Our purpose was to identify an experimental procedure using PCR that provides a reliable genotype at a microsatellite locus using only a few picograms of template DNA. Under these circumstances, it is possible (i) that one allele of a heterozygous individual will not be detected and (ii) that PCR-generated alleles or 'false alleles' will arise. A mathematical model has been developed to account for stochastic events when pipetting template DNA in a very dilute DNA extract and computer simulations have been performed. Laboratory experiments were also carried out using DNA extracted from a bear feces sample to determine if experimental results correlate with the mathematical model. The results of 150 typing experiments are consistent with the proposed model. Based on this model and the level of observed false alleles, an experimental procedure using the multiple tubes approach is proposed to obtain reliable genotypes with a confidence level of 99%. This multiple tubes procedure should be systematically used when genotyping nuclear loci of ancient or forensic samples, museum specimens and hair or feces of free ranging animals.

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Pyrenean brown bears Ursus arctos are threatened with extinction. Management efforts to preserve this population require a comprehensive knowledge of the number and sex of the remaining individuals and their respective home ranges. This goal has been achieved using a combination of noninvasive genetic sampling of hair and faeces collected in the field and corresponding track size data. Genotypic data were collected at 24 microsatellite loci using a rigorous multiple-tubes approach to avoid genotyping errors associated with low quantities of DNA. Based on field and genetic data, the Pyrenean population was shown to be composed at least of one yearling, three adult males, and one adult female. These data indicate that extinction of the Pyrenean brown bear population is imminent without population augmentation. To preserve the remaining Pyrenean gene pool and increase genetic diversity, we suggest that managers consider population augmentation using only females. This study demonstrates that comprehensive knowledge of endangered small populations of mammals can be obtained using noninvasive genetic sampling.

Understanding the factors that influence the rate at which natural populations lose genetic diversity is a central aspect of conservation genetics because of the importance of genetic diversity in maintaining evolutionary potential and individual fitness. Concerns about loss of genetic diversity are particularly relevant to large carnivores, such as brown bears ( Ursus arctos ), that are distributed at low densities and are highly susceptible to human-caused population fragmentation. We used eight highly variable nuclear microsatellite markers to study current levels of genetic variation across the North American range of brown bears. The highest levels of within-population genetic diversity ( H e ϭ 0.76) were found in northern populations in the core of the North American distribution. Diversity was significantly lower in populations at the southern fringe of the distribution, in the Northwest Territories, and in southwest Alaska. Diversity was lower still in the Yellowstone Ecosystem population ( H e ϭ 0.55), an isolated remnant of the larger distribution that recently extended south from the Canadian border into Mexico. The insular population on the Kodiak Archipelago had very low genetic diversity ( H e ϭ 0.26). The Yellowstone and Kodiak data suggest that the effective population size for brown bears is much smaller than previously suspected. These results indicate that the levels of diversity in most undisturbed populations can be maintained only through connections to populations on the scale of the current North American distribution. At the same time, the Kodiak data demonstrate that populations well under the size recommended for long-term conservation can persist and thrive for thousands of years, although the probability of such persistence remains unknown. Variación en la Diversidad Genética a lo largo del Rango de Distribución del Oso Café de NorteaméricaResumen: Entender los factores que influeyen en la tasa a la cual las poblaciones naturales pierden diversidad genética es un aspecto central de la genética de la conservación debido a la importancia de la diversidad genética en el mantenimiento del potencial evolutivo y la condición individual. Las preocupaciones sobre la pérdida de la diversidad genética tiene particular relevancia para los carnivoros mayores, como lo es el oso café ( Ursus arctos ), que se distribuye a bajas densidades y que es altamente susceptible a la fragmentación poblacional causada por humanos. Para estudiar los niveles actuales de variación genética a lo largo del rango de distribución de los osos cafés, usamos ocho marcadores microsatélite nucleares altamente variables. Los niveles mas altos de diversidad genética intrapoblacional ( H e ϭ 0.76) se encontraron en poblaciones del Norte en el centro de la distribución en Norteamérica. La diversidad fue significativamente menor en poblaciones limítrofes sureñas, en los territorios del Noroeste y el Suroeste de Alaska. La diversidad fue mas baja aún en la población de Yellowstone ( H e ϭ 0.55), un remanente de una distribución...

Hybridization presents a unique challenge for conservation biologists and managers. While hybridization is an important evolutionary process, hybridization is also a threat formany native species. The endangered species recovery effort for the red wolf Canis rufus is a classic system for understanding and addressing the challenges of hybridization. From 1987?1993, 63 red wolves were released from captivity in eastern North Carolina, USA, to establish a free-ranging, non-essential experimental population. By 1999, managers recognized hybridization with invasive coyotes Canis latrans was the single greatest threat to successful recovery, and an adaptive management plan was adopted with innovative approaches for managing the threat of hybridization. Here we review the application and results of the adaptive management efforts from 1993 to 2013 by comparing: (1) the numbers of wolves, coyotes, and hybrids captured, (2) the numbers of territorial social groups with presumed breeding capabilities, (3) the number of red wolf and hybrid litters documented each year and (4) the degree of coyote introgression into the wild red wolf gene pool. We documented substantial increases in the number of known red wolves and red wolf social groups from 1987–2004 followed by a plateau and slight decline by 2013.The number of red wolf litters exceeded hybrid litters each year and the proportion of hybrid litters per year averaged 21%. The genetic composition of the wild red wolf population is estimated to include < 4% coyote ancestry from recent introgression since reintroduction. We conclude that the adaptive management plan was effective at reducing the introgression of coyote genes into the red wolf population, but population recovery of red wolves will require continuation of the current management plan, or alternative approaches, for the foreseeable future. More broadly, we discuss the lessons learned from red wolf adaptive management that could assist other endangered species recovery efforts facing the challenge of minimizing hybridization.

The population concept is central in evolutionary and conservation biology, but identifying the boundaries of natural populations is often challenging. Here, we present a new approach for assessing spatial genetic structure without the a priori assumptions on the locations of populations made by adopting an individual-centred approach. Our method is based on assignment tests applied in a moving window over an extensively sampled study area. For each individual, a spatially explicit probability surface is constructed, showing the estimated probability of finding its multilocus genotype across the landscape, and identifying putative migrants. Population boundaries are localized by estimating the mean slope of these probability surfaces over all individuals to identify areas with genetic discontinuities. The significance of the genetic discontinuities is assessed by permutation tests. This new approach has the potential to reveal cryptic population structure and to improve our ability to understand gene flow dynamics across landscapes. We illustrate our approach by simulations and by analysing two empirical datasets: microsatellite data of Ursus arctos in Scandinavia, and amplified fragment length polymorphism (AFLP) data of Rhododendron ferrugineum in the Alps.

The historical distribution of the brown bear ( Ursus arctos ) in North America included Alaska, western Canada, the western and midwestern states, plus northern Mexico. Currently, the brown bear is limited to Alaska, the Canadian provinces of the Yukon, Northwest Territories, British Columbia, and Alberta, and six threatened subpopulations in the lower 48 states. To examine the evolutionary history of U. arctos in North America and to assess the genetic divergence between individuals from different geographic regions, we obtained 294 nucleotides of mitochondrial DNA sequence data from the control region for 317 free-ranging brown bears. Twenty-eight unique sequences, or mitochondrial DNA haplotypes were detected. The average sequence divergence between haplotypes was high (4.3%), and some haplotypes differed by as many as 23 nucleotides. Phylogenetic analyses using maximum parsimony revealed four major mitochondrial DNA phylogeographic groups, or clades. The significant phylogeographic structure detected in brown bears strongly contrasts with results obtained for other large carnivores and suggests limited female-mediated gene flow. The mitochondrial DNA phylogeographic clades do not correlate with taxonomic classifications for U. arctos , and we hypothesize that the clades were formed prior to migration of this species into North America. We suggest evolutionarily significant units for conservation in three Filogenia del ADN Mitocondrial del Oso Café de Norte América y sus Implicaciones en la Conservación Resumen: La distribución histórica del oso caf é ( Ursus arctos ) en Norte Am é ica includía Alaska, oeste de Canada, oeste y medio oeste de los Estados Unidos y Norte de M é xico. Actualmete, el oso caf é esta limitado a Alaska, las provincias Canadienses de Yukon, Northwest territories, British Columbia, Alberta y seis subpoblaciones amenazadas en 48 estados de los Estados Unidos. Obtuvimos datos de secuencias de 294 nucleótidos de ADN mitocondrial de la región control de 317 osos cafes para examinar la historia evolutiva de U. arctos en Norte Am é rica y evaluar la divergencia gen é tica entre individuos de diferentes regiones geográficas. Fueron detectadas veintiocho secuencias únicas o haplotipos de ADN mitocondrial. El porcentaje de secuencia divergente entre haplotipos fue alto (4.3%) y algunos haplotipos difirieron por tantos como 23 nucleótidos. Análisis filogen é ticos usando maxima parsimonia revelaron cuatro grupos mayores de ADN mitocondrial filogeográfico. La estructura filogeografica significativa detectada en osos caf é s contrasta fuertemente con resultados obtenidos de otros carnivoros mayores y sugiere flujo genético limitado, mediado por las hembras. Los grupos de ADN mitocondrial filogeográfico no se correlacionan con clasificaciones taxonómicas de U. arctos , nosotros hipotetizamos que los grupos fueron formados antes de las migraciones de esta especie hacia Norte América. Sugerimos unidades evolutivamente significativas para su conservación en tres regiones geográficas: 1) las islas Admi...

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