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The availability of public genomic resources can greatly assist biodiversity assessment, conservation, and restoration efforts by providing evidence for scientifically informed management decisions. Here we survey the main approaches and applications in biodiversity and conservation genomics, considering practical factors, such as cost, time, prerequisite skills, and current shortcomings of applications. Most approaches perform best in combination with reference genomes from the target species or closely related species. We review case studies to illustrate how reference genomes can facilitate biodiversity research and conservation across the tree of life. We conclude that the time is ripe to view reference genomes as fundamental resources and to integrate their use as a best practice in conservation genomics., Trends in Genetics, 39 (7), ISSN:0168-9525

Pleistocene environmental changes are generally assumed to have dramatically affected species’ demography via changes in habitat availability, but this is challenging to investigate due to our limited knowledge of how Pleistocene ecosystems changed through time. Here, we tracked changes in shallow marine habitat availability resulting from Pleistocene sea level fluctuations throughout the last glacial cycle (120–14 thousand years ago; kya) and assessed correlations with past changes in genetic diversity inferred from genome-wide SNPs, obtained via ddRAD sequencing, in Caribbean hawksbill turtles, which feed in coral reefs commonly found in shallow tropical waters. We found sea level regression resulted in an average 75% reduction in shallow marine habitat availability during the last glacial cycle. Changes in shallow marine habitat availability correlated strongly with past changes in hawksbill turtle genetic diversity, which gradually declined to ~1/4th of present-day levels during the Last Glacial Maximum (LGM; 26–19 kya). Shallow marine habitat availability and genetic diversity rapidly increased after the LGM, signifying a population expansion in response to warming environmental conditions. Our results suggest a positive correlation between Pleistocene environmental changes, habitat availability and species’ demography, and that demographic changes in hawksbill turtles were potentially driven by feeding habitat availability. However, we also identified challenges associated with disentangling the potential environmental drivers of past demographic changes, which highlights the need for integrative approaches. Our conclusions underline the role of habitat availability on species’ demography and biodiversity, and that the consequences of ongoing habitat loss should not be underestimated.

The occasional westward transport of warm water of the Agulhas Current, ‘Agulhas leakage’, around southern Africa has been suggested to facilitate tropical marine connectivity between the Atlantic and Indian oceans, but the ‘Agulhas leakage’ hypothesis doesn’t explain the signatures of eastward gene flow observed in many tropical marine fauna. We investigated an alternative hypothesis: the establishment of a warm-water corridor during comparatively warm interglacial periods. The ‘warm-water corridor’ hypothesis was investigated by studying the population genomic structure of Atlantic and Southwest Indian Ocean green turtles (N = 27) using 12,035 genome-wide single nucleotide polymorphisms (SNPs) obtained via ddRAD sequencing. Model-based and multivariate clustering suggested a hierarchical population structure with two main Atlantic and Southwest Indian Ocean clusters, and a Caribbean and East Atlantic sub-cluster nested within the Atlantic cluster. Coalescent-based model selection supported an ancestral position of the East Atlantic from which the Southwest Indian Ocean and Caribbean populations diverged during the transition from the last interglacial period (130 – 115 thousand years ago; kya) to the last glacial period (115 – 90 kya). The onset of the last glaciation appeared to isolate Atlantic and Southwest Indian Ocean green turtles into three refugia, which subsequently came into secondary contact in the Caribbean and Southwest Indian Ocean when global temperatures increased after the Last Glacial Maximum. Our findings support the establishment of a warm-water corridor facilitating tropical marine connectivity between the Atlantic and Southwest Indian Ocean during warm interglacials.

Progress in genome sequencing now enables the large-scale generation of reference genomes. Various international initiatives aim to generate reference genomes representing global biodiversity. These genomes provide unique insights into genomic diversity and architecture, thereby enabling comprehensive analyses of population and functional genomics, and are expected to revolutionize conservation genomics., Trends in Ecology & Evolution, 37 (3), ISSN:0169-5347, ISSN:1872-8383

Currently, three stocks of sei whales (Balaenoptera borealis) are defined in the North Atlantic; the Nova Scotian, Iceland-Denmark Strait and Eastern North Atlantic stocks, which are mainly based upon historical catch and sighting data. We analyzed mitochondrial control region DNA (mtDNA) sequences and genotypes from 7 to 11 microsatellite loci in 87 samples from three sites in the North Atlantic; Iceland, the Gulf of Maine and the Azores, and compared against the North Pacific using 489 previously published samples. No statistically significant deviations from homogeneity were detected among the North Atlantic samples at mtDNA or microsatellite loci. The genealogy estimated from the mtDNA sequences revealed a clear division of the haplotypes into a North Atlantic and a North Pacific clade, with the exception of one haplotype detected in a single sample from the Azores, which was included in the North Pacific clade. Significant genetic divergence between the North Atlantic and North Pacific Oceans was detected (mtDNA ΦST = 0.72, microsatellite Weir and Cockerham’s ϴ = 0.20; p

Understanding how habitat quality in heterogeneous landscapes governs the distribution and fitness of individuals is a fundamental aspect of ecology. While mean individual fitness is generally considered a key to assessing habitat quality, a comprehensive understanding of habitat quality in heterogeneous landscapes requires estimates of dispersal rates among habitat types. The increasing accessibility of genomic approaches, combined with field-based demographic methods, provides novel opportunities for incorporating dispersal estimation into assessments of habitat quality. In this study, we integrated genomic kinship approaches with field-based estimates of fitness components and Approximate Bayesian Computation (ABC) procedures to estimate habitat-specific dispersal rates and characterize habitat quality in two-toed sloths (Choloepus hoffmanni) occurring in a Costa Rican agricultural ecosystem. Field-based observations indicated that birth and survival rates were similar in a sparsely-shaded cacao farm and adjacent cattle pasture-forest mosaic. Sloth density was threefold higher in pasture compared to cacao, whereas home range size and overlap were greater in cacao compared to pasture. Dispersal rates were similar between the two habitats, as estimated using ABC procedures applied to the spatial distribution of pairs of related individuals identified using 3,431 SNP and 11 microsatellite locus genotypes. Our results indicate that crops produced under a sparse overstory can, in some cases, constitute lower quality habitat than pasture-forest mosaics for sloths, perhaps because of differences in food resources or predator communities. Finally, our study demonstrates that integrating field-based demographic approaches with genomic methods can provide a powerful means for characterizing habitat quality for animal populations occurring in heterogeneous landscapes. This article is protected by copyright. All rights reserved.

HighlightsMitochondrial monophyly is commonly employed to define evolutionary significant units.Monophyly may be caused by insufficient sampling or a recent common ancestor.Mitogenomic studies are generally based on few samples and prone to sampling issues.Expanded mitogenome sampling negates previous monophyly in fin whales.AbstractThe advent of massive parallel sequencing technologies has resulted in an increase of studies based upon complete mitochondrial genome DNA sequences that revisit the taxonomic status within and among species. Spatially distinct monophyly in mitogenomic genealogies, i.e., the sharing of a recent common ancestor among con-specific samples collected in the same region has been viewed as evidence for subspecies. Several recent studies in cetaceans have employed this criterion to suggest subsequent intraspecific taxonomic revisions. We reason that employing intra-specific, spatially distinct monophyly at non-recombining, clonally inherited genomes is an unsatisfactory criterion for defining subspecies based upon theoretical (genetic drift) and practical (sampling effort) arguments. This point is illustrated by a re-analysis of a global mitogenomic assessment of fin whales,Balaenoptera physalusspp., published by Archer et al. (2013) which proposed to further subdivide the Northern Hemisphere fin whale subspecies,B. p. physalus. The proposed revision was based upon the detection of spatially distinct monophyly among North Atlantic and North Pacific fin whales in a genealogy based upon complete mitochondrial genome DNA sequences. The extended analysis conducted in this study (1,676 mitochondrial control region, 162 complete mitochondrial genome DNA sequences and 20 microsatellite loci genotyped in 358 samples) revealed that the apparent monophyly among North Atlantic fin whales reported by Archer et al. (2013) to be due to low sample sizes. In conclusion, defining sub-species from monophyly (i.e., the absence of para-or polyphyly) can lead to erroneous conclusions due to relatively “trivial” aspects, such as sampling. Basic population genetic processes (i.e., genetic drift and migration) also affect the time to most recent common ancestor and hence the probability that individuals in a sample are monophyletic.

Cetaceans are a clade of highly specialized aquatic mammals that include the largest animals that have ever lived. The largest whales can have ∼1,000× more cells than a human, with long lifespans, leaving them theoretically susceptible to cancer. However, large-bodied and long-lived animals do not suffer higher risks of cancer mortality than humans—an observation known as Peto’s Paradox. To investigate the genomic bases of gigantism and other cetacean adaptations, we generated a de novo genome assembly for the humpback whale (Megaptera novaeangliae) and incorporated the genomes of ten cetacean species in a comparative analysis. We found further evidence that rorquals (family Balaenopteridae) radiated during the Miocene or earlier, and inferred that perturbations in abundance and/or the interocean connectivity of North Atlantic humpback whale populations likely occurred throughout the Pleistocene. Our comparative genomic results suggest that the evolution of cetacean gigantism was accompanied by strong selection on pathways that are directly linked to cancer. Large segmental duplications in whale genomes contained genes controlling the apoptotic pathway, and genes inferred to be under accelerated evolution and positive selection in cetaceans were enriched for biological processes such as cell cycle checkpoint, cell signaling, and proliferation. We also inferred positive selection on genes controlling the mammalian appendicular and cranial skeletal elements in the cetacean lineage, which are relevant to extensive anatomical changes during cetacean evolution. Genomic analyses shed light on the molecular mechanisms underlying cetacean traits, including gigantism, and will contribute to the development of future targets for human cancer therapies.

Restriction-enzyme-based sequencing methods enable the genotyping of thousands of single nucleotide polymorphism (SNP) loci in non-model organisms. However, in contrast to traditional genetic markers, genotyping error rates in SNPs derived from restriction-enzyme-based methods remain largely unknown. Here, we estimated genotyping error rates in SNPs genotyped with double digest RAD sequencing from Mendelian incompatibilities in known mother-offspring dyads of Hoffman's two-toed sloth (Choloepus hoffmanni) across a range of coverage and sequence quality criteria, for both reference-aligned and de novo-assembled datasets. Genotyping error rates were more sensitive to coverage than sequence quality and low coverage yielded high error rates, particularly in de novo-assembled datasets. For example, coverage ≥5 yielded median genotyping error rates of ≥0.03 and ≥0.11 in reference-aligned- and de novo-assembled datasets, respectively. Genotyping error rates declined to ≤0.01 in reference-aligned datasets with a coverage >30, but remained >0.04 in the de novo-assembled datasets. We observed approximately 10- and 13-fold declines in the number of loci sampled in the reference-aligned and de novo-assembled datasets when coverage was increased from >5 to >30 at quality score ≥30, respectively. Finally, we assessed the effects of genotyping coverage on a common population genetic application, parentage assignments, and showed that the proportion of incorrectly assigned maternities was relatively high at low coverage. Overall, our results suggest that the tradeoff between sample size and genotyping error rates be considered prior to building sequencing libraries, reporting genotyping error rates become standard practice, and that effects of genotyping errors on inference be evaluated in restriction-enzyme-based SNP studies. This article is protected by copyright. All rights reserved.

Characterizing how frequently, and at what life stages and spatial scales, dispersal occurs can be difficult, especially for species with cryptic juvenile periods and long reproductive life spans. Using a combination of mark-recapture information, microsatellite genetic data, and demographic simulations, we characterize natal and breeding dispersal patterns in the long-lived, slow-maturing, and endangered Blanding's turtle (Emydoidea blandingii), focusing on nesting females. We captured and genotyped 310 individual Blanding's turtles (including 220 nesting females) in a central Wisconsin population from 2010 to 2013, with additional information on movements among 3 focal nesting areas within this population available from carapace-marking conducted from 2001 to 2009. Mark-recapture analyses indicated that dispersal among the 3 focal nesting areas was infrequent (

Kinship analyses are important pillars of ecological and conservation genetic studies with potentially far-reaching implications. There is a need for power analyses that address a range of possible relationships. Nevertheless, such analyses are rarely applied, and studies that use genetic-data-based-kinship inference often ignore the influence of intrinsic population characteristics. We investigated 11 questions regarding the correct classification rate of dyads to relatedness categories (relatedness category assignments; RCA) using an individual-based model with realistic life history parameters. We investigated the effects of the number of genetic markers; marker type (microsatellite, single nucleotide polymorphism SNP, or both); minor allele frequency; typing error; mating system; and the number of overlapping generations under different demographic conditions. We found that (i) an increasing number of genetic markers increased the correct classification rate of the RCA so that up to >80% first cousins can be correctly assigned; (ii) the minimum number of genetic markers required for assignments with 80 and 95% correct classifications differed between relatedness categories, mating systems, and the number of overlapping generations; (iii) the correct classification rate was improved by adding additional relatedness categories and age and mitochondrial DNA data; and (iv) a combination of microsatellite and single-nucleotide polymorphism data increased the correct classification rate if

The appeal of genetic inference methods to assess population genetic structure and guide management efforts is grounded in the correlation between the genetic similarity and gene flow among populations. Effects of such gene flow are typically genomewide; however, some loci may appear as outliers, displaying above or below average genetic divergence relative to the genomewide level. Above average population, genetic divergence may be due to divergent selection as a result of local adaptation. Consequently, substantial efforts have been directed toward such outlying loci in order to identify traits subject to local adaptation. Here, we report the results of an investigation into the molecular basis of the substantial degree of genetic divergence previously reported at allozyme loci among North Atlantic fin whale (Balaenoptera physalus) populations. We sequenced the exons encoding for the two most divergent allozyme loci (MDH-1 and MPI) and failed to detect any nonsynonymous substitutions. Following extensive error checking and analysis of additional bioinformatic and morphological data, we hypothesize that the observed allozyme polymorphisms may reflect phenotypic plasticity at the cellular level, perhaps as a response to nutritional stress. While such plasticity is intriguing in itself, and of fundamental evolutionary interest, our key finding is that the observed allozyme variation does not appear to be a result of genetic drift, migration, or selection on the MDH-1 and MPI exons themselves, stressing the importance of interpreting allozyme data with caution. As for North Atlantic fin whale population structure, our findings support the low levels of differentiation found in previous analyses of DNA nucleotide loci.

Inferring the demographic history of species and their populations is crucial to understand their contemporary distribution, abundance and adaptations. The high computational overhead of likelihood-based inference approaches severely restricts their applicability to large data sets or complex models. In response to these restrictions, approximate Bayesian computation (ABC) methods have been developed to infer the demographic past of populations and species. Here, we present the results of an evaluation of the ABC-based approach implemented in the popular software package diyabc using simulated data sets (mitochondrial DNA sequences, microsatellite genotypes and single nucleotide polymorphisms). We simulated population genetic data under five different simple, single-population models to assess the model recovery rates as well as the bias and error of the parameter estimates. The ability of diyabc to recover the correct model was relatively low (0.49): 0.6 for the simplest models and 0.3 for the more complex models. The recovery rate improved significantly when reducing the number of candidate models from five to three (from 0.57 to 0.71). Among the parameters of interest, the effective population size was estimated at a higher accuracy compared to the timing of events. Increased amounts of genetic data did not significantly improve the accuracy of the parameter estimates. Some gains in accuracy and decreases in error were observed for scaled parameters (e.g., N-e) compared to unscaled parameters (e.g., N-e and ). We concluded that diyabc-based assessments are not suited to capture a detailed demographic history, but might be efficient at capturing simple, major demographic changes.

The Greenlandic and west-central Siberian breeding populations of Sanderlings Calidris alba are separated by ca. 2000 km during the breeding season, but mix in Europe to some extent during migration. However, the number of Siberian Sanderlings that spend the non- breeding season along the East Atlantic Flyway (extending from western Europe to South Africa), if any, is unknown. Although both populations are considered part of the nominate subspecies C. a. alba based on morphology, population structure in Sanderlings has yet to be described with molecular methods. We examined genetic differenti- ation at the mtDNA control region (CR) and seven microsatellite loci between Greenland- and Siberia-breed- ing Sanderlings in order to: (1) develop a diagnostic tool for assessing the breeding origin of Sanderlings along the East Atlantic Flyway, and (2) provide a comparison with the co-distributed and ecologically similar Red Knot, in which CR differentiation of geographically analogous populations (C. canutus islandica and C. c. canutus) has indicated isolation of lineages near the time of the last glacial maximum. By contrast, we found only weak dif- ferentiation between the Sanderling breeding populations at the CR, and no differentiation at microsatellite loci. These results suggest that the assignment of breeding ori- gin of Sanderlings on Afro-European flyways will not be possible with simple and inexpensive genetic methods, and imply that Sanderlings and Red Knots have very different post-glacial phylogeographic histories.

The identification of population bottlenecks is critical in conservation because populations that have experienced significant reductions in abundance are subject to a variety of genetic and demographic processes that can hasten extinction. Genetic bottleneck tests constitute an appealing and popular approach for determining if a population decline has occurred because they only require sampling at a single point in time, yet reflect demographic history over multiple generations. However, a review of the published literature indicates that, as typically applied, microsatellite-based bottleneck tests often do not detect bottlenecks in vertebrate populations known to have experienced declines. This observation was supported by simulations that revealed that bottleneck tests can have limited statistical power to detect bottlenecks largely as a result of limited sample sizes typically used in published studies. Moreover, commonly assumed values for mutation model parameters do not appear to encompass variation in microsatellite evolution observed in vertebrates and, on average, the proportion of multi-step mutations is underestimated by a factor of approximately two. As a result, bottleneck tests can have a higher probability of 'detecting' bottlenecks in stable populations than expected based on the nominal significance level. We provide recommendations that could add rigor to inferences drawn from future bottleneck tests and highlight new directions for the characterization of demographic history.

Although single-nucleotide polymorphisms (SNPs) have become the marker of choice in the field of human genetics, these markers are only slowly emerging in ecological, evolutionary and conservation genetic analyses of nonmodel species. This is partly because of difficulties associated with the discovery and characterization of SNP markers. Herein, we adopted a simple straightforward approach to identifying SNPs, based on screening of a random genomic library. In total, we identified 768 SNPs in the ringed seal, Pusa hispida hispida, in samples from Greenland and Svalbard. Using three seal samples, SNPs were discovered at a rate of one SNP per 402 bp, whereas re-sequencing of 96 seals increased the density to one SNP per 29 bp. Although applicable to any species of interest, the approach is especially well suited for SNP discovery in nonmodel organisms and is easily implemented in any standard genetics laboratory, circumventing the need for prior genomic data and use of next-generation sequencing facilities.

Identifying population structure is one of the most common and important objectives of spatial analyses using population genetic data. Population structure is detected either by rejecting the null hypothesis of a homogenous distribution of genetic variation, or by estimating low migration rates. Issues arise with most current population genetic inference methods when the genetic divergence is low among putative populations. Low levels of genetic divergence may be as a result of either high ongoing migration or historic high migration but no current, ongoing migration. We direct attention to recent developments in the use of the tempo-spatial distribution of closely related individuals to detect population structure or estimate current migration rates. These 'kinship-based' approaches complement more traditional population-based genetic inference methods by providing a means to detect population structure and estimate current migration rates when genetic divergence is low. However, for kinship-based methods to become widely adopted, formal estimation procedures applicable to a range of species life histories are needed.

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.

Genetic assignment methods provide an appealing approach for characterizing dispersal patterns on ecological time scales, but require sufficient genetic differentiation to accurately identify migrants and a large enough sample size of migrants to, for example, compare dispersal between sexes or age classes. We demonstrate that assignment methods can be rigorously used to characterize dispersal patterns in a marbled murrelet (Brachyramphus marmoratus) population from central California that numbers approximately 600 individuals and is only moderately differentiated (F(ST)similar to 0.03) from larger populations to the north. We used coalescent simulations to select a significance level that resulted in a low and approximately equal expected number of type I and II errors and then used this significance level to identify a population of origin for 589 individuals genotyped at 13 microsatellite loci. The proportion of migrants in central California was greatest during winter when 83% of individuals were classified as migrants compared to lower proportions during the breeding (6%) and post-breeding (8%) seasons. Dispersal was also biased toward young and female individuals, as is typical in birds. Migrants were rarely members of parent-offspring pairs, suggesting that they contributed few young to the central California population. A greater number of migrants than expected under equilibrium conditions, a lack of individuals with mixed ancestry, and a small number of potential source populations (two), likely allowed us to use assignment methods to rigorously characterize dispersal patterns for a population that was larger and less differentiated than typically thought required for the identification of migrants.

How do populations of highly mobile species inhabiting open environments become reproductively isolated and evolve into new species? We test the hypothesis that elevated ocean-surface temperatures can facilitate allopatry among pelagic populations and thus promote speciation. Oceanographic modelling has shown that increasing surface temperatures cause localization and reduction of upwelling, leading to fragmentation of feeding areas critical to pelagic species. We test our hypothesis by genetic analyses of populations of two closely related baleen whales, the Antarctic minke whale (Balaenoptera bonaerensis) and common minke whale (Balaenoptera acutorostrata) whose current distributions and migration patterns extent are largely determined by areas of consistent upwelling with high primary production. Phylogeographic and population genetic analyses of mitochondrial DNA control-region nucleotide sequences collected from 467 whales sampled in four different ocean basins were employed to infer the evolutionary relationship among populations of B. acutorostrata by rooting an intraspecific phylogeny with a population of B. bonaerensis. Our findings suggest that the two species diverged in the Southern Hemisphere less than 5 million years ago (Ma). This estimate places the speciation event during a period of extended global warming in the Pliocene. We propose that elevated ocean temperatures in the period facilitated allopatric speciation by disrupting the continuous belt of upwelling maintained by the Antarctic Circumpolar Current. Our analyses revealed that the current populations of B. acutorostrata likely diverged after the Pliocene some 1.5 Ma when global temperatures had decreased and presumably coinciding with the re-establishment of the polar-equatorial temperature gradient that ultimately drives upwelling. In most population samples, we detected genetic signatures of exponential population expansions, consistent with the notion of increasing carrying capacity after the Pliocene. Our hypothesis that prolonged periods of global warming facilitate speciation in pelagic marine species that depend on upwelling should be tested by comparative analyses in other pelagic species.

During the austral winter, adult female southern right whales Eubalaena australis enter the South African coastal waters to give birth and raise their young. Most births take place over a 4-month period, when the females congregate in specific coastal areas or nursery grounds for up to a recorded maximum of 105 days. At this time, the density of cowtextendashcalf pairs in nursery areas can reach as high as 3.2 pairs/km2 over 26 km of coastline. Although a single young is born and suckled exclusively for 7 months to a year, recent observations on nursery grounds include 3 incidents where apparently abandoned/orphaned calves-of-the-year have been seen associating with a minimum of 2textendash3 different cowtextendashcalf pairs over periods of 11textendash38 days. Attempts to suckle from these females have been noted in 2 of the cases, with the response of the female varying from extreme avoidance to apparent tolerance. In one instance where the observations of the same trio extended over 21 days, the non-offspring appeared to compete at least equally with the offspring, even though the mother directed her evasive tactics more at the non-offspring than her own calf. At the same time, both of the calves exhibited some growth in length when compared with the size of the adult female: their subsequent survival is unknown. Non-offspring nursing in monotocous species is generally rare, and the costs to the female potentially high: this is certainly the case for seasonally feeding mysticetes such as the right whale, where the costs of lactation cannot be recovered until the cow resumes feeding about 4 months after parturition. Hence, it is perhaps not surprising that these are the first recorded observations of contemporaneous nursing attempts by offspring and non-offspring calves of any mysticete.Key Words

Extractions from non-invasive hair samples usually yield low amounts of highly degraded DNA. Previously developed mammal molecular sexing methods were not designed with such sub-optimal conditions in mind. We developed a simple and reliable PCR-based sexing method aimed at degraded, low yield DNA extractions from the giant panda (Ailuropoda melanoleuca). Comparisons of this new primer set with others showed that the reliability of sex determination from low-yield, degraded DNA extractions was improved if; amplification products were short (

Global warming is predicted to affect the evolutionary potential of natural populations. We assessed genetic diversity of 25 populations of desert bighorn sheep ( Ovis canadensis nelsoni ) in southeastern California, where temperatures have increased and precipitation has decreased during the 20th century. Populations in low-elevation habitats had lower genetic diversity, presumably reflecting more fluctuations in population sizes and founder effects. Higher-elevation habitats acted as reservoirs of genetic diversity. However, genetic diversity was also affected by population connectivity, which has been disrupted by human development. Restoring population connectivity may be necessary to buffer the effects of climate change on this desert-adapted ungulate.

Key words: baleen whale, kinship, Mysteceti, STR lociInconservationgenetics,itisbecomingincreasinglyclear that parameters derived over evolutionarytime scales may not apply to much shorter ecolog-ical timescales (Palsboll 1999). Molecular geneticmethods can be employed to estimate parametersonanecological timescale ifthe focusis aimedonlyat recently diverged lineages, i.e., among individu-als as opposed to among populations. Theseextensions of current approaches are vital to alignthe application of molecular genetics to contem-porary issues in conservation. In order to estimatethe degree of kinship in a reliable manner, an ade-quate number of loci must be analyzed per indi-vidual. Ensuring an adequate number of locidecreases/eliminates the interference of other levelsof relatives, and compensates for the exponentialincrease in the number of pair-wise comparisonswhen the sample size increases. Towards this end,we presented, in this note, an additional 17 poly-morphic microsatellite loci, which, originated froma di-, tri- and tetra-nucleotide microsatellite locienriched library constructed from humpbackwhale, Megaptera novaeangliae, and fin whale,Balaenoptera physalus, genomic DNA, using theprotocol previously described in Palsboll et al.(1997) and Be´rube´et al. (2000).More specifically, the microsatellite loci wereisolated from size-selected total-cell DNAextracted from humpback whale and fin whaleusing the approach described by Rassmann andcolleagues (1991). Recombinant colonies (dotblotted to a ZetaProbe

For many years, researchers have speculatedthat fin whales are year-round residents in theSea of Cortez (= Gulf of California). Previouswork by Berube and co-workers has shownthat the degree of genetic diversity among finwhales in the Sea of Cortez at nuclear andmitochondrial loci is highly reduced. However,the relatively unobstructed connection with theNorth Pacific Ocean argues that Sea of Cortezfin whales are part of a much larger easternNorth Pacific population given the extensivemigratory ranges observed in fin whales andbaleen whales in general. The low degree ofgenetic variation might thus simply be due tohistoric fluctuations in the effectivepopulation size of an eastern North Pacificpopulation. In order to test if the reducedgenetic variation detected among fin whales inthe Sea of Cortez is due to small populationsize or a past bottleneck in an otherwise largeeastern North Pacific population, we analyzedthe geographic distribution of geneticvariation at a single mitochondrial (controlregion) and 16 nuclear loci in samplescollected from fin whales in the eastern NorthPacific (n = 12) as well as the Sea of Cortez(n = 77). Our results showed that fin whalesobserved in the Sea of Cortez constitute ahighly isolated and thus evolutionary uniquepopulation, which warrants special conservationmeasures given the current low estimate ofabundance of approximately 400 individuals.

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.

It is believed that short interspersed elements (SINEs) are irreversibly inserted into genomes. We use this concept to try to deduce the evolution of whales using sequence and hybridization studies. The observation that microsatellites are associated with SINEs lead us to screen sequences surrounding cetacean microsatellites for artiodactyl-derived SINEs. Two sequences that were thought to be cetacean SINEs and the bovine SINE were aligned for comparison to sequences flanking microsatellites from ungulates. The bovine SINE was observed only in ruminants while CetSINE1 and 2 were found in mammals. Hybridization studies using these three SINEs revealed that CetSINE1 was found in all ungulates and cetaceans with the strongest hybridization signal observed in the hippopotamus and beluga; CetSINE2 hybridized to all ungulate suborders, while the bovine SINE was only observed in Ruminantia. It is proposed that these putative SINEs are not 'generic' SINEs but mammalian-wide interspersed repeats (MIRs). Caution is urged: what initially appears to be a SINE may instead be a MIR and have reduced evolutionary resolving power.

We developed a simple and reliable method to identify carnivore scats to species using PCR and RFLP of a portion of the mtDNA cytochrome b gene, which works for seven of the most common carnivores in western North America. We identified a short (196 bp) polymorphic region of cytochrome b which would be easily amplifiable even from degraded DNA, developed a primer set, and isolated a set of three restriction enzymes (HpaII, DdeI, HpyCH4V) that would identify the seven target species. In order to test whether this protocol would effectively identify scats obtained in the field we collected 243 carnivore scats from 12 sites in the San Francisco Bay area. Eighty five percent (206) of our samples successfully amplified and were subsequently identified to species using our RFLP protocol. We selected 108 of these samples to sequence; our species identifications based on sequencing were identical to those obtained using our PCR-RFLP method. Our PCR-RFLP method is a simple and efficient means to identify carnivore scats to species, eliminating the need for sequencing, which is costly and requires more laboratory equipment. The technique can also be modified depending on the species present at a particular site. It allows for rapid and noninvasive assessment of multiple carnivore taxa and is particularly useful for surveying populations across many sites.

Hyalopterus pruni is an invasive aphid pest in California. To study the population biology of this pest both in California and its native Mediterranean region, we have developed 11 di- and tri-nucleotide repeat microsatellite markers. Each locus amplified in individuals representing the full range of geographical regions and host plants where Hyalopterus is found. Polymorphism was high, ranging from six to 22 alleles per locus in the individuals screened. These loci represent the first microsatellites developed for Hyalopterus and they should be of great value in studying the invasion biology and population structure of this insect pest.

In response to our review of the use of genetic bottleneck tests in the conservation literature (Peery etal. 2012, Molecular Ecology, 21, 3403-3418), Hoban etal. (2013, Molecular Ecology, in press) conducted population genetic simulations to show that the statistical power of genetic bottleneck tests can be increased substantially by sampling large numbers of microsatellite loci, as they suggest is now possible in the age of genomics. While we agree with Hoban and co-workers in principle, sampling large numbers of microsatellite loci can dramatically increase the probability of committing type 1 errors (i.e. detecting a bottleneck in a stable population) when the mutation model is incorrectly assumed. Using conservative values for mutation model parameters can reduce the probability of committing type 1 errors, but doing so can result in significant losses in statistical power. Moreover, we believe that practical limitations associated with developing large numbers of high-quality microsatellite loci continue to constrain sample sizes, a belief supported by a literature review of recent studies using next generation sequencing methods to develop microsatellite libraries. conclusion, we maintain that researchers employing genetic bottleneck tests should proceed with caution and carefully assess both statistical power and type 1 error rates associated with their study design.

Background Telomeres, the protective cap of chromosomes, have emerged as powerful markers of biological age and life history in model and non-model species. The qPCR method for telomere length estimation is one of the most common methods for telomere length estimation, but has received recent critique for being too error-prone and yielding unreliable results. This critique coincides with an increasing awareness of the potentials and limitations of the qPCR technique in general and the proposal of a general set of guidelines (MIQE) for standardization of experimental, analytical, and reporting steps of qPCR. In order to evaluate the utility of the qPCR method for telomere length estimation in non-model species, we carried out four different qPCR assays directed at humpback whale telomeres, and subsequently performed a rigorous quality control to evaluate the performance of each assay. Results Performance differed substantially among assays and only one assay was found useful for telomere length estimation in humpback whales. The most notable factors causing these inter-assay differences were primer design and choice of using singleplex or multiplex assays. Inferred amplification efficiencies differed by up to 40% depending on assay and quantification method, however this variation only affected telomere length estimates in the worst performing assays. Conclusion Our results suggest that seemingly well performing qPCR assays may contain biases that will only be detected by extensive quality control. Moreover, we show that the qPCR method for telomere length estimation can be highly precise and accurate, and thus suitable for telomere measurement in non-model species, if effort is devoted to optimization at all experimental and analytical steps. We conclude by highlighting a set of quality controls which may serve for further standardization of the qPCR method for telomere length estimation, and discuss some of the factors that may cause variation in qPCR experiments.

We determined the gender of a variety of cetacean species, including both ondotocetes and mysticetes, using the polymerase chain reaction for amplification of the sex chromosome specific regions ZFY/ZFX and SRY. This quick and simple method requires extremely small amounts of tissue, and therefore allows gender to be determined from skin biopsies taken from free-ranging specimens. In the fin whale, Balaenoptera physalus, no gender-specific bands were observed when the ZFY/ZFX system was used, but when the SRY system was used, sex was accurately determined. Previous studies in other mammals have also shown the SRY system to be more reliable in sex determination. We therefore recommend amplification of the SRY region alone or in parallel with the ZFY/ZFX regions, as described here, as a test for gender in cetaceans and other mammals.

Genetic divergence among conspecific subpopulations can be due to either low recurrent gene flow or recent divergence and no gene flow. Here we present a modification of an earlier method developed by Nielsen and Wakeley (2001), which accommodates a finite-site mutation model, to assess which of the two models of divergence is most likely given the observed data. We apply the method to nucleotide sequence data collected from the variable part of the mitochondrial control region in fin whales (Balaenoptera physalus) from the Atlantic coast off Spain and the Mediterranean Sea. Our estimations strongly favor a model of recurrent gene flow over a model of recent divergence and zero gene flow. We estimated the migration rate at two females per generation. While the estimated rate is high by evolutionary standards, exchange rates of this order of magnitude is low from an ecological and conservation perspective and entirely consistent with the current paucity of fin whale sightings in the Strait of Gibraltar today. Intensive commercial shore-based whaling during the 1920s removed substantial numbers of fin whales in the Strait of Gibraltar and this local population has seemingly since failed to recover.

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 repeat. (C) 1999 Academic Press.

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.

Mitochondrial DNA haplotypes of humpback whales show strong segregation between oceanic populations and between feeding grounds within oceans, but this highly structured pattern does not exclude the possibility of extensive nuclear gene flow. Here we present allele frequency data for four microsatellite loci typed across samples from four major oceanic regions: the North Atlantic (two mitochondrially distinct populations), the North Pacific, and two widely separated Antarctic regions, East Australia and the Antarctic Peninsula. Allelic diversity is a little greater in the two Antarctic samples, probably indicating historically greater population sizes. Population subdivision was examined using a wide range of measures, including F-st, various alternative forms of Slatkin's R(st), Goldstein and colleagues' Delta mu, and a Monte Carlo approximation to Fisher's exact test. The exact test revealed significant heterogeneity in all but one of the pairwise comparisons between geographically adjacent populations, including the comparison between the two North Atlantic populations, suggesting that, gene flow between oceans is minimal and that dispersal patterns may sometimes be restricted even in the absence of obvious barriers, such as land masses, warm water belts, and antitropical migration behavior. The only comparison where heterogeneity was not detected was the one between the two Antarctic population samples. It is unclear whether failure to find a difference here reflects gene flow between the regions or merely lack of statistical power arising from the small size of the Antarctic Peninsula sample. Our comparison between measures of population subdivision revealed major discrepancies between methods, with little agreement about which populations were most and least separated. We suggest that unbiased R(st) (UR(st), see Goodman 1995) is currently the most reliable statistic, probably because, unlike the other methods, it allows for unequal sample sizes. However, in view of the fact that these alternative measures often contradict one another, we urge caution in the use of microsatellite data to quantify genetic distance.

We sequenced 540 nucleotides of the last exon in the ZFY/ZFX gene in two males and two females for eight cetacean species; four odontocetes (toothed whales) and four mysticetes (baleen whales). Based upon the obtained nucleotide sequences, we designed two sets of oligonucleotide primers for specific amplification of the ZFX and the ZFY sequence in odontocetes and mysticetes, respectively. Each primer set consisted of three oligonucleotides; one forward-orientated primer, which anneals to the ZFY as well as the ZFX sequence, and two reverse-orientated primers that anneal to either the ZFX or or the ZFY sequence. The resulting two amplification products (specific for the ZFY and ZFX sequences) can be distinguished by gel-electrophoresis through 2% NuSieve(TM). The accuracy of the technique was tested by determination of gender in 214 individuals of known sex. Finally we applied the technique to determine the sex of 3570 cetacean specimens; 2284 humpback whales, 315 fin whales, 37 blue whales, 7 minke whales, as well as 592 belugas, 335 narwhals and 25 harbour porpoises.

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. Fil: Carroll, E. L.. University of St. Andrews; Reino Unido Fil: Alderman, R.. Water and Environment; Australia Fil: Bannister, J. L.. The Western Australian Museum; Australia Fil: Bérubé, M.. University of Groningen; Países Bajos Fil: Best, P. B.. No especifica; Fil: Boren, L.. New Zealand Department of Conservation; Nueva Zelanda Fil: Baker, C. S.. State University of Oregon; Estados Unidos. University of Auckland; Nueva Zelanda Fil: Constantine, R.. University of Auckland; Nueva Zelanda Fil: Findlay, K.. Cape Peninsula University of Technology; Sudáfrica Fil: Harcourt, R.. Macquarie University; Australia Fil: Lemaire, L.. University of St. Andrews; Reino Unido Fil: Palsbøll, P. J.. University of Groningen; Países Bajos Fil: Patenaude, N. J.. Collégial International Sainte-Anne; Canadá Fil: Rowntree, V. J.. University of Utah; Estados Unidos Fil: Seger, J.. University of Utah; Estados Unidos Fil: Steel, D.. State University of Oregon; Estados Unidos Fil: Valenzuela, Luciano Oscar. Universidad Nacional del Centro de la Provincia de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Utah; Estados Unidos Fil: Watson, M.. Water and Planning; Australia Fil: Gaggiotti, O. E.. University of St. Andrews; Reino Unido

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 pair-wise 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.

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.