Genetic structure of mountain lion (Puma concolor) populations in California

Analysis of 12 microsatellite loci from431 mountain lions (Puma concolor)revealed distinct genetic subdivision that wasassociated with geographic barriers andisolation by distance in California. Levels ofgenetic variation differed among geographicregions, and mountain lions that inhabitedcoastal areas exhibited less heterozygositythan those sampled inland. The San FranciscoBay and Sacramento-San Joaquin River Delta, theCentral Valley, and the Los Angeles Basinappeared to be substantial barriers to geneflow, and allele frequencies of populationsseparated by those features differedsubstantially. A partial barrier to gene flowappeared to exist along the crest of the SierraNevada. Estimated gene flow was high amongmountain lions inhabiting the Modoc Plateau,the western Sierra Nevada, and northern sectionof the eastern Sierra Nevada. SouthernCalifornia mountain lion populations mayfunction as a metapopulation; however, humandevelopments threaten to eliminate habitat andmovement corridors. While north-south geneflow along the western Sierra Nevada wasestimated to be very high, projected loss andfragmentation of foothill habitat may reducegene flow and subdivide populations. Preservation of existing movement corridorsamong regions could prevent population declinesand loss of genetic variation. This studyshows that mountain lion management andconservation efforts should be individualizedaccording to region and incorporatelandscape-level considerations to protecthabitat connectivity.     

Differentiating between Steller sea lion (Eumetopias jubatus) and northern fur seal (Callorhinus ursinus) scats through analysis of faecal DNA

We describe a method to determine the species of pinniped from faeces collected from sympatric Steller sea lion (Eumetopias jubatus) and northern fur seal (Callorhinus ursinus) rookeries using newly developed species-specific primers that amplify a 667-669-base pair segment from the mitochondrial DNA (mtDNA) cytochrome B (cytB) gene region. The primers yielded the correct species in 100% of tissue samples from 10 known animals and 100% of faecal samples from 13 known animals. Species could be identified unequivocally for 87.7% of faecal samples from 122 unknown individuals. The ability to differentiate between scats of sympatrically breeding Steller sea lions and northern fur seals will contribute to the range-wide knowledge of the foraging strategies of both species as well as allow researchers to examine the niche partitioning and potential resource competition between the two predators.     

Estimating abundance of mountain lions from unstructured spatial sampling

Mountain lions (Puma concolor) are often difficult to monitor because of their low capture probabilities, extensive movements, and large territories. Methods for estimating the abundance of this species are needed to assess population status, determine harvest levels, evaluate the impacts of management actions on populations, and derive conservation and management strategies. Traditional mark–recapture methods do not explicitly account for differences in individual capture probabilities due to the spatial distribution of individuals in relation to survey effort (or trap locations). However, recent advances in the analysis of capture–recapture data have produced methods estimating abundance and density of animals from spatially explicit capture–recapture data that account for heterogeneity in capture probabilities due to the spatial organization of individuals and traps. We adapt recently developed spatial capture–recapture models to estimate density and abundance of mountain lions in western Montana. Volunteers and state agency personnel collected mountain lion DNA samples in portions of the Blackfoot drainage (7,908 km²) in west-central Montana using 2 methods: snow back-tracking mountain lion tracks to collect hair samples and biopsy darting treed mountain lions to obtain tissue samples. Overall, we recorded 72 individual capture events, including captures both with and without tissue sample collection and hair samples resulting in the identification of 50 individual mountain lions (30 females, 19 males, and 1 unknown sex individual). We estimated lion densities from 8 models containing effects of distance, sex, and survey effort on detection probability. Our population density estimates ranged from a minimum of 3.7 mountain lions/100 km² (95% CI 2.3–5.7) under the distance only model (including only an effect of distance on detection probability) to 6.7 (95% CI 3.1–11.0) under the full model (including effects of distance, sex, survey effort, and distance × sex on detection probability). These numbers translate to a total estimate of 293 mountain lions (95% CI 182–451) to 529 (95% CI 245–870) within the Blackfoot drainage. Results from the distance model are similar to previous estimates of 3.6 mountain lions/100 km² for the study area; however, results from all other models indicated greater numbers of mountain lions. Our results indicate that unstructured spatial sampling combined with spatial capture–recapture analysis can be an effective method for estimating large carnivore densities. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.     

Genetic marker investigation of the source and impact of predation on a highly endangered species

In September and October 2000, the remains of a number of apparently predated northern hairy-nosed wombats (Lasiorhinus krefftii) were discovered in Epping Forest National Park, the site of the only known population of this highly endangered species. Analysis of DNA recovered from six carcasses and a section of intestine found nearby was carried out using microsatellite and Y-specific primers. This identified seven individual wombats, the identity of three of which was inferred from a genotype database prepared from animals sampled during trapping programmes. Six victims were male and one female, suggesting that female-biased predation rates are unlikely to be the cause of the current male-biased population sex ratio. DNA isolated from four canid faeces found in the vicinity revealed three distinct canid microsatellite genotypes with very high probabilities of belonging to dingoes (Canis familiaris dingo). A wombat genotype matching that of one of the dead individuals was identified from scats of two of the dingoes. In addition, two macropod microsatellites were amplified from two dingo scats. These observations provided vital information regarding predation on northern hairy-nosed wombats, and prompted the permanent exclusion of dingoes from the park by the erection of a dingo-proof fence.     

Techniques for application of faecal DNA methods to field studies of Ursids

We describe methods for the preservation, extraction and amplification of DNA from faeces that facilitate field applications of faecal DNA technology. Mitochondrial, protein encoding and microsatellite nuclear DNA extracted and amplified from faeces of Malayan sun bears and North American black bears is shown to be identical to that extracted and amplified from the same individual's tissue or blood. A simple drying agent, silica beads, is shown to be a particularly effective preservative, allowing easy and safe transport of samples from the field. Methods are also developed to eliminate the risk of faecal DNA contamination from hair present in faeces.     

Nondestructive DNA sampling from bumblebee faeces

Genetic studies provide valuable data to inform conservation strategies for species with small or declining populations. In these circumstances, obtaining DNA samples without harming the study organisms is highly desirable. Excrements are increasingly being used as a source of DNA in such studies, but such approaches have rarely been applied to arthropods. Bumblebees are ecologically and economically important as pollinators; however, some species have recently suffered severe declines and range contractions across much of Western Europe and North America. We investigated whether bumblebee faeces could be used for the extraction of DNA suitable for genotyping using microsatellite markers. We found that DNA could be extracted using a Chelex method from faecal samples collected either in microcapillary tubes or on filter paper, directly from captured individuals. Our results show that genotypes scored from faecal samples are identical to those from tissue samples. This study describes a reliable, consistent and efficient noninvasive method of obtaining DNA from bumblebees for use in population genetic studies. This approach should prove particularly useful in breeding and conservation programs for bumblebees and may be broadly applicable across insect taxa.     

Leader of the pack: faecal pellet deposition order impacts PCR amplification in wombats

DNA sourced from faeces is notoriously less reliable than that from tissue. Hence, understanding whether faecal pellet quality varies within faecal piles may be important for sample selection. We hypothesized that the order in which faecal pellets are deposited may influence microsatellite polymerase chain reaction (PCR) amplification success from sampled faeces, more specifically, that first pellets deposited will have signatures of greater success than later ones. In a first test of the hypothesis, first and later-deposited pellets, as determined from the direction of footprints, were collected from fresh (overnight) faecal piles of northern hairy-nosed wombats (Lasiorhinus krefftii). DNA extracts were typed for seven microsatellite loci. We found that faecal deposition order significantly affected optical density of bands on autoradiographs (a measure of PCR amplification success) when the first faecal pellet was compared with the last one, but not when the first pellet was only distinguishable from later ones. The absence of a difference in amplification rate between first and later pellets is likely a reflection of the overall high amplification success in this study. That first pellets deposited yield more product suggests they contain more intestinal cells. Although further comparisons are needed, these results may inform sample selection in species for which success of microsatellite PCR amplification of faecal DNA is low. Deposition order may have more of an impact on amplification success and genotyping errors as faecal age increases.     

Effects of storage type and time on DNA amplification success in tropical ungulate faeces

The present study compares the effect of three storage media (silica, RNAlater®, ethanol) and time to extraction (1 week, 1 month and 3 months) on mitochondrial and nuclear marker amplification success in faecal DNA extracts from a sympatric community of small to medium‐sized Central African forest ungulates (genera Cephalophus, Tragelaphus, Hyemoschus). The effect of storage type and time on nuclear DNA concentrations, genotyping errors and percentage recovery of consensus genotypes was also examined. Regardless of storage method, mitochondrial and nuclear amplification success was high in DNA extracted within the first week after collection. Over longer storage periods, RNAlater yielded better amplification success rates in the mitochondrial assay. However, samples stored on silica showed (i) highest nuclear DNA concentrations, (ii) best microsatellite genotyping success, (iii) lowest genotyping errors, and (iv) greatest percentage recovery of the consensus genotype. The quantity of nuclear DNA was generally a good predictor of microsatellite performance with 83% amplification success or greater achieved with sample DNA concentrations of ≥ 50 pg/µL. If faecal DNA samples are to be used for nuclear microsatellite analyses, we recommend silica as the best storage method. However, for maximum mitochondrial amplification success, RNAlater appears to be the best storage medium. In contrast, ethanol appeared inferior to the other two methods examined here and should not be used to store tropical ungulate faeces. Regardless of storage method, samples should be extracted as soon as possible after collection to ensure optimal recovery of DNA.     

Population size estimation in Yellowstone wolves with error-prone noninvasive microsatellite genotypes

Determining population sizes can be difficult, but is essential for conservation. By counting distinct microsatellite genotypes, DNA from noninvasive samples (hair, faeces) allows estimation of population size. Problems arise because genotypes from noninvasive samples are error-prone, but genotyping errors can be reduced by multiple polymerase chain reaction (PCR). For faecal genotypes from wolves in Yellowstone National Park, error rates varied substantially among samples, often above the 'worst-case threshold' suggested by simulation. Consequently, a substantial proportion of multilocus genotypes held one or more errors, despite multiple PCR. These genotyping errors created several genotypes per individual and caused overestimation (up to 5.5-fold) of population size. We propose a 'matching approach' to eliminate this overestimation bias.     

Estimating Mountain Lion Abundance in Arizona Using Statistical Population Reconstruction

Directly monitoring abundance of cryptic species, such as mountain lions (Puma concolor), over large areas is a challenge for wildlife managers because traditional population estimation techniques may be impractical and expensive. We generated annual estimates of mountain lion abundance in Arizona, USA, for 2004–2018 by employing statistical population reconstruction methods, which use available age-at-harvest data and auxiliary information such as estimated survival rates, harvest probabilities, and hunter effort. Using PopRecon 2.0 software, we estimated that the statewide abundance of all mountain lions including kittens ranged from 1,848 (95% CI = 650–3,046) to 4,661 (95% CI = 393–9,030) during 2004–2018. Abundance for subadults and adults was more stable and precisely estimated, ranging from 1,166 (95% CI = 622–1,709) to 1,715 (95% CI = 872–2,558). Our results suggest a stable statewide mountain lion population. This approach provides a practical and cost-effective option for monitoring Arizona's mountain lion population, and will improve the ability of managers to monitor the population annually to respond to changes in abundance and to evaluate factors that influence mountain lion abundance. © 2019 The Authors. Journal of Wildlife Management published by Wiley Periodicals, Inc. on behalf of The Wildlife Society.     

Estimating population size by genotyping faeces.

Population size is a fundamental biological parameter that is difficult to estimate. By genotyping coyote (Canis latrans) faeces systematically collected in the Santa Monica Mountains near Los Angeles, California, we exemplify a general, non-invasive method to census large mammals. Four steps are involved in the estimation. First, presumed coyote faeces are collected along paths or roadways where coyotes, like most carnivores, often defaecate and mark territorial boundaries. Second, DNA is extracted from the faeces and species identity and sex is determined by mitochondrial DNA and Y-chromosome typing. Third, hypervariable microsatellite loci are typed from the faeces. Lastly, rarefaction analysis is used to estimate population size from faecal genotypes. This method readily provides a point count estimate of population size and sex ratio. Additionally, we show that home range use paternity and kinship can be inferred from the distribution and relatedness patterns of faecal genotypes.     

Abundance and characteristics of the recreational water quality indicator bacteria Escherichia coli and enterococci in gull faeces

AIMS: To evaluate the numbers and selected phenotypic and genotypic characteristics of the faecal indicator bacteria Escherichia coli and enterococci in gull faeces at representative Great Lakes swimming beaches in the United States. METHODS AND RESULTS: E. coli and enterococci were enumerated in gull faeces by membrane filtration. E. coli genotypes (rep-PCR genomic profiles) and E. coli (Vitek GNI+) and enterococci (API rapid ID 32 Strep and resistance to streptomycin, gentamicin, vancomycin, tetracycline and ampicillin) phenotypes were determined for isolates obtained from gull faeces both early and late in the swimming season. Identical E. coli genotypes were obtained only from single gull faecal samples but most faecal samples yielded more than one genotype (median of eight genotypes for samples with 10 isolates). E. coli isolates from the same site that clustered at >/=85% similarity were from the same sampling date and shared phenotypic characteristics, and at this similarity level there was population overlap between the two geographically isolated beach sites. Enterococcus API(R) profiles varied with sampling date. Gull enterococci displayed wide variation in antibiotic resistance patterns, and high-level resistance to some antibiotics. CONCLUSIONS: Gull faeces could be a major contributor of E. coli (10(5)-10(9) CFU g(-1)) and enterococci (10(4)-10(8) CFU g(-)1) to Great Lakes recreational waters. E. coli and enterococci in gull faeces are highly variable with respect to their genotypic and phenotypic characteristics and may exhibit temporal or geographic trends in these features. SIGNIFICANCE AND IMPACT OF THE STUDY: The high degree of variation in genotypic or phenotypic characteristics of E. coli or enterococci populations within gull hosts will require extensive sampling for adequate characterization, and will influence methods that use these characteristics to determine faecal contamination sources for recreational waters.     

Faecal DNA amplification in Pacific walruses (Odobenus rosmarus divergens)

Dietary information is critical for assessing the population status of seals, sea lions and walruses—and is determined for most species of pinnipeds using non-invasive methods. However, diets of walruses continue to be described from the stomach contents of dead individuals. Our goal was to assess whether DNA could be extracted from the faeces of Pacific walruses (Odobenus rosmarus divergens) collected at haulout sites, and whether potential prey species or taxa could be amplified from that DNA. We extracted DNA from 70 faecal samples collected from ice pans in the Bering Sea during the spring of 2008 and 2009 (with between 4.6 and 308.9 ng/μl of DNA in every sample). We also extracted DNA from 12 potential prey species or taxa collected by bottom-grabs in 2009 to identify positive controls for primers and to test the ability of previously published taxon-specific and species-specific primers to correctly identify the prey using conventional PCR. We tested primers that successfully amplified DNA from the tissue of at least one potential prey species or taxon on all 70 walrus faecal samples. We found that two sets of primers successfully amplified many of the potential prey species or taxa using DNA from their tissue, and that one of these primer sets produced positive amplification in 4 of the 70 faecal samples. The band size that was produced for prey organisms and in the faecal samples was consistent with expectations, although prey identities were not verified with sequencing. Our pilot study demonstrates that DNA can be successfully extracted and amplified from walrus faeces, providing a stepping stone towards describing the diets of walruses from faecal DNA.     

Differential Expression of Immune Response Genes in Steller Sea Lions (Eumetopias jubatus): An Indicator of Ecosystem Health?

Characterization of the polygenic and polymorphic features of the Steller sea lion major histocompatibility complex (MHC) provides an ideal window for evaluating immunologic vigor of the population and identifying emergence of new genotypes that reflect ecosystem pressures. MHC genotyping can be used to measure the potential immunologic vigor of a population. However, since ecosystem-induced changes to MHC genotype can be slow to emerge, measurement of differential expression of these genes can potentially provide real-time evidence of immunologic perturbations. MHC DRB genes were cloned and sequenced using peripheral blood mononuclear leukocytes derived from 10 Steller sea lions from Southeast Alaska, Prince William Sound, and the Aleutian Islands. Nine unique DRB gene sequences were represented in each of 10 animals. MHC DRB gene expression was measured in a subset of six sea lions. Although DRB in genomic DNA was identical in all individuals, relative levels of expressed DRB mRNA was highly variable. Selective suppression of MHC DRB genes could be indicative of geographically disparate environmental pressures, thereby serving as an immediate and sensitive indicator of population and ecosystem health.     

Twenty polymorphic microsatellite markers in the Asiatic lion (Panthera leo persica)

The Asiatic lion (Panthera leo persica) is driven to a single habitat in Gir forests in India for its survival. In order to devise adequate conservation and management strategies for this critically endangered species, it is important to characterize its genetic diversity and understand its population structure. Here we report twenty microsatellite loci, in addition to seven reported earlier, from the genome of a pure Asiatic lion. The microsatellite loci described here will provide potentially useful markers for the assessment of genetic variability in the only existing wild population of the Asiatic lions and other big cat species.     

Low genotyping error rates in wild ungulate faeces sampled in winter

We show that Alpine ibex (Capra ibex) and Corsican mouflon (Ovis musimon) faeces yield useful DNA for microsatellite analysis, however, we detected higher genotyping error rates for spring faeces than for winter faeces. We quantified the genotyping error rate by repeatedly genotyping four microsatellites. Respectively, 99 and 95% of mouflon and ibex genotyping repetitions provided a correct genotype using winter samples, whereas spring samples provided only 52 and 59% correct genotypes. Thus, before starting a noninvasive study, we recommend that researchers conduct a pilot study to quantify genotyping error rates for each season, population and species to be studied.     

DNA-Based Individual and Sex Identification from Wolverine (Gulo Gulo) Faeces and Urine

Non-invasive genetic analyses are important for studies of species that are rare, sensitive or at risk of extinction. This study investigates the possibility of using faeces and urine to obtain microsatellite genotypes for individual identification of wolverines (Gulo gulo). The reliability of the employed method was assessed by analysing independent amplifications of non-invasive samples (a multiple-tube approach) and by comparing genotypes obtained from faeces to genotypes obtained from blood or tissue of the same individual. Ten microsatellite markers were successfully amplified in 65% of the faecal samples (n = 32) and 40% of the urine samples (n = 22). Allelic dropout was found in 12 and 14% of the amplifications from extracts of faeces and urine, respectively. Nevertheless, all multi-locus genotypes were correct, as judged from comparison to data from tissue or blood samples, after three replicates. These results suggest that a non-invasive approach based on DNA-analysis of faeces can be a powerful tool in population monitoring of wolverines, potentially providing reliable estimates of population size and immigration rate. A second objective of the study was to develop markers for DNA-based sex identification in wolverines using non-invasive samples. We developed two Y-linked markers, one that was specific to wolverine and one that also successfully identified sex in another mustelid. Importantly, none of the markers amplified potential prey species such as reindeer or rodents.     

Characterization of eight microsatellite loci in Steller sea lions (Eumetopias jubatus)

Steller sea lions (Eumetopias jubatus) are listed as an endangered species in western Alaska and have exhibited a significant population decline throughout their range. Eight microsatellite loci were isolated from genomic DNA libraries. In addition, all these markers were found to be variable in nine individuals of the California sea lion (Zalophus californicus). This panel of markers was developed to analyse population structure in Steller sea lions throughout their range.     

Reliable microsatellite genotyping of dolphin DNA from faeces

Noninvasive samples have proved useful in genotyping studies of free‐ranging mammals. However, potential genotyping errors associated with such samples dictate the need for validation studies. This pilot study demonstrates the use of dolphin faeces in multilocus microsatellite genotyping studies. An empirical approach to calculating the rate of genotyping error was applied to data from matched pairs of blood or tissue and faecal samples from both captive and wild bottlenose dolphins. Microsatellite genotypes were assigned to dolphin faecal extracts with greater than 95% confidence by using a multiple tube approach, and at least two independent replicate genotypings.     

Single-tube HotSHOT technique for the collection, preservation and PCR-ready DNA preparation of faecal samples: the threatened Cabrera's vole as a model

A cost-effective, reliable and efficient method of obtaining DNA samples is essential in large-scale genetic analyses. This study examines the possibility of using a threatened vole species, Microtus cabrerae, as a model for the collection and preservation of faecal samples for subsequent DNA extraction with a protocol based on the HotSHOT technique. Through the examination of the probability of multi-copies (mitochondrial) and single copy (microsatellite) loci amplification (including the genotype error) and of the DNA yield (estimated by real-time qPCR), the new protocol was compared with both the frequently employed methods that successfully use ethanol to preserve faecal samples and with commercial kit-based DNA extraction. The single-tube HotSHOT-based protocol is a user-friendly, non-polluting, time-saving and inexpensive method of faeces sample collection, preservation and PCR-quality gDNA preparation. This technique therefore provides researchers with a new approach that can be employed in high-throughput, noninvasive genetic analyses of wild animal populations.