Which future for the French Pyrenean brown bear (Ursus arctos) population? An approach using stage-structured deterministic and stochastic models

The Pyrenean brown bear (Ursus arctos) population is considered as one of the most seriously threatened with extinction in Western Europe. To assess its viability and possible needs of augmentation, we develop deterministic and stochastic stage-structured demographic models. The deterministic model reveals that a bear population cannot have a high annual growth rate and is particularly sensitive to breeder survival. High demographic parameters appear to be crucial to population persistence, especially for a small population that remains vulnerable to demographic and environmental stochasticities. The Pyrenean population cannot therefore be considered as viable. Successful conservation strategies for this population would require releasing more bears in both sub-populations in the near future.     

Genetic diversity, structure, and size of an endangered brown bear population threatened by highway construction in the Pindos Mountains, Greece

One of the major negative effects of roads is the creation of barriers to the movement of wildlife, ultimately disconnecting populations and increasing extinction risk. We collected genetic data from a threatened brown bear population in the central part of the Pindos mountain range in northwestern Greece to provide information about this, as yet genetically undescribed, population and to evaluate its status prior to the construction of a major highway. We used noninvasive genetic sampling methods and microsatellite analysis to investigate nuclear genetic diversity, population genetic structure, demographic history, relatedness within the population and estimated effective and total population size. Brown bears in the study area were found to possess a relatively high level of nuclear genetic diversity and low levels of inbreeding; the population did not show any signs of substructuring but seems to have gone through a genetic bottleneck in the recent past. The estimated effective population size was 29, and the total population size estimate obtained by two different methods was 33 and 51 individuals, respectively. Our results indicate a good conservation status of this bear population and provide baseline genetic data for the future evaluation of the effects on bears from the construction of a major highway, for monitoring the genetic status of this and other bear populations in Greece and for assessing gene flow in bear populations in southern Europe.     

Estimating survival in the Apennine brown bear accounting for uncertainty in age classification

For most rare and elusive species, estimating age-specific survival is a challenging task, although it is an important requirement to understand the drivers of population dynamics, and to inform conservation actions. Apennine brown bears Ursus arctos marsicanus are a small, isolated population under a severe risk of extinction, for which the main demographic mechanisms underlying population dynamics are still unknown, and population trends have not been formally assessed. We present a 12-year analysis of their survival rates using non-invasive genetic sampling data collected through four different sampling techniques. By using multi-event capture–recapture models, we estimated survival probabilities for two broadly defined age classes (cubs and older individuals), even though the age of the majority of sampled bears was unknown. We also applied the Pradel model to provide a preliminary assessment of population trend during the study period. Survival was different between cubs [ϕ = 0.51, 95% CI (0.22, 0.79)], adult males [ϕ = 0.85, 95% CI (0.76, 0.91)] and adult females [ϕ = 0.92, 95% CI (0.87, 0.95)], no temporal variation in survival emerged, suggesting that bear survival remained substantially stable throughout the study period. The Pradel analysis of population trend yielded an estimate of λ = 1.009 [SE = 0.018; 95% CI (0.974, 1.046)]. Our results indicate that, despite the status of full legal protection, the basically stable demography of this relict population is compatible with the observed lack of range expansion, and that a relatively high cub mortality could be among the main factors depressing recruitment and hence population growth.     

Environmental DNA from Residual Saliva for Efficient Noninvasive Genetic Monitoring of Brown Bears (Ursus arctos)

Noninvasive genetic sampling is an important tool in wildlife ecology and management, typically relying on hair snaring or scat sampling techniques, but hair snaring is labor and cost intensive, and scats yield relatively low quality DNA. New approaches utilizing environmental DNA (eDNA) may provide supplementary, cost-effective tools for noninvasive genetic sampling. We tested whether eDNA from residual saliva on partially-consumed Pacific salmon (Oncorhynchus spp.) carcasses might yield suitable DNA quality for noninvasive monitoring of brown bears (Ursus arctos). We compared the efficiency of monitoring brown bear populations using both fecal DNA and salivary eDNA collected from partially-consumed salmon carcasses in Southeast Alaska. We swabbed a range of tissue types from 156 partially-consumed salmon carcasses from a midseason run of lakeshore-spawning sockeye (O. nerka) and a late season run of stream-spawning chum (O. keta) salmon in 2014. We also swabbed a total of 272 scats from the same locations. Saliva swabs collected from the braincases of salmon had the best amplification rate, followed by swabs taken from individual bite holes. Saliva collected from salmon carcasses identified unique individuals more quickly and required much less labor to locate than scat samples. Salmon carcass swabbing is a promising method to aid in efficient and affordable monitoring of bear populations, and suggests that the swabbing of food remains or consumed baits from other animals may be an additional cost-effective and valuable tool in the study of the ecology and population biology of many elusive and/or wide-ranging species.     

Microsatellite diversity and structure of Carpathian brown bears (Ursus arctos): consequences of human caused fragmentation

The formerly large, continuous brown bear population of the Carpathians has experienced a radical decrease in population size due to human activities which have resulted in splitting the population into the larger Eastern Carpathian and the smaller Western Carpathian subpopulations. In the Western Carpathians, brown bears came close to extinction at the beginning of 1930s, but thanks to both conservation and management efforts the bear population has begun to recover. In contrast, the Eastern Carpathian subpopulation in Romania has never dropped below 800 individuals, potentially preserving the original amount of genetic variation. In this paper we present results of a genetic study of brown bear subpopulations distributed in the Slovak and Romanian sections of the Carpathians using 13 nuclear microsatellites. The documented level of genetic differentiation between the Western and Eastern Carpathian subpopulations reflects the isolation which lasted almost 100 years. Furthermore, the existence of two, different, genetic clusters within the Western Carpathians despite close geographic proximity indicates that human-caused fragmentation and isolation have resulted in significant genetic divergence. Although the subpopulations display an indication of genetic bottleneck, the level of genetic diversity is within the range commonly observed in different brown bear populations. The results presented here point out the significance of human exploitation to the population structure of this large carnivore species. Future management efforts should be aimed at securing and restoring the connectivity of forested habitats, in order to preserve the genetic variation of the Carpathian brown bear subpopulations and to support the gene flow between them.     

The power of genetic monitoring for studying demography,ecology and genetics of a reintroduced brown bear population

Genetic monitoring has rarely been used for wildlife translocations despite the potential benefits this approach offers, compared to traditional field‐based methods. We applied genetic monitoring to the reintroduced brown bear population in northern Italy. From 2002 to 2008, 2781 hair and faecal samples collected noninvasively plus 12 samples obtained from captured or dead bears were used to follow the demographic and geographical expansion and changes in genetic composition. Individual genotypes were used to reconstruct the wild pedigree and revealed that the population increased rapidly, from nine founders to >27 individuals in 2008 (λ = 1.17–1.19). Spatial mapping of bear samples indicated that most bears were distributed in the region surrounding the translocation site; however, individual bears were found up to 163 km away. Genetic diversity in the population was high, with expected heterozygosity of 0.74–0.79 and allelic richness of 4.55–5.41. However, multi‐year genetic monitoring data showed that mortality rates were elevated, immigration did not occur, one dominant male sired all cubs born from 2002 to 2005, genetic diversity declined, relatedness increased, inbreeding occurred, and the effective population size was extremely small (Ne = 3.03, ecological method). The comprehensive information collected through genetic monitoring is critical for implementing future conservation plans for the brown bear population in the Italian Alps. This study provides a model for other reintroduction programmes by demonstrating how genetic monitoring can be implemented to uncover aspects of the demography, ecology and genetics of small and reintroduced populations that will advance our understanding of the processes influencing their viability, evolution, and successful restoration.     

Genetic diversity, structure and conservation of the endangered Cantabrian Capercaillie in a unique peripheral habitat

Populations at the rear edge of the species’ range are often at a high risk of extinction due to their isolation, fragmentation and small population sizes. However, these populations also play a relevant role in the conservation of biodiversity since they may represent a valuable genetic resource. The endangered Cantabrian Capercaillie (Tetrao urogallus cantabricus) inhabits deciduous forests of the Cantabrian Mountains of Spain, at the southwestern limit of the species’ range. Recently, nine Cantabrian Capercaillie leks were discovered in Pyrenean oak forests of the southern slope of the Cantabrian range, where the subspecies historically occurred. To elucidate if the origin of this peripheral population nucleus is the result of a historical range contraction or a recent re-colonization from the core population, we sampled moulted feathers from all the known leks in the southern peripheral forests and from the adjacent main core population, based on nine microsatellite loci genotypes. No significant genetic differentiation was detected between main core and peripheral forests suggesting that gene flow is not interrupted between these nuclei. Contrary to expected, peripheral forests did not represent sink populations, since gene flow mainly occurred from southern peripheral to northern main core forests. Therefore, the origin of these birds inhabiting the peripheral nucleus seems not to be a recent colonization but relicts from the former distribution range that have remained unnoticed in a drier and warmer environment than described so far for the species. Cantabrian Capercaillie faces a high risk of extinction in the southernmost forests of its distribution, not only because of its peripheral location but also due to its small population size, low genetic diversity and low incoming gene flow. According to our results, this peripheral nucleus could represent an expanding edge for the population if Pyrenean oak forests continue to spread out southwards and consequently stress the need for conservation programs to preserve habitat availability and forest connectivity.     

Nuclear DNA microsatellite analysis of genetic diversity and gene flow in the Scandinavian brown bear (Ursus arctos)

In the 1930s, the Scandinavian brown bear was close to extinction due to vigorous extermination programmes in Norway and Sweden. Increased protection of the brown bear in Scandinavia has resulted in the recovery of four subpopulations, which currently contain close to 1000 individuals. Effective conservation and management of the Scandinavian brown bear requires knowledge of the current levels of genetic diversity and gene flow among the four subpopulations. Earlier studies of mitochondrial DNA (mtDNA) diversity revealed extremely low levels of genetic variation, and population structure that grouped the three northern subpopulations in one genetic clade and the southernmost subpopulation in a second highly divergent clade. In this study, we extended the analysis of genetic diversity and gene flow in the Scandinavian brown bear using data from 19 nuclear DNA microsatellite loci. Results from the nuclear loci were strikingly different than the mtDNA results. Genetic diversity levels in the four subpopulations were equivalent to diversity levels in nonbottlenecked populations from North America, and significantly higher than levels in other bottlenecked and isolated brown bear populations. Gene flow levels between subpopulations ranged from low to moderate and were correlated with geographical distance. The substantial difference in results obtained using mtDNA and nuclear DNA markers stresses the importance of collecting data from both types of genetic markers before interpreting data and making recommendations for the conservation and management of natural populations. Based on the results from the mtDNA and nuclear DNA data sets, we propose one evolutionarily significant unit and four management units for the brown bear in Scandinavia.     

Genetic data confirm critical status of the reintroduced Dinaric population of Eurasian lynx

Eurasian lynx (Lynx lynx) reintroduction to the Dinaric Mountains is considered one of the most successful reintroductions of a large predator. Six reintroduced animals founded the population, which rapidly expanded from Slovenia, through Croatia, and all the way to Bosnia and Herzegovina. However, a decrease of the population size has been observed during the last 10–15 years. Considering that possible inbreeding depression would be additive to threats like poaching, traffic mortality and prey base depletion, another extinction of this species from the Dinaric Mountains is a real possibility. We analyzed 204 samples collected between 1979 and 2010 using twenty microsatellite loci and 900-bp mitochondrial DNA control region sequence to evaluate conservation genetics aspects of this endangered population. Both markers confirmed low genetic variability of the Dinaric lynx population, and considerable effective inbreeding (0.3) compared to the source Carpathian population. Our analysis of effective population size and microsatellite variability supported field observations of decreasing population number. As a natural recolonization is a very remote possibility, we recommend population augmentation from a large source population.     

Review of capture-recapture methods applicable to noninvasive genetic sampling

The use of noninvasive genetic sampling to identify individual animals for capture-recapture studies has become widespread in the past decade. Strong emphasis has been placed on the field protocols and genetic analyses with fruitful results. Little attention has been paid to the capture-recapture application for this specific type of data beyond stating the effects of assumption violations. Here, we review the broad class of capture-recapture methods that are available for use with DNA-based capture-recapture data, noting the array of biologically interesting parameters such as survival, emigration rates, state transition rates and the finite rate of population change that can be estimated from such data. We highlight recent developments in capture-recapture theory specifically designed for noninvasive genetic sampling data.     

Sexing free-ranging brown bears Ursus arctos using hairs found in the field

As an aid to the management of the Pyrenean population of the brown bear Ursus arctos , a sexing method based on the amplification of a Y chromosome specific sequence has been developed, and tested using hairs found in the field as a source of DNA. This method involves a two-step polymerase chain reaction (PCR) which allows the detection of a very small amount of DNA, probably a single SRY gene molecule. The sex can reliably be identified using about 50pg of DNA extract as template. It is possible that this approach could, with adjustments, be used to identify the sex of other species of eutherian mammals.     

Genetic sampling for estimating density of common species

Understanding population dynamics requires reliable estimates of population density, yet this basic information is often surprisingly difficult to obtain. With rare or difficult‐to‐capture species, genetic surveys from noninvasive collection of hair or scat has proved cost‐efficient for estimating densities. Here, we explored whether noninvasive genetic sampling (NGS) also offers promise for sampling a relatively common species, the snowshoe hare (Lepus americanus Erxleben, 1777), in comparison with traditional live trapping. We optimized a protocol for single‐session NGS sampling of hares. We compared spatial capture–recapture population estimates from live trapping to estimates derived from NGS, and assessed NGS costs. NGS provided population estimates similar to those derived from live trapping, but a higher density of sampling plots was required for NGS. The optimal NGS protocol for our study entailed deploying 160 sampling plots for 4 days and genotyping one pellet per plot. NGS laboratory costs ranged from approximately $670 to $3000 USD per field site. While live trapping does not incur laboratory costs, its field costs can be considerably higher than for NGS, especially when study sites are difficult to access. We conclude that NGS can work for common species, but that it will require field and laboratory pilot testing to develop cost‐effective sampling protocols.     

Conserving small and fragmented populations of large mammals: Non-invasive genetic sampling in an isolated population of Asian elephants in Nepal

The Terai is one of the world's most spectacular landscapes, encompassing parts of Nepal and northern India. This area used to harbour large and continuous populations of charismatic species like elephants, tigers and rhinoceros. However, recent habitat fragmentation reduced these populations into small, partially or completely isolated remnants. The largest of these fragments in Nepal is the Bardia National Park. Here, the elephant population was functionally extinct in the early 1970s and -80s, but was rescued by a considerable number of immigrants in 1994. In order to assess population size, sex ratio, age structure, and levels of genetic variation, we carried out non-invasive genetic sampling, using elephant dung as the source of DNA. A capture-mark-recapture estimate of population size suggested that there were 57 individuals in the study area, which agrees well with field observations. Notably, a strongly male-biased sex ratio was evident among sub-adult individuals. This observation suggests the presence of sub-adult immigrants in the population, which was supported by formal migrant detection analysis. Genetic variation was quite high and the evidence for male immigrants suggests that there are good prospects for maintenance of genetic diversity. A decade ago a large-scale project was initiated in the Terai region to link remaining populations of large mammals through dispersal corridors. The program is basically founded on the assumption that habitat fragments are isolated with little or no migration between them. Our results indicate that this may not be the case, at least not for the Asian elephant in western Nepal, which therefore reduces the alleged extinction risk from genetic erosion and stochastic demographic events.     

A machine learning approach to investigate the reasons behind species extinction

Species extinction is one of the most important phenomena in conservation biology. Many factors are involved in the disappearance of species, including stochastic population fluctuations, habitat change, resource depletion, and inbreeding. Due to the complexity of the interactions between these various factors and the lengthy time period required to make empirical observations, studying the phenomenon of species extinction can prove to be very difficult in nature. On the other hand, an investigation of the various features involved in species extinction using individual-based simulation modeling and machine learning techniques can be accomplished in a reasonably short period of time. Thus, the aim of this paper is to investigate multiple factors involved in species extinction using computer simulation modeling. We apply several machine learning techniques to the data generated by EcoSim, a predator–prey ecosystem simulation, in order to select the most prominent features involved in species extinction, along with extracting rules that outline conditions that have the potential to be used for predicting extinction. In particular, we used five feature selection methods resulting in the selection of 25 features followed by a reduction of these to 14 features using correlation analysis. Each of the remaining features was placed in one of three broad categories, viz., genetic, environmental, or demographic. The experimental results suggest that factors such as population fluctuation, reproductive age, and genetic distance are important in the occurrence of species extinction in EcoSim, similar to what is observed in nature. We argue that the study of the behavior of species through Individual-Based Modeling has the potential to give rise to new insights into the central factors involved in extinction for real ecosystems. This approach has the potential to help with the detection of early signals of species extinction that could in turn lead to conservation policies to help prevent extinction.     

Conservation genetics of maned wolves in a highly impacted area of the Brazilian Cerrado biome

Maned wolves are large canids currently considered vulnerable to extinction due to habitat loss. They are still commonly found within the urban mesh inside the Brazilian Federal District (Distrito Federal—DF), in nearby Protected Areas (PAs), and in surrounding farms. We evaluated the genetic diversity of maned wolves in three PAs of the DF, using both invasive and noninvasive techniques to obtain DNA that was later amplified for five microsatellite markers. We sampled 23 wolves: 10 with the noninvasive method, three captured in traps, six road-killed, and four rescued in urban areas. In águas Emendadas Ecological Station (ESECAE) we also used samples from six specimens captured between 1997 and 1998 for a temporal comparison. For maned wolves, non-invasive techniques are affordable and easier to conduct in the field, while laboratory costs are much lower for invasive samples. Hence, a sampling strategy combining both techniques may provide an interesting approach for molecular ecology studies requiring comprehensive coverage of local individuals. On the basis of such integrated sampling scheme, our analyses indicated that none of the investigated populations currently present deviations from Hardy–Weinberg expectations or indication of inbreeding. Furthermore, in ESECAE there was no reduction in genetic diversity during the last 9 years. Overall, maned wolves did not present evidence of genetic structuring among the three sampled PAs. These results thus indicate that individual exchange among PAs is still occurring at sufficient rates to avoid differentiation, and/or that the recent fragmentation in the region has not yet produced measurable effects in the genetic diversity of maned wolves.     

Noninvasive genetic sampling reveals intrasex territoriality in wolverines

Due to its conspicuous manifestations and its capacity to shape the configuration and dynamics of wild populations, territorial behavior has long intrigued ecologists. Territoriality and other animal interactions in situ have traditionally been studied via direct observations and telemetry. Here, we explore whether noninvasive genetic sampling, which is increasingly supplementing traditional field methods in ecological research, can reveal territorial behavior in an elusive carnivore, the wolverine (Gulo gulo). Using the locations of genotyped wolverine scat samples collected annually over a period of 12 years in central Norway, we test three predictions: (1) male home ranges constructed from noninvasive genetic sampling data are larger than those of females, (2) individuals avoid areas used by other conspecifics of the same sex (intrasexual territoriality), and (3) avoidance of same‐sex territories diminishes or disappears after the territory owner's death. Each of these predictions is substantiated by our results: sex‐specific differences in home range size and intrasexual territoriality in wolverine are patently reflected in the spatial and temporal configuration of noninvasively collected genetic samples. Our study confirms that wildlife monitoring programs can utilize the spatial information in noninvasive genetic sampling data to detect and quantify home ranges and social organization.     

Genetic evidence of recent population contraction in the southernmost population of giant pandas

Anthropogenic habitat loss and fragmentation have been implicated in the endangerment and extinction of many species. Here we assess genetic variation and demographic history in the southernmost population of giant pandas (Ailuropoda melanoleuca) that continues to be threatened by habitat degradation and fragmentation, using noninvasive genetic sampling, mitochondrial control region sequence and 12 microsatellite loci. Compared to other giant panda populations, this population has medium-level genetic diversity based on the measure of both mitochondrial and nuclear markers. Mitochondrial DNA-based demographic analyses revealed that no historical population expansion or contraction has occurred, indicating a relatively stable population size. However, a Bayesian-coalescent method based on the observed allele distribution and allele frequencies of microsatellite clearly did detect, quantify and date a recent decrease in population size. Overall, the results indicate that a population contraction in the order of 95-96% has taken place over the last 910-999 years and is most likely due to anthropogenic habitat loss. These findings highlight the need for a greater focus on habitat protection and restoration for the long-term survival of this giant panda population.     

Evidence for recent population bottlenecks in northern spotted owls (<Emphasis Type="Italic">Strix occidentalis caurina</Emphasis>)

The northern spotted owl (Strix occidentalis caurina) is one of the most controversial threatened subspecies ever listed under the US Endangered Species Act. Despite protection of its remaining forest habitat, recent field studies show continued declines of northern spotted owls. One potential threat to northern spotted owls which has not yet been shown is loss of genetic variation from population bottlenecks. Bottlenecks can increase the probability of mating among related individuals, potentially causing inbreeding depression, and can decrease adaptive potential. Here we report evidence for recent bottlenecks in northern spotted owls using a large genetic dataset (352 individuals and 10 microsatellite loci). The signature of bottlenecks was strongest in the Washington Cascade Mountains, in agreement with field data. Our results provide independent evidence that northern spotted owls have recently declined, and suggest that loss of genetic variation is an emerging threat to the subspecies’ persistence. Reduced effective population size (N _e) shown here in addition to field evidence for demographic decline highlights the increasing vulnerability of this bird to extinction.     

Estimating population size of endangered brush-tailed rock-wallaby (Petrogale penicillata) colonies using faecal DNA

The brush-tailed rock-wallaby (Petrogale penicillata) is an endangered species in southeastern Australia and many of the remaining populations are declining. The steep rocky habitat and shy nature of the species make it difficult to obtain data on population parameters such as abundance and recruitment. Faecal pellet counts from scat plots are commonly used to monitor population trends but these are imprecise and difficult to relate to absolute population size. We conducted a noninvasive genetic sampling 'mark-recapture' study over a 2-year period to identify individuals from faecal DNA samples and estimate the population size of four brush-tailed rock-wallaby colonies located in Wollemi National Park, New South Wales. Scat plots in rock-wallaby colonies were used as sample collection points for this study. Two separate population estimates were carried out for three of the colonies to determine if we could detect recruitment and changes in population size. We determined that there was one large colony of an estimated 67 individuals (95% confidence interval: 55-91) and three smaller colonies. Monitoring of the smaller colonies also detected possible population size increases in all three. Our results indicate that faecal DNA analysis may be a promising method for estimating and monitoring population trends in this species particularly when used with a traditional field survey method.     

Addressing challenges in non invasive capture-recapture based estimates of small populations: a pilot study on the Apennine brown bear

It is often difficult to determine optimal sampling design for non-invasive genetic sampling, especially when dealing with rare or elusive species depleted of genetic diversity. To address this problem, we ran a hair-snag pilot study on the remnant Apennine brown bear population. We used occupancy models to estimate the performance of an improved field protocol, a meta-analysis approach to indirectly model capture probability, and simulations to evaluate the effect of genotyping errors on the accuracy of capture-recapture population estimates. In spring 2007 we collected 70 bear hair samples in 15 5 × 5 km cells, using 5 10-day trapping sessions. Bear detectability was higher in 2007 than in a previous attempt on the same population in 2004, reflecting improved field protocols and sampling design. However, individual capture probability was 0.136 (95% CI = 0.120–0.152), still below the minimum requirements of capture-mark-recapture closed population models. We genotyped hair samples (n = 63) at 9 microsatellite loci, obtaining 94% Polymerase Chain Reaction success, and 13 bear genotypes. Estimated P_IDsib was 0.00594, and per-genotype error rate was 0.13, corresponding to a 99% probability of correct individual identification. Simulation studies showed that the effect of non-corrected or filtered genetic errors on the accuracy of population estimates was negligible only when individual capture probability was >0.2. Our results underline how the interaction among field protocols, sampling strategies and genotyping errors may affect the accuracy of DNA-based estimates of small and genetically depleted populations, and warned us about the feasibility of a survey using only traditional hair-snag sampling. In this and similar cases, indications from pilot studies can provide cost-effective means to evaluate the efficiency of designed sampling and modelling procedures.