuals in a study of humpback whales in the North Atlantic Ocean. Analysis of six microsatellite loci among 3,060 skin samples collected throughout this ocean allowed the unequivocal identification of individuals. Analysis of 692 'recaptures', identified by their genotype, revealed individual local and migratory movements of up to 10,000 km, limited exchange among summer feeding grounds, and mixing in winter breeding areas, and also allowed the first estimates of animal abundance based solely on genotypic data. Our study demonstrates that genetic tagging is not only feasible, but generates data (for example, on sex) that can be valuable when interpreting the results of tagging experiments.

Population structure and seasonal movements of narwhals, Monodon monoceros, determined from mtDNA analysis.

We determined the nucleotide sequence of the first 287 base pairs in the mitochondrial control region from 74 narwhals, Monodon monoceros, collected in the North-west Atlantic. We detected four polymorphic sites that defined five haplotypes, two of which were found in single specimens. The same DNA sequence was characterized in an additional 353 specimens by digestion with two restriction endonucleases. In this manner each specimen could be assigned to one of the three most common haplotypes. The nucleotide diversity for the total sample (as well as the sequenced subset) was estimated as 0.0017 and pairwise genetic distances between haplotypes ranged from 0.0035-0.0070. The low nucleotide diversity and the low average pairwise genetic distance between haplotypes suggest a recent expansion in abundance from a small founding population. Despite the low degree of variation, frequencies of the common haplotypes differed markedly between areas. The results indicate isolation, even between geographically close areas, as well as fidelity to specific summer and autumn feeding grounds. Heterogeneity within a presumed single breeding ground suggests mixing of pods with different haplotypic composition.

Molecular analysis of paternity shows promiscuous mating in female humpback whales (Megaptera novaeangliae, Borowski).

It is widely assumed that the mating system of the humpback whale. Magaptera novaeangliae, is similar to that of most mammals in that it represents some form of polygyny or promiscuity, but this cannot be tested without observations of copulation or data on paternity of offspring. Microsatellite DNA markers were used to examine the paternity of calves born to individually identified mature female humpback whales from the Gulf of Maine. Skin biopsies were obtained from three females, and several (range: three to five) of their known offspring. Multiple paternity of offspring, indicated by the presence of at least three different paternal alleles, was evident in all three females at either three or four of the six microsatellite loci surveyed. Such promiscuous mating is expected given current knowledge of the social ecology of this species. It is also consistent with resightings of individually identified female humpbacks with different male associates during two or more breeding seasons.