GENETICS: MAMMALS

Puechmaille S. J., Mathy G., Petit E. J. (2007): Good DNA from bat droppings. Acta Chiropterologica 9: 269-276.
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Amplification of a mitochondrial DNA fragment was used to compare the efficiency of five methods for extracting DNA from bat droppings. The Qiagen DNA Stool Kit, which yielded > 90% mtDNA amplification success, was chosen to extract DNA from 586 samples taken over two years in three French colonies of the lesser horseshoe bat (Rhinolophus hipposideros). Samples, for which mtDNA amplification was successful, were subject to the multiplex amplification of eight microsatellite loci. This resulted in > 95% amplification success over 12,592 PCRs. Allelic dropout (ADO) and false allele (FA) rates were low, and consequently, sample and locus quality indexes (QI) were high. These results demonstrate that large scale noninvasive studies of bat colonies are possible.

Lindsay D. L., Guan X., Farrington H. L., Blake M. D., Barker N. D., Carr M. R., Nvarro P. E., Lance R. F. (2015): Genetic structure among hibernacula of the endangered gray bat (Myotis grisescens). Acta Chiropterologica 17: 293-306.
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In an attempt to fill knowledge gaps relating to genetic structure in the endangered gray bat (Myotis grisescens), we investigated geographic patterns in multilocus microsatellite DNA (msDNA) genotypes and mitochondrial DNA (mtDNA) haplotype frequencies across eight primary hibernacula. Isolation-by-distance (IBD) was absent in the msDNA data and no bottlenecks were detected, with genotypic diversity (A R = 6.52, H O = 0.64) and overall genetic differentiation (F ST = 0.024, P < 0.001) being comparable to other Myotis and North American vespertilionids. Genetic structure (φ CT = 0.063, P < 0.001) among groups of populations (K = 2) was observed. We also identified a pattern of slight east-west regional genetic structure, likely attributable to the natural barrier of the Mississippi River Alluvial Plain, in the haplotype data (φ CT = 0.086, P < 0.05), along with moderate IBD (r = 0.486, P < 0.05). Though genetic differentiation among populations was generally low, significant interpopulation genetic structure, likely arising from some degree of philopatric behavior and a lack of hibernacula-associated mating sites within the Mississippi River Alluvial Plain, was observed.

Viglino A., Caniglia R., Ruiz-Gonzalez A., Russo D., Galaverni M., Toffoli R., Culasso P., De Bernardi P., Patriarca E., Agnelli P., Farina F. (2016): What can we learn from faeces? Assessing genotyping success and genetic variability in three mouse-eared bat species from non-invasive genetic sampling. Hystrix 27: 150-157.
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In the last century bat populations significantly declined mainly due to habitat fragmentation and degradation. As management-dependent species, bats need appropriate monitoring programs for the implementation of sound conservation strategies. However, bats’ small size, high mobility, elusiveness and nocturnal lifestyle make them difficult to survey. Non-Invasive Genetic Sampling (NIGS) may offer safe and cost-effective solutions, but requires well-planned sampling strategies, informative molecular markers and reliable laboratory protocols. Here we developed a NIGS protocol for species and individual identification of three mouse-eared bats, the Geoffroy’s bat (Myotis emarginatus), the long-fingered bat (Myotis capaccinii) and the Daubenton’s bat (Myotis daubentonii). Species identification was accomplished by mitochondrial (mt) DNA sequencing of reference tissue (n=49) and droppings (n=285) from Central-Northern Italy. In addition, we optimized a multiplex panel of seven microsatellites suitable for species and individual identification of the three species from droppings. We obtained a good success with mtDNA sequencing (245/285; 86%) and microsatellite genotyping (129/245; 53%). All microsatellites were successfully amplified with low error rates, and were polymorphic in the three Myotis species, with probabilities of identity 6 0.001 and observed heterozygosities of Ho=0.48 in M. emarginatus, 0.62 in M. capaccinii and 0.71 in M. daubentonii. Our protocol represents a useful tool for population genetic studies on mouse-eared bats that could likely be extended to other bat species and provide useful information to implement effective conservation plans.

Player D., Lausen C., Zaitlin B., Harrison J., Paetkau D., Harmston E. (2017): An alternative minimally invasive technique for genetic sampling of bats: Wing swabs yield species identification. Wildlife Society Bulletin 41: 590-596.
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Bat species are traditionally identified morphologically, but in some cases, species can be difficult to differentiate. Wing punches (biopsies) of wing or tail membranes are commonly used to collect tissue for DNA analysis, but less invasive techniques are preferable. As such, DNA acquired using buccal and wing swabs or from fecal pellets are increasingly being employed. We compared a dry swabbing technique with the wing biopsy technique for DNA collection. We compared species identification between tissue biopsies and wing swabs collected from bats in Alberta and British Columbia, Canada, between April and November, 2014, and September and October 2015. Species identification was achieved with varying methods of field collection and lab processing. DNA was extracted, sequenced, and compared with reference sequences and field identifications. We concluded that wing swabs are an effective way to identify bat species genetically and far less invasive than biopsy techniques. These methods should be considered for genetically sampling bats, especially during seasons when wounds from biopsy are slow to heal.

Oyler-McCance S. J., Fike J. A., Lukacs P. M., Sparks D. W., O’Shea T. J., Whitaker Jr J. O. (2018): Genetic mark–recapture improves estimates of maternity colony size for Indiana bats. Journal of Fish and Wildlife Management 9: 25-35.
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Genetic mark–recapture methods are increasingly being used to estimate demographic parameters in species where traditional techniques are problematic or imprecise. The federally endangered Indiana bat Myotis sodalis has declined dramatically and threats such as white-nose syndrome continue to afflict this species. To date, important demographic information for Indiana bats has been difficult to estimate precisely using traditional techniques such as emergence counts. Successful management and protection of Indiana bats requires better methods to estimate population sizes and survival rates throughout the year, particularly during summer when these bats reproduce and are widely dispersed away from their winter hibernacula. In addition, the familial makeup of maternity colonies is unknown, yet important for understanding local and regional population dynamics. We had four objectives in this study. For the first two objectives we investigated the potential use of DNA from fecal samples (fecal DNA) collected at roosts to obtain genetically based mark–recapture estimates of 1) colony size and 2) survival rates, for an Indiana bat maternity colony in Indianapolis, Indiana. The third objective was to compare our genetically based colony-size estimates with emergence counts conducted at the same roost tree to evaluate the genetic mark–recapture method. Our fourth objective was to use fecal DNA to estimate levels of relatedness among individuals sampled at the roost. In the summer of 2008, we collected fecal pellets and conducted emergence counts at a prominent roost tree during three time periods each lasting 7 or 8 d. We genotyped fecal DNA using five highly polymorphic microsatellite loci to identify individuals and used a robust-design mark–recapture approach to estimate survival rates as well as colony size at the roost tree. Emergence count estimates at the roost tree ranged from 100 to 215, whereas genetic mark–recapture estimates were higher, ranging from 122 to 266 and more precise. Apparent survival was 0.994 (SE = 0.04) between sampling periods suggesting that few bats died or permanently emigrated during the course of the study. Relatedness estimates, r, between all pairs of individuals averaged 0.055 ranging from 0 to 0.779, indicating that most individuals were not closely related. We demonstrate here the promise of using fecal DNA to estimate demographic information for Indiana bats and potentially other bat species.

Swift J. F., Lance R. F., Guan X., Britzke E. R., Lindsay D. L., Edwards C. E. (2018): Multifaceted DNA metabarcoding: Validation of a noninvasive, next‐generation approach to studying bat populations. Evolutionary Applications 11: 1120-1138.
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As multiple species of bats are currently experiencing dramatic declines in populations due to white-nose syndrome (WNS) and other factors, conservation managers have an urgent need for data on the ecology and overall status of populations of once-common bat species. Standard approaches to obtain data on bat populations often involve capture and handling, requiring extensive expertise and unavoidably resulting in stress to the bats. New methods to rapidly obtain critical data are needed that minimize both the stress on bats and the spread of WNS. Guano provides a noninvasive source of DNA that includes information from the bat, but also dietary items, parasites, and pathogens. DNA metabarcoding is a high-throughput, DNA-based identification technique to assess the biodiversity of environmental or fecal samples. We investigated the use of multifaceted DNA metabarcoding (MDM), a technique combining next-generation DNA sequencing (NGS), DNA barcodes, and bioinformatic analysis, to simultaneously collect data on multiple parameters of interest (bat species composition, individual genotype, sex ratios, diet, parasites, and presence of WNS) from fecal samples using a single NGS run. We tested the accuracy of each MDM assay using samples in which these parameters were previously determined using conventional approaches. We found that assays for bat species identification, insect diet, parasite diversity, and genotype were both sensitive and accurate, the assay to detect WNS was highly sensitive but requires careful sample processing steps to ensure the reliability of results, while assays for nectivorous diet and sex showed lower sensitivity. MDM was able to quantify multiple data classes from fecal samples simultaneously, and results were consistent whether we included assays for a single data class or multiple data classes. Overall, MDM is a useful approach that employs noninvasive sampling and a customizable suite of assays to gain important and largely accurate information on bat ecology and population dynamics.

Zarzoso‐Lacoste D., Jan P. L., Lehnen L., Girard T., Besnard A. L., Puechmaille S. J., Petit E. J. (2018): Combining noninvasive genetics and a new mammalian sex‐linked marker provides new tools to investigate population size, structure and individual behaviour: an application to bats. Molecular Ecology Resources 18: 217-228.
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Monitoring wild populations is crucial for their effective management. Noninvasive genetic methods provide robust data from individual free‐ranging animals, which can be used in capture–mark–recapture (CMR) models to estimate demographic parameters without capturing or disturbing them. However, sex‐ and status‐specific behaviour, which may lead to differences in detection probabilities, is rarely considered in monitoring. Here, we investigated population size, sex ratio, sex‐ and status‐related behaviour in 19 Rhinolophus hipposideros maternity colonies (Northern France) with a noninvasive genetic CMR approach (using faeces) combined with parentage assignments. The use of the DDX3X/Y‐Mam sexual marker designed in this study, which shows inter‐ and intrachromosomal length polymorphism across placental mammals, together with eight polymorphic microsatellite markers, produced high‐quality genetic data with limited genotyping errors and allowed us to reliably distinguish different categories of individuals (males, reproductive and nonreproductive females) and to estimate population sizes. We showed that visual counts represent well‐adult female numbers and that population composition in maternity colonies changes dynamically during the summer. Before parturition, colonies mainly harbour pregnant and nonpregnant females with a few visiting males, whereas after parturition, colonies are mainly composed of mothers and their offspring with a few visiting nonmothers and males. Our approach gives deeper insight into sex‐ and status‐specific behaviour, a prerequisite for understanding population dynamics and developing effective monitoring and management strategies. Provided sufficient samples can be obtained, this approach can be readily applied to a wide range of species.

Harrington A. P., O’Meara D. B., Aughney T., McAneyc K., Schofield H., Collins A., Deenen H., O’Reilly C. (2019): Novel real-time PCR species identification assays for British and Irish bats and their application to a non-invasive survey of bat roosts in Ireland. Mammalian Biology 99: 109-118.
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Detection and monitoring of extant bat populations are crucial for conservation success. Non-invasive genetic analysis of bat droppings collected at roosts could be very useful in this respect as a rapid, cost-efficient monitoring tool. We developed species-specific real-time PCR assays for 18 British and Irish bat species to enable non-invasive, large-scale distribution monitoring, which were then applied to a field survey in Ireland. One hundred and sixty-four DNA samples were collected from 95 bat roosts, of which 73% of samples were identified to species, and the resident bat species were identified at 89% of roosts. However, identification success varied between roost types, ranging from 22% for underground sites to 92% for bat boxes. This panel of DNA tests will be especially useful in cases where roosts contain multiple species, where the number of bats present is small, or bats are otherwise difficult to directly observe. The methodology could be applied to the surveillance of proposed development sites, post development mitigation measures, distribution surveys, bat box schemes and the evaluation of agri-environmental bat box schemes.

Ibouroi M. T., Arnal V., Cheha A., Dhurham S. A. O., Montgelard C., Besnard A. (2021): Noninvasive genetic sampling for flying foxes: a valuable method for monitoring demographic parameters. Ecosphere 12: e03327.
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Establishing effective wildlife conservation measures requires accurate demographic information such as population size and survival probability: parameters that can be extremely difficult to obtain. This is especially the case for threatened species, which are often rare and sometimes occupy inaccessible areas. While noninvasive genetic sampling (NIGS) techniques are promising tools for providing demographic data, these methods may be unreliable in certain situations. For instance, fecal samples of frugivorous species in tropical areas degrade rapidly, affecting the usability of the genetic material. In this study, we compared (1) NIGS capture–mark–recapture (NIGS-CMR) with conventional CMR to determine their potential in estimating demographic parameters of fruit bats, and (2) the precision of these demographic parameters and the associated costs given varying sampling designs through simulations. Using Livingstone’s fruit bats (Pteropus livingstonii) fecal samples, microsatellite markers were tested and genotyping success and error rates were assessed. The average genotyping success rate was 77%, and the total genotyping error rate for all loci was low (allelic dropout rate = 0.089, false alleles rate = 0.018). Our results suggested that five loci were required to identify individuals. Simulations showed that monitoring the species over a 9-yr period with a recapture rate of 0.20 or over a 6-yr period with a recapture rate of 0.30 seems appropriate to obtain valuable demographic parameters. Overall, in comparison to conventional CMR, NIGS-CMR offers a better method for estimating demographic parameters and subsequently for conducting long-term population monitoring in flying foxes due to the fact that (1) sample collection is easy and the level of genotyping errors in the laboratory is low and (2) it is cheaper, less time-consuming, and less disturbing to individual animals. We strongly advocate an approach that couples a pilot study with simulations as done in this study in order to choose the most efficient monitoring method for a given species or context.

Lehnen L., Jan P. L., Besnard A. L., Fourcy D., Kerth G., Biedermann M., Nyssen P., Schorcht W., Petit E. J.  Puechmaille S. J. (2021): Genetic diversity in a long‐lived mammal is explained by the past’s demographic shadow and current connectivity. Molecular Ecology 30: 5048-5063.
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Within-species genetic diversity is crucial for the persistence and integrity of populations and ecosystems. Conservation actions require an understanding of factors influencing genetic diversity, especially in the context of global change. Both population size and connectivity are factors greatly influencing genetic diversity; the relative importance of these factors can, however, change through time. Hence, quantifying the degree to which population size or genetic connectivity are shaping genetic diversity, and at which ecological time scale (past or present), is challenging, yet essential for the development of efficient conservation strategies. In this study, we estimated the genetic diversity of 42 colonies of Rhinolophus hipposideros, a long-lived mammal vulnerable to global change, sampling locations spanning its continental northern range. Here, we present an integrative approach that disentangles and quantifies the contribution of different connectivity measures in addition to contemporary colony size and historic bottlenecks in shaping genetic diversity. In our study, the best model explained 64% of the variation in genetic diversity. It included historic bottlenecks, contemporary colony size, connectivity and a negative interaction between the latter two. Contemporary connectivity explained most genetic diversity when considering a 65 km radius around the focal colonies, emphasizing the large geographic scale at which the positive impact of connectivity on genetic diversity is most profound and hence, the minimum scale at which conservation should be planned. Our results highlight that the relative importance of the two main factors shaping genetic diversity varies through time, emphasizing the relevance of disentangling them to ensure appropriate conservation strategies.

CARNIVORES

Mowat G., Strobeck C. (2000): Estimating population size of grizzly bears using hair capture, DNA profiling, and mark-recapture analysis. The Journal of Wildlife Management 64: 183-193.
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We used DNA analysis to estimate grizzly bear (Ursus arctos) population size in a 9,866-km2 area in southeast British Columbia and a 5,030-km2 area in southwest Alberta. We sampled bears by removing hair at bait sites surrounded by a single strand of barbed wire. DNA profiling with microsatellites of the root portion of the hair was used to identify individuals. We collected hair from 109 different bears and had 25 recaptures in 5 10-day trapping sessions in British Columbia. In Alberta we collected hair from 37 bears and had 9 recaptures in 4 14-day sessions. A model in program CAPTURE that accommodates heterogeneity in individual capture probabilities estimated the population size in British Columbia as 262 (95% CI = 224-313) and in Alberta as 74 (60-100). We believe that hair capture combined with DNA profiling is a promising technique for estimating distribution and abundance of bears and potentially many other species. This approach is of special interest to management biologists because it can be applied at the scale conservation and management decisions are made.

Bellemain E. V. A., Swenson J. E., Tallmon D., Brunberg S., Taberlet P. (2005): Estimating population size of elusive animals with DNA from hunter‐collected feces: four methods for brown bears. Conservation Biology 19: 150-161.
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Noninvasive genetic methods can be used to estimate animal abundances and offer several advantages over conventional methods. Few attempts have been made, however, to evaluate the accuracy and precision of the estimates. We compared four methods of estimating population size based on fecal sampling. Two methods used rarefaction indices and two were based on capture‐mark‐recapture (CMR) estimators, one combining genetic and field data. Volunteer hunters and others collected 1904 fecal samples over 2 consecutive years in a large area containing a well‐studied population of brown bears (Ursus arctos). On our 49,000‐km2 study area in south‐central Sweden, population size estimates ranged from 378 to 572 bears in 2001 and 273 to 433 bears in 2002, depending on the method of estimation used. The estimates from the best model in the program MARK appeared to be the most accurate, based on the minimum population size estimate from radio‐marked bears in a subsection of our sampling area. In addition, MARK models included heterogeneity and temporal variation in detection probabilities, which appeared to be present in our samples. All methods, though, incorrectly suggested a biased sex ratio, probably because of sex differences in detection probabilities and low overall detection probabilities. The population size of elusive animals can be estimated reliably over large areas with noninvasive genetic methods, but we stress the importance of an adequate and well‐distributed sampling effort. In cases of biased sampling, calibration with independent estimates may be necessary. We recommend that this noninvasive genetic approach, using the MARK models, be used in the future in areas where sufficient numbers of volunteers can be mobilized.

Scandura M., Capitani C., Iacolina L., Marco A. (2006): An empirical approach for reliable microsatellite genotyping of wolf DNA from multiple noninvasive sources. Conservation Genetics 7: 813-823.
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Wildlife management and conservation take advantage of the possibility to study free-living populations by collecting and analysing noninvasive samples. Nevertheless, the commonly adopted approaches, aimed at preventing results being affected by genotyping errors, considerably limit the applicability of noninvasive genotyping. An empirical approach is presented for achieving a reliable data set of wolf (Canis lupus) genotypes from multiple sources of DNA collected in a monitored population. This method relies on the relationship between sample quality and amplification outcome, which is ultimately related to the occurrence of typing errors (allelic dropout, false alleles). After DNA extraction, templates are amplified once at each locus and a conservative rating system (Q-score) is adopted to define the quality of single-locus amplifications. A significant relationship was found between quality scores and error rate (ER) (r 2=0.982). Thus it was possible to predict the chance a genotype has of being affected by errors on the basis of its Q-score. Genotypes not reaching a satisfactory confidence level can either be replicated to become reliable or excluded from the data set. Accordingly, in the present case study, 48–73% of all single-locus and 51–53% of all multilocus (ML) genotypes reached a sufficient (99 and 95%, respectively) reliability level after a single amplification per locus. Despite the possible decrease in overall yield, this method could provide a good compromise between accuracy in genotyping and effectiveness in screening large data sets for long-term or large-scale population surveys. However, to achieve complete and reliable data sets, replicated amplifications are necessary for those samples and loci providing poor results.

Schmidt K., Kowalczyk R. (2006): Using scent‐marking stations to collect hair samples to monitor Eurasian lynx populations. Wildlife Society Bulletin 34: 462-466.
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Noninvasive sampling of mammalian hairs for surveying their populations and for providing density estimations is widely applicable in wildlife ecology and management. However, the efficiency of the method may differ depending on the species or local circumstances. We modified a method of hair trapping from free‐ranging Eurasian lynx (Lynx lynx) to collect DNA samples to work in a low‐density population. We constructed hair traps based on a device developed for Canada lynx and assessed their effectiveness in Bialowieźa Forest, Poland. We set 153 hair traps baited with beaver (Castor canadensis) castoreum and catnip oil at points previously used by lynx for scent‐marking. We conducted the study in 2 consecutive winter and summer seasons during 2003–2004. Lynx rubbed 22–46% of the hair traps in 5 different trapping sessions. Lynx were more likely to rub hair traps set directly at scent‐marking points on conspicuous marked objects than when they were set some distance (1–3 m) from the marked objects. Efficiency of hair‐trapping sessions increased from 30.1 to 46.4% after selecting the most likely points. The percentage of traps visited and rubbed by lynx was higher in winter than summer in 2 consecutive years (30.1 vs. 22.2% and 46.4 vs. 23.3%, respectively), which may be related to mating behavior. This method proved efficient for monitoring low‐density Eurasian lynx populations.

Swanson B. J., Kelly B. P., Maddox C. K., Moran J. R. (2006): Shed skin as a source of DNA for genotyping seals. Molecular Ecology Notes 6: 1006-1009.
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Obtaining a sufficient number of DNA samples from ice‐breeding marine phocids, in a noninvasive manner, has proven difficult and has limited the ability to use molecular genetics on these species. We evaluate the ability to genotype ringed seals using a novel source of DNA, skin cells shed by the seal as it moults on sea ice. We found that shed skin samples yielded a lower quantity and purity of DNA compared to tissue samples. Nevertheless, the shed skin cells were a viable source of DNA for microsatellite analysis; we found no significant difference in allelic diversity or heterozygosities between tissue samples and shed skin cells. This source of DNA should allow the rapid collection of a large number of noninvasively collected DNA samples in ice‐breeding phocids.

Hausknecht R., Gula R., Pirga B., Kuehn R. (2007): Urine – a source for noninvasive genetic monitoring in wildlife. Molecular Ecology Notes 7: 208-212.
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Noninvasive samples are of increasing importance to study wild populations. In this study, we investigate the applicability of urine samples as the sole source of DNA for routine noninvasive genetic monitoring of wildlife using wolves (Canis lupus) as an example. Within the scope of a long‐term wolf population survey, we collected during winter snow tracking in Bieszczady Mountains, Poland 41 urine samples considered as utilizable for genetic analyses. DNA concentration was determined by quantitative real‐time polymerase chain reaction (qPCR) and six microsatellite loci were genotyped in threefold repeated genotyping experiments to assess the reliability of genetic analyses of urine. DNA concentration of 33 urine samples was successfully quantified and of 14 samples, we obtained congruent results for all analysed loci and all repeated genotyping experiments. The gender of urine samples was identified with a Y‐chromosome‐linked marker. Considering the high discovery rate of urine in conjunction with its genotype reliability, our study confirms that urine is a valuable source in noninvasive genetic monitoring. Additionally, preselection of samples via qPCR proved to be a powerful tool contributing to a beneficial cost‐value ratio of genetic analyses by minimizing genotyping errors.

Saito M., Yamauchi K., Aoi T. (2008): Individual identification of Asiatic black bears using extracted DNA from damaged crops. Ursus 19: 162-167.
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To reduce crop damage by Asiatic black bears (Ursus thibetanus), we developed a method to identify individual bears that damaged corn crops based on microsatellite analysis using bear DNA obtained from damaged corn. During summer 2004 in Iwate prefecture, Japan, 99 corn-bite samples were collected, of which 30 (30%) yielded sufficient DNA for 6 complete microsatellite loci. We detected that at least 21 individuals (16 males, 1 female, and 4 of unknown sex) had damaged dent corn in 5 fields. Results enabled individual identification of bears from the samples, but more accurate analysis is needed. Moreover, the sex ratio of nuisance individuals was extremely biased to males compared to that of bears killed through control programs.

Malherbe G. P., Maude G., Bastos A. D. S. (2009): Genetic clues from olfactory cues: brown hyaena scent marks provide a non-invasive source of DNA for genetic profiling. Conservation Genetics 10: 759-762.
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Brown hyaena scent marks were tested for their potential to provide DNA suitable for PCR. Up to 100% of scent marks were successfully amplified. The approach can potentially be adapted to other species with similar pasting behaviour and scent mark morphology, or to samples that are environmentally exposed/degraded/inhibitor-rich or where there are very limited amounts of sample.

Pérez T., Vázquez F., Naves J., Fernández A., Corao A., Albornoz J., Domínguez A. (2009): Non-invasive genetic study of the endangered Cantabrian brown bear (Ursus arctos). Conservation Genetics 10: 291-301.
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The Brown Bear (Ursus arctos) population present in the Cantabrian Mountains has suffered a dramatic decline in recent centuries and is now threatened with extinction. This situation has led to the development and implementation of a species recovery plan. To accomplish this plan, we need to improve our knowledge about the ecology, demography and genetics of this population. This paper presents the genetic analysis of the Cantabrian brown bear population using non-invasive samples (faeces and hairs) collected between 2004 and 2006. It was necessary to optimize a set of 18 microsatellite loci and a sex marker (several new multiplex reactions were developed) to obtain a suitable probability of identity among genotypes to work with this small, deeply structured population. Genotyping of 48 individuals was carried out using a two-step PCR protocol to increase the quality of the multilocus genotypes. Validation of genotypes was performed using a multi-tube approach combined with different software programmes to measure their error rate and reliability. Diversity in the Cantabrian population was low (He = 0.51) and the population was markedly subdivided into two subpopulations (western and eastern) without current gene flow between them. The level of divergence between the two subpopulations (Fst = 0.41) and the extremely low diversity in the eastern group (He = 0.25) indicate that this has had an extremely low effective population size and had been isolated from the main group during the last century. Connectivity between the two subpopulations will be of prime importance for the long-term survival of this species in the Cantabrian Mountains.

Williams B. W., Etter D. R., Linden D. W., Millenbah K. F., Winterstein S. R., Scribner K. T. (2009): Noninvasive hair sampling and genetic tagging of co‐distributed fishers and American martens. The Journal of Wildlife Management 73: 26-34.
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Estimation of abundance is important for assessing population responses to management actions. Accurate abundance estimates are particularly critical for monitoring temporal variation following reintroductions when the management goal is to attain population sizes capable of sustaining harvest. Numerous reintroductions have taken place in the Great Lakes region of North America, including efforts to restore extirpated fishers (Martes pennanti) and American martens (M. americana). We used a DNA‐based noninvasive hair‐snaring method based on one trap design and trapping ‐grid configuration, and evaluated capture—mark—recapture (CMR) analytical approaches to simultaneously estimate population size for co‐distributed fishers and American martens in a 671‐km2 area of the Ottawa National Forest in the western Upper Peninsula of Michigan, USA. We included harvest as a final recapture period to increase probability of recapture and to evaluate potential violations of geographic closure assumptions. We used microsatellite markers to identify target species, eliminate congener species, and provide individual identity for estimation of abundance. Population estimates for fishers and martens on the study area ranged from 35 to 60 and 8 to 28, respectively. Estimators incorporating harvest data resulted in up to a 40% increase in abundance estimates relative to estimators without harvest. We considered population estimates not including harvest data the most appropriate for the study due to timing of sampling and environmental factors, but inclusion of harvested individuals was shown to be useful as a means to detect violations of the assumption of geographic closure. We suggest improvements on future CMR sampling designs for larger landscape scales of relevance to management through incorporation of habitat or historical harvest data. Noninvasive genetic methods that simultaneously estimate the numerical abundance of co‐distributed species can greatly decrease assessment costs relative to traditional methods, and increase resulting demographic and ecological information.

Clevenger A. P., Sawaya M. A. (2010): Piloting a non-invasive genetic sampling method for evaluating population-level benefits of wildlife crossing structures. Ecology and Society 15: 7.
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Intuitively, wildlife crossing structures should enhance the viability of wildlife populations. Previous research has demonstrated that a broad range of species will use crossing structures, however, questions remain as to whether these measures actually provide benefits to populations. To assess this, studies will need to determine the number of individuals using crossings, their sex, and their genetic relationships. Obtaining empirical data demonstrating population-level benefits for some species can be problematic and challenging at best. Molecular techniques now make it possible to identify species, individuals, their sex, and their genetic relatedness from hair samples collected through non-invasive genetic sampling (NGS). We describe efforts to pilot a method to assess potential population-level benefits of wildlife crossing structures. We tested the feasibility of a prototype NGS system designed to sample hair from black bears (Ursus americanus) and grizzly bears (U. arctos) at two wildlife underpasses. The piloted hair-sampling method did not deter animal use of the trial underpasses and was effective at sampling hair from more than 90% of the bear crossing events at the underpasses. Hair samples were also obtained from non-target carnivore species, including three out of five (60%) cougar (Puma concolor) crossing events. Individual identification analysis revealed that three female and two male grizzly bears used one wildlife underpass, whereas two female and three male black bears were identified as using the other underpass. Of the 36 hair samples from bears analyzed, five failed, resulting in an 87% extraction success rate, and six more were only identified to species. Overall, 70% of the hair samples from bears collected in the field had sufficient DNA for extraction purposes. Preliminary data from our NGS suggest the technique can be a reliable method to assess the population-level benefits of Banff wildlife crossings. Furthermore, NGS can be an important tool for the conservation value of wildlife crossings for other taxa, and we urge others to carry out evaluations of this emerging methodology.

Cullingham C. I., Curteanu M., Ball M. C., Manseau M. (2010): Feasibility and recommendations for swift fox fecal DNA profiling. The Journal of Wildlife Management 74: 849-859.
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Genetic profiling using fecal samples collected noninvasively in the wild can provide managers with an alternative to live‐trapping. However, before embarking on a large‐scale survey, feasibility of this methodology should be assessed for the focal species. Costs associated with fecal genotyping can be high because of the need for multiple amplifications to prevent and detect errors. Assessing the relative amount of target DNA before genotyping means samples can be eliminated where error rates will be high or amplification success will be low, thereby reducing costs. We collected fecal samples from an endangered population of swift fox (Vulpes velox) and employed target‐DNA quantification and a screening protocol to assess sample quality before genetic profiling. Quantification was critical in identifying samples of low quality (68%, <0.2 ng/μL). Comparison of the amplification, from a subset of loci in 25 samples that did not meet the screening criteria, confirmed the effectiveness of this method. The protocol, however, used a considerable amount of DNA, and an assessment of the locus and sample variability allowed us to refine it for future population surveys. Although we did not use <50% of the samples we collected, the remaining samples provided 36 unique genotypes, which corresponded to approximately 70% of animals estimated to be present in the study area. Although obtaining fecal DNA from small carnivores is challenging, our protocol, including the quantification and qualification of DNA, the selection of markers, and the use of postgenotyping analyses, such as DROPOUT, CAPWIRE, and geographic information, provides a more cost‐effective way to produce reliable results. The method we have developed is an informative approach that wildlife managers can use to conduct population surveys where the collection of feces is possible without the need for live‐trapping.

Haag T., Santos A. S., Valdez F. P., Sana D. A., Silveira L., Cullen L., De Angelo C., Morato R. G., Crawshaw P. G., Salzano F. M., Eizirik E. (2010): Molecular tracking of jaguar melanism using faecal DNA. Conservation Genetics 11: 1239-1242.
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Major evolutionary questions remain elusive due to persistent difficulties in directly studying the genetics of variable phenotypes in natural populations. Many phenotypic variants may be of adaptive relevance, and thus important to consider in the context of conservation genetics. However, since the dynamics of these traits is usually poorly understood in the wild, their incorporation in conservation strategies is difficult to accomplish. For animals which exhibit intriguing phenotypic variation but are difficult to track in the wild, innovative approaches are required to investigate such issues. Here we demonstrate that non-invasive DNA sampling can be used to study the genetics and ecology of melanism in the jaguar, by directly genotyping the molecular polymorphism underlying this coloration trait. These results open new prospects for the in-depth investigation of this polymorphism, and highlight the broader potential of non-invasive DNA-based phenotype tracking for wildlife in general.

Karamanlidis A. A., Drosopoulou E., de Gabriel Hernando M., Georgiadis L., Krambokoukis L., Pllaha S., Zedrosser A., Scouras Z. (2010): Noninvasive genetic studies of brown bears using power poles. European Journal of Wildlife Research 56: 693-702.
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One difficulty in the conservation of endangered wildlife is the lack of reliable information on its status. This lack of knowledge can often be attributed to financial and logistic constraints as well as the lack of trained personnel to collect data. We test a simple method to study bears in the southern Balkans by inspecting power poles, which are used by bears for marking and rubbing purposes. We created a network of barbed-wire fitted poles for the collection of hair samples, evenly distributed throughout six study areas. During 87 sampling sessions in the main study area, we collected 191 samples and identified six microsatellite loci that were variable enough for individual bear identification. The most and best-quality hair samples were collected during the mating period, and DNA was most successfully extracted from samples remaining <4 weeks in the field. In the six study areas, we identified 47 bears. An advantage of using power poles for hair sampling is their availability and accessibility; no bait is required, and the network can be easily set up. A drawback may be an unequal capture probability of sex and age classes of bears. Despite this limitation, using power poles proved to be a simple and cheap method for the noninvasive genetic study of bears that did not require any prior knowledge on habitat use and activity patterns. The method is suitable for large-scale surveys to estimate distribution and relative densities of bears and could also be applied for studying other species.

Ausband D. E., Young J., Fannin B., Mitchell M. S., Stenglein J. L., Waits L. P., Shivik J. A. (2011): Hair of the dog: Obtaining samples from coyotes and wolves noninvasively. Wildlife Society Bulletin 35: 105-111.
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Canids can be difficult to detect and their populations difficult to monitor. We tested whether hair samples could be collected from coyotes (Canis latrans) in Texas, USA and gray wolves (C. lupus) in Montana, USA using lure to elicit rubbing behavior at both man‐made and natural collection devices. We used mitochondrial and nuclear DNA to determine whether collected hair samples were from coyote, wolf, or nontarget species. Both coyotes and wolves rubbed on man‐made barbed surfaces but coyotes in Texas seldom rubbed on hanging barbed surfaces. Wolves in Montana showed a tendency to rub at stations where natural‐material collection devices (sticks and debris) were present. Time to detection was relatively short (5 nights and 4 nights for coyotes and wolves, respectively) with nontarget and unknown species comprising approximately 26% of the detections in both locations. Eliciting rubbing behavior from coyotes and wolves using lures has advantages over opportunistic genetic sampling methods (e.g., scat transects) because it elicits a behavior that deposits a hair sample at a fixed sampling location, thereby increasing the efficiency of sampling for these canids. Hair samples from rub stations could be used to provide estimates of abundance, measures of genetic diversity and health, and detection–nondetection data useful for cost‐effective population monitoring.

Borthakur U., Barman R. D., Das C., Basumatary A., Talukdar A., Ahmed M. F., Talukdar B. K., Bharali R. (2011): Noninvasive genetic monitoring of tiger (Panthera tigris tigris) population of Orang National Park in the Brahmaputra floodplain, Assam, India. European Journal of Wildlife Research 57: 603-613.
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The Brahmaputra Valley of Assam, India, is one of the prime habitats for the endangered Royal Bengal tiger Panthera tigris tigris. With dwindling global population, estimation of the minimum number of tigers has always been a curiosity to wildlife researchers as well as to protected area managers. In the present study, DNA-based techniques were used for identifying individual tigers present in Orang National Park of Assam, from 57 faecal samples collected during February 2009. Orang National Park stands as an island of a single forest patch along the north bank of river Brahmaputra. The present study confirms the presence of 17 individual tigers in Orang National Park, with five male and 12 female. DNA-based capture–recapture analysis yielded minimum range estimate of 18 and 19 individuals, with possible overestimates of population size following two models of capture probability in CAPWIRE. The results of our genetic counting of tigers are compared with the estimates of 19 tigers based on pugmark analysis by the state Forest Department in 2000 and an independent capture–recapture estimate of 14 (±3.6) individuals based on photographic identity study in 2009. Looking at high mortality of tigers in the area, with 19 reported deaths during 2000 to 2009, our results indicate high individual turnover in the area. This study shows that Orang National Park harbours a healthy breeding population of tigers. However, the possibility of a source-sink dynamics operating in the landscape could not be ruled out, with possible immigration from nearby Kaziranga National Park on the south bank of Brahmaputra, which has the highest reported density of the species in the world.

Guo Y., Hu Y., Qi D., Zhan X., Bruford M. W., Wei F. (2011): Genotyping faeces of red pandas (Ailurus fulgens): implications for population estimation. European Journal of Wildlife Research 57: 1231-1235.
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The red panda (Ailurus fulgens) is an endangered species distributed in the Himalaya and Hengduan Mountains and extremely difficult to monitor because it is elusive, wary and nocturnal. However, recent advances in noninvasive genetics are allowing conservationists to indirectly estimate population size of this animal. Here, we present a pilot study of individual identification of wild red pandas using DNA extracted from faeces. A chain of optimal steps in noninvasive studies were used to maximize genotyping success and minimize error rate across sampling, selection of microsatellite loci, DNA extraction and amplification and data checking. As a result, 18 individual red pandas were identified successfully from 33 faecal samples collected in the field using nine red panda-specific microsatellite loci with a low probability of identity of 1.249 × 10−3 for full siblings. Multiple methods of tracking genotyping error showed that the faecal genetic profiles possessed very few genotyping errors, with an overall error rate of 1.12 × 10−5. Our findings demonstrate the feasibility and reliability of using faeces as an effective source of DNA for estimating and monitoring wild red panda populations.

Kindberg J., Swenson J. E., Ericsson G., Bellemain E., Miquel C., Taberlet P. (2011): Estimating population size and trends of the Swedish brown bear Ursus arctos population. Wildlife Biology 17: 114-123.
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Estimating population size and trends are key issues in the conservation and management of large carnivores. The rebounding brown bear Ursus arctos population in Sweden is monitored by two different systems, both relying on voluntary resources. Population estimates have been calculated using Capture-Mark-Recapture methods, based on DNA-based scat surveys in five of the six Swedish counties with established bear populations. A total of 1,358 genotypes were identified using DNA extracted from collected scats. An independent ongoing programme, the Large Carnivore Observation Index (LCOI), was initiated in 1998. The LCOI uses effort-corrected observations of bears by moose Alces alces hunters during the moose hunt (> 2 million observation hours/year) and has shown a good correlation with relative population density of bears using the DNA-based method. From this, we have calculated population trends during the period 1998-2007. Using an exponential model, we estimated the yearly increase in the bear population to be 4.5% at the national level, varying between 0 and 10.2% in different counties. We used the regional population estimates and the trends from the LCOI, taking the variation from both systems into account using parametric bootstrapping, to calculate the regional as well as the national population size in Sweden in fall 2008. In one case (the northernmost county; Norrbotten) a DNA-scat survey was lacking, so we used assumptions based on data from the neighbouring county to estimate population size. We estimated the Swedish brown bear population to be 3,298 individuals (2,968-3,667; 95% confidence intervals) in 2008. Our results suggest that reliable information, necessary for the management of the brown bear population can be obtained from volunteers using standardised methods.

Mowry R. A., Gompper M. E., Beringer J., Eggert L. S. (2011): River otter population size estimation using noninvasive latrine surveys. The Journal of Wildlife Management 75: 1625-1636.
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Across much of North America, river otter (Lontra canadensis) populations were extirpated or greatly reduced by the early 20th century. More recently, reintroductions have resulted in restored populations and the recommencement of managed trapping. Perhaps the best example of these river otter reintroductions occurred in Missouri, regarded as one of the most successful carnivore recovery programs in history. However, abundance estimates for river otter populations are difficult to obtain and often contentious when used to underpin management activities. We assessed the value of latrine site monitoring as a mechanism for quantifying river otter abundance. Analyses of fecal DNA to identify individual animals may result in an improved population estimate and have been used for a variety of mammal species. We optimized laboratory protocols, redesigned existing microsatellite primers, and calculated genotyping error rates to enhance genotyping success for a large quantity of river otter scat samples. We also developed a method for molecular sexing. We then extracted DNA from 1,421 scat samples and anal sac secretions (anal jelly) collected during latrine site counts along 22–34‐km stretches representing 8–77% of 8 rivers in southern Missouri in 2009. Error rates were low for the redesigned microsatellites. We obtained genotypes at 7–10 microsatellite loci for 24% of samples, observing highest success for anal jelly samples (71%) and lowest for fresh samples (collected within 1 day of defecation). We identified 63 otters (41 M, 22 F) in the 8 rivers, ranging from 2 to 14 otters per river. Analyses using program CAPWIRE resulted in population estimates similar to the minimum genotyping estimate. Density estimates averaged 0.24 otters/km. We used linear regression to develop and contrast models predicting population size based on latrine site and scat count indices, which are easily collected in the field. Population size was best predicted by a combination of scats per latrine and latrines per kilometer. Our results provide methodological approaches to guide wildlife managers seeking to initiate similar river otter fecal genotyping studies, as well as to estimate and monitor river otter population sizes.

Naidu A., Smythe L. A., Thompson R. W., Culver M. (2011): Genetic analysis of scats reveals minimum number and sex of recently documented mountain lions. Journal of Fish and Wildlife Management 2: 106-111.
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Recent records of mountain lions Puma concolor and concurrent declines in desert bighorn sheep Ovis canadensis mexicana on Kofa National Wildlife Refuge in Arizona, United States, have prompted investigations to estimate the number of mountain lions occurring there. We performed noninvasive genetic analyses and identified species, individuals, and sex from scat samples collected from the Kofa and Castle Dome Mountains. From 105 scats collected, we identified a minimum of 11 individual mountain lions. These individuals consisted of six males, two females and three of unknown sex. Three of the 11 mountain lions were identified multiple times over the study period. These estimates supplement previously recorded information on mountain lions in an area where they were historically considered only transient. We demonstrate that noninvasive genetic techniques, especially when used in conjunction with camera-trap and radiocollaring methods, can provide additional and reliable information to wildlife managers, particularly on secretive species like the mountain lion.

Sawaya M. A., Ruth T. K., Creel S., Rotella J. J., Stetz J. B., Quigley H. B., Kalinowski S. T. (2011): Evaluation of noninvasive genetic sampling methods for cougars in Yellowstone National Park. The Journal of Wildlife Management 75: 612-622.
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Conventional methods for monitoring cougar, Puma concolor, populations involve capture, tagging, and radio‐collaring, but these methods are time‐consuming, expensive, and logistically challenging. For difficult‐to‐study species such as cougars, noninvasive genetic sampling (NGS) may be a useful alternative. The ability to identify individuals from samples collected through NGS methods provides many opportunities for developing population‐monitoring tools, but the utility of these survey methods is dependent upon collection of samples and accurate genotyping of those samples. In January 2003, we initiated a 3‐yr evaluation of NGS methods for cougars using a radio‐collared population in Yellowstone National Park (YNP), USA. Our goals were to: 1) determine which DNA collection method, hair snares or snow tracking, provided a better method for obtaining samples for genetic analysis, 2) evaluate reliability of the genetic data derived from hair samples collected in the field, and 3) evaluate the potential of NGS for demographic monitoring of cougar populations. Snow tracking yielded more hair samples and was more cost effective than snagging hair with rub pads. Samples collected from bed sites and natural hair snags (e.g., branch tips, thorn bushes) while snow tracking accurately identified and sexed 22 individuals (9 F, 13 M). The ratio of the count from snow tracking to the count from radio‐telemetry was 15:24 in winter 2004, 13:12 in 2005, and 22:29 for both years combined. Annual capture probabilities for obtaining DNA from snow tracking varied considerably between years for females (0.42 in 2004 and 0.88 in 2005) but were more consistent for males (0.77 in 2004 and 0.88 in 2005). Our results indicate that snow tracking can be an efficient, reliable NGS method for cougars in YNP and has potential for estimating demographic and genetic parameters of other carnivore populations in similar climates.

Chaves P. B., Graeff V. G., Lion M. B., Oliveira L. R., Eizirik E. (2012): DNA barcoding meets molecular scatology: short mtDNA sequences for standardized species assignment of carnivore noninvasive samples. Molecular Ecology Resources 12: 18-35.
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Although species assignment of scats is important to study carnivore biology, there is still no standardized assay for the identification of carnivores worldwide, which would allow large‐scale routine assessments and reliable cross‐comparison of results. Here, we evaluate the potential of two short mtDNA fragments [ATP6 (126 bp) and cytochrome oxidase I gene (COI) (187 bp)] to serve as standard markers for the Carnivora. Samples of 66 species were sequenced for one or both of these segments. Alignments were complemented with archival sequences and analysed with three approaches (tree‐based, distance‐based and character‐based). Intraspecific genetic distances were generally lower than between‐species distances, resulting in diagnosable clusters for 86% (ATP6) and 85% (COI) of the species. Notable exceptions were recently diverged species, most of which could still be identified using diagnostic characters and uniqueness of haplotypes or by reducing the geographic scope of the comparison. In silico analyses were also performed for a 110‐bp cytochrome b (cytb) segment, whose identification success was lower (70%), possibly due to the smaller number of informative sites and/or the influence of misidentified sequences obtained from GenBank. Finally, we performed case studies with faecal samples, which supported the suitability of our two focal markers for poor‐quality DNA and allowed an assessment of prey DNA co‐amplification. No evidence of prey DNA contamination was found for ATP6, while some cases were observed for COI and subsequently eliminated by the design of more specific primers. Overall, our results indicate that these segments hold good potential as standard markers for accurate species‐level identification in the Carnivora.

De Groot P. V. C., Wong P. B., Harris C., Dyck M. G., Kamookak L., Pagès M., Michaux J., Boag P. T. (2013): Toward a non‐invasive inuit polar bear survey: genetic data from polar bear hair snags. Wildlife Society Bulletin 37: 394-401.
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We explore the feasibility of collecting microsatellite genotypes of non‐invasively collected hair from free‐ranging polar bears (Ursus maritimus) in M’Clintock Channel, Nunavut, Canada. We estimate the minimum number of individuals in a region of the M’Clintock Channel population and evaluate potential sampling biases associated with corral sampling stations. We optimized 6 variable microsatellite loci for genotyping hair‐snag DNA with low genotyping error (mean allelic dropout and false allele error rates <5%). In May of 4 sequential years (2006–2009), we collected 595 hair‐snag samples from 145 baited corral sampling stations, from which 319 hair snags were used to detect 59–82 individuals using 4–6 microsatellite loci; we also genetically sexed these individuals. Although genetic sex estimates of matching genotypes are generally in agreement, the estimated sex ratio differs from that previously reported from aerial mark–recapture, which suggests a potential male bias in our sampling stations. These noninvasive methods of identifying individual and sex of bears hold promise for frequent and inexpensive estimates of polar bear population activity informed by Inuit hunters.

Brzeski K. E., Gunther M. S., Black J. M. (2013): Evaluating river otter demography using noninvasive genetic methods. The Journal of Wildlife Management 77: 1523-1531.
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The decline in river otter (Lontra canadensis) populations during the late nineteenth and early twentieth centuries throughout North America has led to protective status and strict harvest regulations. Despite sustained interest in protecting river otters, relatively few contemporary studies have evaluated the current status of populations, especially in California. An effective way to estimate river otter population sizes and facilitate monitoring is with noninvasive genetic methods. Our objective was to establish baseline demographic information for an understudied river otter population in northern California by estimating river otter abundance using noninvasive genetic sampling, supplemented with data obtained from an observation‐based citizen science project focusing on river otters. We extracted DNA from scat and genotyped samples at 6 microsatellite loci. We used Program MARK to build mark‐recapture models to estimate river otter abundance and pooled visual observations from a citizen science project maintained in Humboldt County, California to supplement population estimates with information regarding pups and group size. Between 41 and 44 river otters were in the study area based on noninvasive genetic samples, and group sizes ranged 1–7 or 2–12 based on observational and genetic data, respectively. The Humboldt Bay region had a high density of river otters (0.93 otters/km) as compared to other coastal systems; resource attributes of Humboldt Bay could contribute to this difference. Results reported herein demonstrate methods appropriate for establishing baseline river otter demographics.

Hanke P. U., Dickman C. R. (2013): Sniffing out the stakes: hair-snares for wild cats in arid environments. Wildlife Research 40: 45-51.
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Wild cats (Felis spp.) are difficult to monitor because of their cryptic lifestyle and usually low numbers. Hair-snaring is a promising non-invasive method being used increasingly to estimate mammal populations. Our aim was to carry out pilot trials of a simple hair-snare designed to capture hair from wild cats in arid environments. Roughened wooden stakes were set at multiple sites on the crests of sand dunes and in swales in western Queensland, Australia, and in mostly sandy habitats of the Namib and Kalahari Deserts, Namibia. In Australia, stakes were sprayed with cat urine, extracts of catnip or valerian herbs as lures, or left untreated; in Namibia, alternate stakes were sprayed with a food lure of tuna emulsion oil. The stakes were checked for hair, usually daily, for 2–14 days, and the surrounding ground was inspected for tracks. Remote cameras also were used at some sites to confirm the identity of visitors to stakes. In Australia, feral cats (Felis catus) were attracted to, and left hairs on, stakes sprayed with cat urine six times more frequently than to unsprayed stakes irrespective of whether snares were on dune crests or in swales, and showed no response to catnip or valerian. Tracks and photos showed that cats, dingoes or wild dogs (Canis lupus ssp.) and foxes (Vulpes vulpes) also approached and sniffed the stakes. In Namibia, F. catus, F. lybica and F. nigripes left hair on stakes, with deposition rates two and a half-fold higher at stakes with the food lure than without it. At least five other species of predators visited the hair-snare sites. Simple wooden stakes provide a cheap and simple method of snaring hairs from wild cats, especially if used in conjunction with appropriate lures. Our results broadly support previous work, and extend the utility of the method to different Felis spp. in arid habitats. Further research is needed on snares to investigate the seasonal efficiency of different lures. If DNA also is to be extracted to identify individuals, more work is needed to confirm that snares yield hair of sufficient quality to allow this.

DeMatteo K. E., Rinas M. A., Argüelles C. F., Zurano J. P., Selleski N., Bitetti M. S. D., Eggert L. S. (2014): Noninvasive techniques provide novel insights for the elusive bush dog (Speothos venaticus). Wildlife Society Bulletin 38: 862-873.
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The bush dog (Speothos venaticus), a small and rarely seen canid from Central and South America, has proven extremely challenging to locate and study in the wild, making the development of species‐specific and comprehensive carnivore conservation strategies difficult. From May to August 2011, a detection‐dog–handler team located 34 scats from bush dogs in the northern (n = 26) and central (n = 8) zones of Misiones, Argentina. We identified 22 unique genotypes (14 northern and 8 central) and assigned sex to 100% of the genotyped scats. Only half of the scats were located inside 7 protected areas (4 northern and 3 central); the remaining half were located in 4 sites outside of protected areas (3 northern and 1 central). Results suggest low but significant differentiation between zones for bush dogs (F ST = 0.049, P = 0.010). Bush dogs demonstrated high habitat‐use flexibility and a close association with altered habitat; however, altered habitat may not be optimal for the species because of the potential for lower prey densities and risk of exposure to life threatening diseases by domestic dogs. The effectiveness of noninvasive techniques (detection dogs, genetic analyses of scat, Geographic Information System technology) in studying the ecology of bush dogs not only opens the door for additional studies of a species that has proven difficult to study with standard survey techniques, but also provides an alternative approach that conservationists can use independent of habitat type and presence of humans.

Lerone L., Mengoni C., Carpaneto G. M., Randi E., Loy A. (2014): Procedures to genotype problematic non-invasive otter (Lutra lutra) samples. Acta Theriologica 59: 511-520.
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Non-invasive genetics is a powerful tool in wildlife research and monitoring, especially when dealing with elusive and rare species such as the Eurasian otter (Lutra lutra). Nevertheless, otter DNA obtained from scats and anal secretions appears to be exposed to very quick degradation processes, and the success rate in DNA amplification is lower than in other carnivores. We collected 191 samples from April to September 2011 along the river Sangro basin (Italy) which was recently re-colonized by the Eurasian otter. Using two sets of microsatellite loci (six Lut and seven OT loci), we investigated the influence of sample type and age, collection time, storage time, temperature and humidity on genotyping success and amplification success. We also tested the efficacy of different DNA extraction kits and storage buffer mediums. Finally, we compared amplification success rate, allelic dropout and false allele rates for each locus. We obtained a mean amplification success rate of 79.0 % and a genotyping success rate of 35.1 %. Fresh pure jellies yielded the highest amplification success and genotyping rate. Six microsatellite loci should be theoretically sufficient to distinguish the individual unrelated otters (PID = 0.001), while 13 loci were needed to distinguish sibling otters (PIDsibs = 0.002) in our population. We identified 11 otters, and molecular sexing ascertained the presence of five males, four females and two uncertain individuals. Generalized linear models highlighted a significant influence of sample type and age, temperature and humidity both on genotyping and amplification success.

Nagai T., Murakami T., Masuda R. (2014): Effectiveness of noninvasive DNA analysis to reveal isolated-forest use by the sable Martes zibellina on eastern Hokkaido, Japan. Mammal Study 39: 99-104.
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In order to assess importance of isolated forests as the wintering habitat of the sable (Martes zibellina) in eastern Hokkaido, Japan, we performed DNA analyses on 59 samples noninvasively collected in field (54 fecal, 3 urine and 2 hair samples). Consequently, 46 (41 fecal, 3 urine and 2 hair samples) of the 59 samples were recognized to have dropped from the sable. In addition, we successfully identified the individuality on 36 (35 fecal and 1 urine samples) of the 46 samples, and they consisted of at least 23 individuals. Our new method using sex chromosomal DNA markers showed that the 23 individuals comprised 12 males, 9 females and 2 with unknown sex. In 2010 and 2011, the minimum number of individuals in the study area was 21 (11 males, 8 females and 2 with unknown sex) and 5(1 male and 4 females), respectively. In addition, we found the sexual difference in fidelity to their home range. Thus, our noninvasive DNA technique provided promising information on estimating and monitoring ecological features about the regional sable population.

Nussberger B., Wandeler P., Camenisch G. (2014): A SNP chip to detect introgression in wildcats allows accurate genotyping of single hairs. European Journal of Wildlife Research 60: 405-410.
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Genotyping non-invasively collected samples is challenging. Nevertheless, genetic monitoring of elusive species like the European wildcat (Felis silvestris silvestris) mainly relies on such samples. Wildcats are likely threatened through introgression with domestic cats (F. silvestris catus). To determine introgression based on single cat hairs, we developed a 96.96 Fluidigm single nucleotide polymorphism (SNP) genotyping array chip. To estimate the accuracy of this method, we compared genotypes of 17 cats called with both Sanger sequencing and Fluidigm. When Sanger sequencing genotypes were considered as a reference, the genotyping error rate with Fluidigm was 0.9 %. We subsequently compared 16 hair samples to tissue samples of the same individual. When the tissue samples were used as a reference, the genotyping error rate in hair samples was 1.6 %. This low error rate allowed reliable recognition of individuals and correct assessment of introgression levels. Thus, the genotyping method presented in this paper is suitable for non-invasively collected samples. It will help conservationists to monitor the introgression rate in wildcat populations based on non-invasive hair sampling and subsequently to conduct effective conservation measures.

Roques S., Furtado M., Jácomo A. T., Silveira L., Sollmann R., Torres N. M., Godoy J. A., Palomares F. (2014): Monitoring jaguar populations Panthera onca with non-invasive genetics: a pilot study in Brazilian ecosystems. Oryx 48: 361-369.
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The global population of jaguars Panthera onca has decreased significantly since the beginning of the 20th century. Given the scarcity of demographic and biological information, estimating population parameters is critical for the design of conservation measures. The jaguar’s elusive behaviour makes it impossible to estimate and monitor populations by direct observation. We propose a non-invasive genetic sampling approach and demonstrate its potential for large-scale monitoring. Sex identification was optimized for faecal samples of jaguars and other felids. We also optimized a set of 11 microsatellite markers for reliable identification of individuals. We estimated the effectiveness of faecal sample genotyping in two distinct Brazilian biomes: the Pantanal and the semi-arid Caatinga. Almost 90% of the samples that were molecularly identified as jaguar (n = 90) were successfully genotyped and were assigned to 30 individuals. Genetic diversity was generally high but was significantly lower in the Caatinga population. We show that non-invasive genetic sampling can be a reliable tool to study population parameters and to monitor the genetic status of jaguar populations in different habitats. It may also be useful for future surveys of jaguars that address ecological, behavioural and conservation issues, and could provide a baseline for non-invasive genetic studies of other wild felid populations.

Sheehy E., O’Meara D. B., O’Reilly C., Smart A., Lawton C. (2014): A non-invasive approach to determining pine marten abundance and predation. European Journal of Wildlife Research 60: 223-236.
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A non-invasive approach was used to investigate variation in pine marten (Martes martes) abundance between the midlands and east of Ireland, and to determine the frequency of occurrence of squirrels and other small mammals in the diet. Remotely plucked hair samples were genotyped to differentiate between individual animals, and real-time polymerase chain reaction (PCR) was used to identify predator and prey DNA in scats. Macro analysis of prey remains was carried out on a sub sample of scats and the results from both methods are compared. Non-invasive techniques were successful in determining the presence and relative abundance of the pine marten at woodland level. As expected, abundance was found to be higher in the core population of the midlands than in the east. Pine martens were found to reach higher numbers per km2 of forested habitat in Ireland than their British or European counterparts. Both traditional hard part analysis and molecular dietary analysis of mammalian prey yielded similar results. We provide the first evidence of the European pine marten predating upon the North American grey squirrel (Sciurus carolinensis) in its invasive range. While the grey squirrel was not available as a prey item in any of the midlands sites, it was available in the east, where it featured significantly more frequently in the diet than the native red squirrel. In both the midlands and the east the woodmouse is the most frequently occurring mammal in the diet.

Sugimoto T., Aramilev V. V., Kerley L. L., Nagata J., Miquelle D. G., McCullough D. R. (2014): Noninvasive genetic analyses for estimating population size and genetic diversity of the remaining Far Eastern leopard (Panthera pardus orientalis) population. Conservation Genetics 15: 521-532.
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Understanding and monitoring the population status of endangered species is vital for developing appropriate management interventions. We used noninvasive genetic analyses to obtain ecological and genetic data on the last remaining Far Eastern leopard population in the world. During seven winters from 2000–2001 to 2007–2008, we collected feces, hair, and saliva from most of the leopard habitat. Of the 239 leopard samples collected during the study period, 155 were successfully genotyped at 13 microsatellite loci and 37 individuals (18 males and 19 females) were identified. Population size estimates based on the Capwire model were 28 (95 % CI 19–38) in 2002–03 and 26 (95 % CI 13–33) in 2007–2008. The leopard population had a low level of genetic diversity (expected and observed heterozygosity = 0.43; average number of alleles per locus = 2.62), and effective population size was estimated to be low (N e = 7–16) by two genetic-based methods. We observed little improvement in the genetic diversity during the study period and did find an indication of allele loss compared with individuals from the mid-1990s, suggesting that the remaining population will continue to suffer loss of genetic diversity. Given the small population size and the low genetic diversity, with little expectation of replenishment of the genetic variation by natural immigration, successful expansion of available habitat and development of a second population based on captive individuals may be crucial for persistence of this leopard subspecies in the wild.

Wultsch C., Waits L. P., Kelly M. J. (2014): Noninvasive individual and species identification of jaguars (Panthera onca), pumas (Puma concolor) and ocelots (Leopardus pardalis) in Belize, Central America using cross‐species microsatellites and faecal DNA. Molecular Ecology Resources 14: 1171-1182.
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There is a great need to develop efficient, noninvasive genetic sampling methods to study wild populations of multiple, co‐occurring, threatened felids. This is especially important for molecular scatology studies occurring in challenging tropical environments where DNA degrades quickly and the quality of faecal samples varies greatly. We optimized 14 polymorphic microsatellite loci for jaguars (Panthera onca), pumas (Puma concolor) and ocelots (Leopardus pardalis) and assessed their utility for cross‐species amplification. Additionally, we tested their reliability for species and individual identification using DNA from faeces of wild felids detected by a scat detector dog across Belize in Central America. All microsatellite loci were successfully amplified in the three target species, were polymorphic with average expected heterozygosities of H= 0.60 ± 0.18 (SD) for jaguars, H= 0.65 ± 0.21 (SD) for pumas and H= 0.70 ± 0.13 (SD) for ocelots and had an overall PCR amplification success of 61%. We used this nuclear DNA primer set to successfully identify species and individuals from 49% of 1053 field‐collected scat samples. This set of optimized microsatellite multiplexes represents a powerful tool for future efforts to conduct noninvasive studies on multiple, wild Neotropical felids.

Yamauchi K., Kurakake S., Morosawa T., Kondo M., Uno R., Yuasa T., Tsuruga H., Tamate H. B., Yoneda M. (2014): A pilot study of the hair-trapping method in Asiatic black bears (Ursus thibetanus): determination of optimal survey period for estimating population size. Mammal Study 39: 191-200.
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In order to establish methodology for population estimation of Asiatic black bears using the hair-trapping method, hair-trapping devices were located within a study area, and basic data were collected to determine the optimal survey period for hair sampling. Bear hairs collected were classified as three types (guard hair; G, underfur; U, and intermediate hair; I) depending on hair-bulb thickness. After DNA extraction, six microsatellite loci and amelogenin locus were amplified in order to identify individual bears and sex, respectively. The number of hairs collected by hair-trapping devices decreased dramatically in late August, while those that had lost part of the hair bulb increased after September. The success rate of genetic analysis was over 90% prior to the month of July and rapidly decreased in August. Based on the results of the generalized linear model, it was determined that sampling session had the most significant impact on the success rate of genetic analysis of all the explanatory variables examined. Higher rates of U-type hairs within samples used in DNA extraction resulted in lower success rates of genetic analysis. It was concluded that the optimal survey period for estimating population size using the hair-trapping method was between June and early August.

Dumond M., Boulanger J., Paetkau D. (2015): The estimation of grizzly bear density through hair‐snagging techniques above the tree line. Wildlife Society Bulletin 39: 390-402.
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Assessing grizzly bears’ (Ursus arctos) abundance in the Arctic has been challenging because of the large scale of their movements and the remoteness of field locations. We modified a post sampling method used for wolverines (Gulo gulo) to allow collection of hair samples from grizzly bears in the Canadian tundra. We deployed 1 post/cell in a sampling grid of 393 10 × 10‐km cells sampled in 2008 and 2009 for two 14‐day sessions in July–August of both years. We then compared density estimates from mark–recapture estimators that used telemetry data from previous years with spatially explicit mark–recapture models that used only genetic detections. Over the 2 years of sampling, we detected 98 female and 81 male grizzly bears. We found that the DNA degradation rate was related to collection interval and the number of days between rainfall events and sample collection. Estimates of density were in the order of 5 bears/1,000 km2. The estimates from the 2 methods were statistically similar, but spatially explicit estimates were more precise than those using radiocollar data. Our results provide the first demonstration of the viability of posts as hair‐snagging stations to obtain DNA from grizzly bears, and of spatially explicit mark–recapture methods to estimate population size and density for grizzly bears above the tree line.

Harms V., Nowak C., Carl S., Muñoz-Fuentes V. (2015): Experimental evaluation of genetic predator identification from saliva traces on wildlife kills. Journal of Mammalogy 96: 138-143.
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Identification of predators from saliva traces on game species and/or livestock kills is gaining increasing importance in wildlife management, particularly in areas where direct wildlife–human conflicts regularly occur. When the noninvasive sampling of hairs and scats is difficult, as with rare and elusive predators, saliva samples constitute a potentially useful source of DNA. To test the feasibility of this approach in obtaining an accurate genotype of the predator, we applied an experimental approach. Captive wolves (Canis lupus) and lynxes (Lynx lynx) were allowed to feed on freshly killed roe deer (Capreolus capreolus) pieces for 1min. After removal, pieces were sampled for saliva traces after 1, 24, and 48h. Microsatellite analysis revealed that error rates and amplification failure increased sharply over time. While samples collected after 1 and 24 h yielded > 83% complete genotypes, values dropped to < 50% for samples collected after 48h, of which 7% were incorrect even when consensus genotypes from 9 polymerase chain reactions were obtained. Our results stress the importance of rapid sampling after carcass detection, as well as implementing a multiple-tubes approach when using microsatellite markers for genetic predator identification based on saliva traces.

Kraus R. H., Vonholdt B., Cocchiararo B., Harms V., Bayerl H., Kühn R., Förster D. W., Fickel J., Roos C., Nowak C. (2015): A single‐nucleotide polymorphism‐based approach for rapid and cost‐effective genetic wolf monitoring in Europe based on noninvasively collected samples. Molecular Ecology Resources 15: 295-305.
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Noninvasive genetics based on microsatellite markers has become an indispensable tool for wildlife monitoring and conservation research over the past decades. However, microsatellites have several drawbacks, such as the lack of standardisation between laboratories and high error rates. Here, we propose an alternative single‐nucleotide polymorphism (SNP)‐based marker system for noninvasively collected samples, which promises to solve these problems. Using nanofluidic SNP genotyping technology (Fluidigm), we genotyped 158 wolf samples (tissue, scats, hairs, urine) for 192 SNP loci selected from the Affymetrix v2 Canine SNP Array. We carefully selected an optimised final set of 96 SNPs (and discarded the worse half), based on assay performance and reliability. We found rates of missing data in this SNP set of <10% and genotyping error of ~1%, which improves genotyping accuracy by nearly an order of magnitude when compared to published data for other marker types. Our approach provides a tool for rapid and cost‐effective genotyping of noninvasively collected wildlife samples. The ability to standardise genotype scoring combined with low error rates promises to constitute a major technological advancement and could establish SNP s as a standard marker for future wildlife monitoring.

Lobo D., Godinho R., Álvares F., López-Bao J. V., Rodríguez A. (2015): A new method for noninvasive genetic sampling of saliva in ecological research. Plos One 10: e0139765.
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Noninvasive samples for genetic analyses have become essential to address ecological questions. Popular noninvasive samples such as faeces contain degraded DNA which may compromise genotyping success. Saliva is an excellent alternative DNA source but scarcity of suitable collection methods makes its use anecdotal in field ecological studies. We develop a noninvasive method of collection that combines baits and porous materials able to capture saliva. We report its potential in optimal conditions, using confined dogs and collecting saliva early after deposition. DNA concentration in saliva extracts was generally high (mean 14 ng μl-1). We correctly identified individuals in 78% of samples conservatively using ten microsatellite loci, and 90% of samples using only eight loci. Consensus genotypes closely matched reference genotypes obtained from hair DNA (99% of identification successes and 91% of failures). Mean genotyping effort needed for identification using ten loci was 2.2 replicates. Genotyping errors occurred at a very low frequency (allelic dropout: 2.3%; false alleles: 1.5%). Individual identification success increased with duration of substrate handling inside dog’s mouth and the volume of saliva collected. Low identification success was associated with baits rich in DNA-oxidant polyphenols and DNA concentrations <1 ng μl-1. The procedure performed at least as well as other noninvasive methods, and could advantageously allow detection of socially low-ranked individuals underrepresented in sources of DNA that are involved in marking behaviour (faeces or urine). Once adapted and refined, there is promise for this technique to allow potentially high rates of individual identification in ecological field studies requiring noninvasive sampling of wild vertebrates.

Rodgers T. W., Giacalone J., Heske E. J., Janečka J. E., Jansen P. A., Phillips C. A., Schooley R. L. (2015): Socio-spatial organization and kin structure in ocelots from integration of camera trapping and noninvasive genetics. Journal of Mammalogy 96: 120-128.
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Kin selection theory predicts that under certain conditions animals will tolerate related individuals in their home ranges. We examined the relationship between spatiotemporal overlap and genetic relatedness in ocelots (Leopardus pardalis) to determine if kin selection plays a role in structuring ocelot populations. We used 3 years of camera trapping to examine the spatial organization of an ocelot population on Barro Colorado Island in Panama. We also placed camera traps on ocelot latrines to match photographs of individual ocelots with microsatellite genotypes from feces. Strengths of spatiotemporal overlap between individual ocelots were calculated using a half-weight association index based on how often individuals were photographed at the same camera within 30 days of one another. We calculated relatedness between individuals based on 11 variable microsatellite loci. Male ocelots overlapped with ≤ 11 females, and females overlapped with ≤ 7 males. We detected no clear evidence of strict intersexual territoriality in either sex. Mean overlap among males was more than 5 times greater than overlap among females; however, spatiotemporal overlap was strong between some female pairs. Overall, overlapping individuals were more related to one another than was the sample population as a whole, consistent with the hypothesis that kin selection influences ocelot spatial organization. This finding was driven by relatedness among overlapping females, and by relatedness among overlapping individuals of opposite sex, but not by overlapping males.

Valtonen M., Heino M., Aspi J., Buuri H., Kokkonen T., Kunnasranta M., Palo J. U., Nyman T. (2015): Genetic monitoring of a critically-endangered seal population based on field-collected placentas. Annales Zoologici Fennici 52: 51-65.
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Genetic analyses of non-invasively collected samples are increasingly being used in the monitoring of wildlife populations and individuals. This study is the first describing the use of placentas as non-invasive genetic samples from a natural population. We collected 66 placentas from birth-lair sites of Saimaa ringed seals (Phoca hispida saimensis) after the breeding seasons, with the aim of obtaining DNA from both the pup and the mother. Umbilical cord samples proved to yield the pup genotypes, but mothers could not be genotyped with confidence. Comparisons with existing mtDNA and microsatellite reference data sets showed that placentas can be used for inferring population-level genetic parameters. Our microsatellite panel provided sufficient resolution for genetic identification of individuals but, due to the extremely low variability of the population, parentage and sibship could not be inferred reliably. Field-collected placentas could provide means for genetic monitoring of many other seal species as well.

Wultsch C., Waits L. P., Hallerman E. M., Kelly M. J. (2015): Optimizing collection methods for noninvasive genetic sampling of neotropical felids. Wildlife Society Bulletin 39: 403-412.
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Field‐sampling methods for molecular scatology studies must be optimized, especially when working on elusive species in challenging tropical environments where rates of DNA degradation are elevated because of hot and humid weather conditions. To maximize polymerase chain reaction (PCR) amplification success and genotyping accuracy rates and to minimize genotyping error rates for fecal DNA samples of jaguars (Panthera onca) and co‐occurring Neotropical felids collected in Belize, Central America, we evaluated the performance of two fecal DNA storage techniques (dimethyl sulfoxide saline solution [DETs buffer] and 95% ethanol [EtOH]) suitable for long‐term preservation at remote tropical sites. Additionally, we tested fecal samples collected from 4 different locations on the scat (top, side, bottom, inside) at 2 different tropical forest types (tropical broadleaf and tropical pine forests). DETs buffer was the superior fecal DNA preservation method, with 44% higher PCR amplification success (P = 0.009) and 17% higher genotyping accuracy (P = 0.021) than 95% EtOH‐stored samples. Polymerase chain reaction amplification success of fecal DNA collected at the more open, pine‐forest (Pinus sp.) site differed significantly across locations on the scat, with highest mean success rates obtained from the top (85% ± 6.5% SD), followed by the side (79% ± 9.4% SD), bottom (76% ± 11.9% SD), and inside (69% ± 10.3% SD) of scat samples. Scat samples collected at the more closed‐canopy broadleaf site did not show any significant differences in amplification success rates across scat locations. We recommend that researchers optimize field‐sampling methods, including collection and storage protocols, by conducting a pilot study prior to their molecular scatology research efforts.

Bischof R., Gregersen E. R., Brøseth H., Ellegren H., Flagstad Ø. (2016): Noninvasive genetic sampling reveals intrasex territoriality in wolverines. Ecology and Evolution 6: 1527-1536.
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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.

Bu H., Hopkins III J. B., Zhang D., Li S., Wang R., Yao M., Wang D. (2016): An evaluation of hair-snaring devices for small-bodied carnivores in southwest China. Journal of Mammalogy 97: 589-598.
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Human population growth is a major threat to the biodiversity of mammals in China. Rapid development of infrastructure, pollution, and poor enforcement of environmental laws in China have altered the landscape and accelerated the extinction rates of mammals. To maintain biodiversity and conserve the mammals of China, it is essential to develop monitoring protocols for species of concern that are efficient, cost-effective, and current. It is now common practice for ecologists to survey mammals of interest using noninvasive sampling methods. These sampling techniques are affordable, require no handling of the target animals, and generally yield large samples. In China, such sampling methods have rarely been used and evaluated. For this study, we recorded the behavior of masked palm civets (Paguma larvata) and Siberian weasels (Mustela sibirica) to determine their relative preference for using different hair-snaring devices and evaluated the effectiveness of each device at collecting hair. Our goal was to determine which genetic sampling method(s) should be used to study masked palm civets and Siberian weasels in the future in Southwest China. In March–June of 2014, we monitored 13 hair-snare stations throughout the Main Valley of Laohegou Nature Reserve, Sichuan Province, China. Genetic analysis revealed that we collected 373 hair samples from civets and 47 hair samples from weasels over 104 and 26 sessions, respectively. Both civets and weasels seemed to prefer lure sticks to ground cubbies; however, the latter were more effective at collecting civet hair. Although glue sheets from lure sticks collected large samples of guard hair (> 10), no device in its current design was consistently effective at collecting weasel hair. We suggest researchers in Southwest China use ground cubbies as described in this study to collect hair from masked palm civets and test other cubby designs (e.g., longer gun brushes or smaller in size) for collecting hair from Siberian weasels.

Wang D., Hu Y., Ma T., Nie Y., Xie Y., Wei F. (2016): Noninvasive genetics provides insights into the population size and genetic diversity of an Amur tiger population in China. Integrative Zoology 11: 16-24.
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Understanding population size and genetic diversity is critical for effective conservation of endangered species. The Amur tiger (Panthera tigris altaica) is the largest felid and a flagship species for wildlife conservation. Due to habitat loss and human activities, available habitat and population size are continuously shrinking. However, little is known about the true population size and genetic diversity of wild tiger populations in China. In this study, we collected 55 fecal samples and 1 hair sample to investigate the population size and genetic diversity of wild Amur tigers in Hunchun National Nature Reserve, Jilin Province, China. From the samples, we determined that 23 fecal samples and 1 hair sample were from 7 Amur tigers: 2 males, 4 females and 1 individual of unknown sex. Interestingly, 2 fecal samples that were presumed to be from tigers were from Amur leopards, highlighting the significant advantages of noninvasive genetics over traditional methods in studying rare and elusive animals. Analyses from this sample suggested that the genetic diversity of wild Amur tigers is much lower than that of Bengal tigers, consistent with previous findings. Furthermore, the genetic diversity of this Hunchun population in China was lower than that of the adjoining subpopulation in southwest Primorye Russia, likely due to sampling bias. Considering the small population size and relatively low genetic diversity, it is urgent to protect this endangered local subpopulation in China.

Wheat R. E., Allen J. M., Miller S. D., Wilmers C. C., Levi T. (2016): Environmental DNA from residual saliva for efficient noninvasive genetic monitoring of brown bears (Ursus arctos). Plos One 11: e0165259.
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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.

Biffi D., Williams D. A. (2017): Use of non-invasive techniques to determine population size of the marine otter in two regions of Peru. Mammalian Biology 84: 12-19.
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The marine otter (Lontra felina) can be found on rocky shores from the northern coast of Peru (9° S) to the extreme south of Argentina (56° S). This species is currently classified as endangered but there is little information on population size because marine otters are very difficult to observe and count. Between June and August 2012 we collected 240 samples of marine otter feces from seven localities in Peru. All locations were visited four times. One-hundred and thirty-three samples (55%) were successfully amplified at five to seven microsatellite loci and a sex-linked marker. We identified a minimum of 80 individuals (41 males and 39 females) across all locations for a density estimate of 4.4 otters per km, a value about 2X higher than estimates based on previous visual counts. Estimates using the program CAPWIRE averaged 12.6 otters/km, a value six times higher than estimates based on previous visual counts, although confidence limits were large due to the low number of recaptures. There was a strong positive relationship between the number of fresh scats and the number of unique genotypes, suggesting scat counts might be used to estimate the minimum number of otters at a site. Non-invasive genotyping of marine otter feces and scat counts will be valuable tools for estimating population sizes and monitoring movements of this secretive species.

Espinosa‐de Aquino W., Olvera‐Ramírez A., Arellano‐Carbajal F., Lanz‐Mendoza H., Villagrán‐Herrera E., Acevedo‐Whitehouse K. (2017): Protein and RNA extraction from mucosal swabs: a minimally invasive source of ecological data for studies of natural populations. Methods in Ecology and Evolution 8: 370-378.
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Mucosal swabs have long been used to study various physiological processes in humans. In contrast, systematic sampling of mucosae is rare for wild animals, and except for its use as a source of DNA for population genetics, its potential as a tool to study physiologically relevant processes of natural populations has not been explored fully. We collected swabs from the rectal, genital, nasal and oral mucosae of California sea lion pups and investigated differences in RNA and protein yield, quality and integrity between storage times and mucosal types. Downstream applications were tested on the extracted products to determine the potential value of mucosal sampling in free‐ranging mammals. For most samples, RNA yield was stable regardless of storage time, and RNA quality and integrity were equal for all mucosal types. Ribosomal fragments of sizes expected for mammals, yeast and bacteria were observable in genital and rectal samples, but in nasal and oral samples only bacterial ribosomal fragments were observable. Amplification of selected transcripts was successful for all samples. The protein profile was distinct between mucosae, and samples with high protein yields were useful for antibody detection. Our study demonstrates that mucosal swabbing is a minimally invasive tool that yields useful physiological data for free‐ranging wildlife. Minimum experience is needed to collect samples, processing is inexpensive, and downstream applications for ecological studies are realistic.

Nakamura M., Godinho R., Rio-Maior H., Roque S., Kaliontzopoulou A., Bernardo J., Castro D., Lopes S., Petrucci-Fonseca F., Álvares F. (2017): Evaluating the predictive power of field variables for species and individual molecular identification on wolf noninvasive samples. European Journal of Wildlife Research 63: 53.
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Live-trapping elusive animals is often challenging, hampering the achievement of reasonable sample sizes for molecular studies. In such cases, the use of noninvasive samples (NIS) is critical in many research fields, mostly related to ecology, management and conservation of wild species. We analysed the influence of several variables potentially associated with the quality of wolf NIS – season, weather conditions, and in situ collected site and sample characteristics – on the success rates of species and individual identification performed using mtDNA and 13 microsatellites, respectively. NIS included scats, urine and saliva collected from two areas in Portugal. Scat samples exhibited the highest success rate for both species (81%) and individual identification (59%), compared with urine (63 and 30%, respectively) or saliva samples (48 and 36%, respectively). The success rate of species identification of scats was better explained by season of collection, the presence of mucous, moisture and odour. For samples with successful species identification analysis, individual identification success was best predicted by the presence of odour. Performing a preliminary selection of scat samples with the best characteristics can increase up to 13% the success rates of molecular analysis. Urine collected on snow had a higher success rate of species identification than that collected on vegetation. To our knowledge, this was the first time that wolf urine on vegetation near ground-scratching marks is used as DNA source. Saliva samples collected with different substrate types can also be used for species identification. These results contribute to optimising noninvasive sampling procedures, maximising the success of molecular ecology studies, and ultimately minimising sampling efforts and costs.

von Thaden A., Cocchiararo B., Jarausch A., Jüngling H., Karamanlidis A. A., Tiesmeyer A., Nowak C., Muñoz-Fuentes V. (2017): Assessing SNP genotyping of noninvasively collected wildlife samples using microfluidic arrays. Scientific Reports 7: 10768.
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Noninvasively collected samples are a common source of DNA in wildlife genetic studies. Currently, single nucleotide polymorphism (SNP) genotyping using microfluidic arrays is emerging as an easy-to-use and cost-effective methodology. Here we assessed the performance of microfluidic SNP arrays in genotyping noninvasive samples from grey wolves, European wildcats and brown bears, and we compared results with traditional microsatellite genotyping. We successfully SNP-genotyped 87%, 80% and 97% of the wolf, cat and bear samples, respectively. Genotype recovery was higher based on SNPs, while both marker types identified the same individuals and provided almost identical estimates of pairwise differentiation. We found that samples for which all SNP loci were scored had no disagreements across the three replicates (except one locus in a wolf sample). Thus, we argue that call rate (amplification success) can be used as a proxy for genotype quality, allowing the reduction of replication effort when call rate is high. Furthermore, we used cycle threshold values of real-time PCR to guide the choice of protocols for SNP amplification. Finally, we provide general guidelines for successful SNP genotyping of degraded DNA using microfluidic technology.

Bayerl H., Kraus R. H., Nowak C., Foerster D. W., Fickel J., Kuehn R. (2018): Fast and cost‐effective single nucleotide polymorphism (SNP) detection in the absence of a reference genome using semideep next‐generation Random Amplicon Sequencing (RAM seq). Molecular Ecology Resources 18: 107-117.
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Biodiversity has suffered a dramatic global decline during the past decades, and monitoring tools are urgently needed providing data for the development and evaluation of conservation efforts both on a species and on a genetic level. However, in wild species, the assessment of genetic diversity is often hampered by the lack of suitable genetic markers. In this article, we present Random Amplicon Sequencing (RAMseq), a novel approach for fast and cost‐effective detection of single nucleotide polymorphisms (SNPs) in nonmodel species by semideep sequencing of random amplicons. By applying RAMseq to the Eurasian otter (Lutra lutra), we identified 238 putative SNPs after quality filtering of all candidate loci and were able to validate 32 of 77 loci tested. In a second step, we evaluated the genotyping performance of these SNP loci in noninvasive samples, one of the most challenging genotyping applications, by comparing it with genotyping results of the same faecal samples at microsatellite markers. We compared (i) polymerase chain reaction (PCR) success rate, (ii) genotyping errors and (iii) Mendelian inheritance (population parameters). SNPs produced a significantly higher PCR success rate (75.5% vs. 65.1%) and lower mean allelic error rate (8.8% vs. 13.3%) than microsatellites, but showed a higher allelic dropout rate (29.7% vs. 19.8%). Genotyping results showed no deviations from Mendelian inheritance in any of the SNP loci. Hence, RAMseq appears to be a valuable tool for the detection of genetic markers in nonmodel species, which is a common challenge in conservation genetic studies.

Mengüllüoğlu D., Fickel J., Hofer H., Förster D. W. (2019): Non-invasive faecal sampling reveals spatial organization and improves measures of genetic diversity for the conservation assessment of territorial species: Caucasian lynx as a case species. Plos One 14: e0216549.
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The Caucasian lynx, Lynx lynx dinniki, has one of the southernmost distributions in the Eurasian lynx range, covering Anatolian Turkey, the Caucasus and Iran. Little is known about the biology and the genetic status of this subspecies. To collect baseline genetic, ecological and behavioural data and benefit future conservation of L. l. dinniki, we monitored 11 lynx territories (396 km2) in northwestern Anatolia. We assessed genetic diversity of this population by non-invasively collecting 171 faecal samples and trapped and sampled 12 lynx individuals using box traps. We observed high allelic variation at 11 nuclear microsatellite markers, and found no signs of inbreeding despite the potential isolation of this population. We obtained similar numbers of distinct genotypes from the two sampling sources. Our results indicated that first order female relatives occupy neighbouring territories (female philopatry) and that territorial male lynx were highly unrelated to each other and to female territorial lynx, suggesting long distance male dispersal. Particular male and female resident territorial lynx and their offspring (kittens and subadults) were more likely to be trapped than resident floaters or dispersing (unrelated) lynx. Conversely, we obtained more data for unrelated lynx and higher numbers of territorials using non-invasive sampling (faeces). When invasive and non-invasive samples were analysed separately, the spatial organisation of lynx (in terms of female philopatry and females and males occupying permanent ranges) affected measures of genetic diversity in such a way that estimates of genetic diversity were reduced if only invasive samples were considered. It appears that, at small spatial scales, invasive sampling using box traps may underestimate the genetic diversity in carnivores with permanent ranges and philopatry such as the Eurasian lynx. As non-invasive sampling can also provide additional data on diet and spatial organisation, we advocate the use of such samples for conservation genetic studies of vulnerable, endangered or data deficient territorial species.

Peelle L. E., Wirsing A. J., Pilgrim K. L., Schwartz M. K. (2019): Identifying predators from saliva at kill sites with limited remains. Wildlife Society Bulletin 43: 546-557.
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Kill‐site investigations can yield valuable data about elusive predators and predator–prey interactions, provided the predator species can be definitively identified as the one responsible for the predation event. The traditional method of visually analyzing prey remains to identify predators is prone to observer bias and may be particularly challenging when few remains are present or in systems with congeneric predators. Other potential evidence left by predators, such as scat, may be difficult to reliably link to the responsible predator, whereas tracking requires adequate substrate conditions. Swabbing for predator DNA from saliva is an objective yet underutilized alternative that has primarily been applied to larger carcasses. We demonstrate the usefulness of a saliva‐swabbing method for smaller prey with minimal surface area, including kill sites with almost no prey remains or even just a radiocollar left behind. This study is also the first to compare saliva‐swabbing success by sample type (carcass remains vs. radiocollar). From 2010 to 2014, in the Loomis State Forest and Okanogan National Forest, Washington, USA, we employed forensic techniques to increase certainty about predator species identification at snowshoe hare (Lepus americanus) kill sites for the ultimate purpose of elucidating predator–prey interactions. Predator saliva was sampled from remains and radiocollars with foam buccal swabs, stored in lysis buffer for shelf‐stable preservation, and tested for mitochondrial DNA using polymerase chain reaction and species‐specific primers. This saliva‐swabbing protocol yielded definitive and objective predator species identification for a majority (58.5%) of sampled kill sites (n kill sites = 31/53). Not only were minimal remains often able to yield predator DNA, but swabs from radiocollars provided significantly more predator identifications. Saliva swabbing also provided identifications at 65.5% of kill sites lacking identifiable predator sign and contributed significantly more predator identifications throughout the year when compared with snow‐tracking methods. Expanding saliva‐swabbing methods to smaller prey, radiocollars, and limited remains should allow for more definitive predator identifications at kill sites than have been possible with commonly employed methods, thus augmenting the potential to understand and manage for predators and prey.

Qiao M., Connor T., Shi X., Huang J., Huang Y., Zhang H., Ran J. (2019): Population genetics reveals high connectivity of giant panda populations across human disturbance features in key nature reserve. Ecology and Evolution 9: 1809-1819.
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The giant panda is an example of a species that has faced extensive historical habitat fragmentation, and anthropogenic disturbance and is assumed to be isolated in numerous subpopulations with limited gene flow between them. To investigate the population size, health, and connectivity of pandas in a key habitat area, we noninvasively collected a total of 539 fresh wild giant panda fecal samples for DNA extraction within Wolong Nature Reserve, Sichuan, China. Seven validated tetra‐microsatellite markers were used to analyze each sample, and a total of 142 unique genotypes were identified. Nonspatial and spatial capture–recapture models estimated the population size of the reserve at 164 and 137 individuals (95% confidence intervals 153–175 and 115–163), respectively. Relatively high levels of genetic variation and low levels of inbreeding were estimated, indicating adequate genetic diversity. Surprisingly, no significant genetic boundaries were found within the population despite the national road G350 that bisects the reserve, which is also bordered with patches of development and agricultural land. We attribute this to high rates of migration, with four giant panda road‐crossing events confirmed within a year based on repeated captures of individuals. This likely means that giant panda populations within mountain ranges are better connected than previously thought. Increased development and tourism traffic in the area and throughout the current panda distribution pose a threat of increasing population isolation, however. Maintaining and restoring adequate habitat corridors for dispersal is thus a vital step for preserving the levels of gene flow seen in our analysis and the continued conservation of the giant panda meta‐population in both Wolong and throughout their current range.

Khan A., Patel K., Bhattacharjee S., Sharma S., Chugani A. N., Sivaraman K., Hosawad V., Sahu Y. K., Reddy G. V., Ramakrishnan U. (2020): Are shed hair genomes the most effective noninvasive resource for estimating relationships in the wild? Ecology and Evolution 10: 4583-4594.
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Knowledge of relationships in wild populations is critical for better understanding mating systems and inbreeding scenarios to inform conservation strategies for endangered species. To delineate pedigrees in wild populations, study genetic connectivity, study genotype‐phenotype associations, trace individuals, or track wildlife trade, many identified individuals need to be genotyped at thousands of loci, mostly from noninvasive samples. This requires us to (a) identify the most common noninvasive sample available from identified individuals, (b) assess the ability to acquire genome‐wide data from such samples, and (c) evaluate the quality of such genome‐wide data, and its ability to reconstruct relationships between animals within a population. We followed identified individuals from a wild endangered tiger population and found that shed hair samples were the most common compared to scat samples, opportunistically found carcasses, and opportunistic invasive samples. We extracted DNA from these samples, prepared whole genome sequencing libraries, and sequenced genomes from these. Whole genome sequencing methods resulted in between 25%–98% of the genome sequenced for five such samples. Exploratory population genetic analyses revealed that these data were free of holistic biases and could recover expected population structure and relatedness. Mitochondrial genomes recovered matrilineages in accordance with long‐term monitoring data. Even with just five samples, we were able to uncover the matrilineage for three individuals with unknown ancestry. In summary, we demonstrated that noninvasive shed hair samples yield adequate quality and quantity of DNA in conjunction with sensitive library preparation methods, and provide reliable data from hundreds of thousands of SNPs across the genome. This makes shed hair an ideal noninvasive resource for studying individual‐based genetics of elusive endangered species in the wild.

López-Bao J. V., Godinho R., Rocha R. G., Palomero G., Blanco J. C., Ballesteros F., Jiménez J. (2020): Consistent bear population DNA-based estimates regardless molecular markers type. Biological Conservation 248: 108651.
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Reliability of population size and density estimates is one of the most contentious issues when evaluating the conservation status of species. Non-invasive DNA monitoring, combined with spatially explicit capture-recapture approaches (SCR), is recurrently presented as a reliable procedure to achieve accurate, precise and feasible estimates. However, conservation practitioners frequently face the dilemma of which molecular marker type would perform better for their objectives. Here, using as case study the eastern subpopulation of the endangered Cantabrian brown bear, and the same dataset of non-invasive samples, we evaluated which molecular marker perform better (microsatellites vs. SNPs), and whether the selection of the marker could impact on estimates. Our results show similar posterior estimates for bear density and for the scale parameter, resulting in consistent population estimates, around 50 bears, regardless the molecular marker type selected, microsatellites: 48.4 (95%BCI = 33.8–67.3) or SNPs: 52.7 (95%BCI = 36.0–74.8). Using our dataset, we provide evidence for the importance of running several replicates for both microsatellites and SNPs when genotyping non-invasive samples, and how for the same number of molecular markers, microsatellites were statistically more powerful. The positive trend observed in small large carnivore populations requires the adaptation of monitoring approaches. The quality of indexes commonly used, such as the minimum annual counts of females with cubs of the year in our study case, may be more difficult to maintain over time as populations increase in size and range. We therefore recommend the implementation of a regular monitoring based on non-invasive DNA monitoring and SCR approaches.

Peralta D. M., Ibañez E. A., Lucero S., Cappozzo H. L., Túnez J. I. (2020): A new minimally invasive and inexpensive sampling method for genetic studies in pinnipeds. Mammal Research 65: 11-18.
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The choice of sampling method is essential in the development of a population genetic study. This choice influences the type of samples that can be obtained and, therefore, the quality of the extracted DNA. Sampling methods can be classified into invasive, non-invasive, and minimally invasive. This last category is preferred, as it minimizes handling, damage, and degree of disturbance to animals, while still providing enough DNA for genetic studies. Here, we describe a very low cost and easy to manufacture minimally invasive sampler, developed to obtain hair samples from pinnipeds. The sampler was highly effective both at reaching animals and collecting a good number of hairs. We compared the efficiency of hair samples obtained using our device, versus tissue samples collected from dead animals, by counting successful mtDNA amplifications and sequencing. Hair samples amplified more efficiently compared with tissue samples, but sequencing was equally efficient for both sample types. In conclusion, the sampler described is an excellent option to obtain DNA for genetic studies. This minimally invasive technique can also be used to sample hair for other studies, such as estimation of environmental contaminants, measurement of individual hormone levels, or diet determination through the analysis of stable isotopes, among other potential uses.

Sato Y., Nakamura H., Kyoko K., Sekiguchi M., Ishibashi Y., Itoh T. (2020): Evaluation of the effectiveness of scented wooden posts for DNA hair snagging of brown bears. Mammal Study 45: 213-218.
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Estimating population density and monitoring demographic trends of large carnivores are hard to be done, as they, in general, occur at very low population densities over large ranges and are often elusive. We tested the utility of artificially scented wooden rubbing posts as a tool for DNA hair snagging of brown bears (Ursus arctos), which can be used for noninvasive population monitoring. To determine which type of scents can attract bears, we applied various types of the scent lure [odor of brown bear, α-pinene (turpentine), and wood creosote oil] on wooden posts to compare with an unscented control, using 75 posts in total during 2009–2012. Among them, we confirmed that 23 posts were rubbed by bears at least once. The scent lures were selected as significant variables by a likelihood ratio test. Probability of rubbing by bears was the highest for wooden posts with creosote oil (57.6%). Our study offers a potentially effective means of DNA hair snagging by using artificially scented wooden rubbing posts.

Von Duyke A. L., Crawford J. A., Quakenbush L., Adams J. R., Waits L. P. (2023): Determination of polar bear (Ursus maritimus) individual genotype and sex based on DNA extracted from paw-prints in snow. Frontiers in Conservation Science 4: 1166279.
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Polar bears rely upon sea ice to hunt, travel, and reproduce. Declining sea ice extent and duration has led polar bears to be designated as “threatened” (ESA). Population monitoring is vital to polar bear conservation; but recently, poor sea ice has made traditional aircraft-based methods less viable. These methods largely rely upon the capture and handling of polar bears, and have been criticized over animal welfare concerns. Monitoring polar bears via DNA sampling is a promising option. One common method utilizes biopsy darts delivered from a helicopter to collect DNA, a method that faces similar ice associated challenges to those described above. However, epidermal cells shed from the foot pads of a polar bear into its paw-prints in snow are a source of “environmental DNA” (e-DNA) that can be collected non-invasively on the sea ice or on land for potential use in population monitoring. Mitochondrial DNA (mt-DNA) is used to assess whether polar bear DNA is present within a snow sample, and nuclear DNA (n-DNA) can identify individuals and their sex. The goal of this investigation was to assess the viability of using e-DNA collected from paw-prints in the snow to identify individual polar bears and their sex. Snow was sampled from 13 polar bear trails (10 paw-prints per trail) on the sea ice in the Chukchi and Beaufort seas along the North Slope of Alaska. Species verification was based on a mt-DNA PCR fragment analysis test. Identification of individuals was accomplished by amplifying a multiplex of seven n-DNA microsatellite loci, and sex was determined by the amelogenin gene sex ID marker. Six of the 13 bear trails sampled (46%) yielded consensus genotypes for five unique males and one female. To our knowledge, this is the first time that polar bears have been individually identified by genotype and sex using e-DNA collected from snow. This method is non-invasive, could be integrated into genetic mark-recapture sampling designs, and addresses some of the current challenges arising from poor sea ice conditions. It also can involve, engage, and empower Indigenous communities in the Arctic, which are greatly affected by polar bear management decisions.

CETACEANS

Green M. L., Herzing D. L., Baldwin J. D. (2007): Noninvasive methodology for the sampling and extraction of DNA from free‐ranging Atlantic spotted dolphins (Stenella frontalis). Molecular Ecology Notes 7: 1287-1292.
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Genetic sampling and molecular investigations are important parts of studying wild populations. However, collecting tissues from free‐ranging animals can be difficult or impractical. This study develops a sampling and extraction protocol for template DNA from faecal material collected in a marine environment from small cetaceans. DNA was extracted from faecal material of free‐ranging Atlantic spotted dolphins (Stenella frontalis) and subsequently tested for its suitability in molecular investigations by amplifying both mitochondrial and nuclear DNA. The resulting mitochondrial sequences were found to closely match known S. frontalis haplotypes. Three microsatellite loci were amplified and fall within the expected size range for cetaceans. Mother and calf families previously assigned by observation were genetically confirmed using both mitochondrial haplotype and allele sharing between the mother and offspring. The protocol effectively collects and extracts dolphin DNA from faecal samples and enables species identification as well as confirmation of genetic relatedness and should be considered as a noninvasive alternative to current protocols.

Green M. L., Herzing D. L., Baldwin J. D. (2011): Reproductive success of male Atlantic spotted dolphins (Stenella frontalis) revealed by noninvasive genetic analysis of paternity. Canadian Journal of Zoology 89: 239-253.
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Cetaceans are known to frequently engage in sexual behavior; however, the lack of male parental investment in offspring makes assessment of male reproductive success difficult. We assessed paternity in a small population (mean individuals sighted per year = 93) of Atlantic spotted dolphins (Stenella frontalis (G. Cuvier, 1829)) utilizing noninvasively collected fecal material. Samples (n = 88) were collected from dolphins from four social clusters. Of the 29 offspring tested, 34.5% were assigned paternity, resulting in 10 paternities assigned to seven males. Our study indicates that achieving a certain age is a potential precursor for males to mate successfully, as 18 years was the youngest estimated age of a male at the time of calf conception. In all pairings but one, the males were older than the female (mean age difference = 7.7+ years). Successful males were from two of the four social clusters and males most often mated within their social group or with females from the next geographically closest group. The study combines genetic data with known maternal pedigree information and reveals patterns in the overall mating system in a cetacean species where reproductive success of males was previously unknown.

Richard J. T., Schultz K., Goertz C., Hobbs R., Romano T. A., Sartini B. L. (2017): Assessing the quantity and downstream performance of DNA isolated from beluga (Delphinapterus leucas) blow samples. Aquatic Mammals 43: 398-408.
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Blow (exhale) sampling in cetaceans may provide a minimally invasive alternative to biopsy sampling for genetic analyses that may be favored in vulnerable populations. However, the utility of single-exhale blow samples has not been evaluated, and the relationship between the number of exhales collected and DNA yield and its subsequent performance during polymerase chain reaction (PCR) is unknown. DNA was extracted from 98 blow samples collected from 11 aquarium-housed and 29 wild belugas in Bristol Bay, Alaska. Blow samples consisted of one, two, or four successive exhales, with at least nine samples per type from both aquarium-housed and wild belugas. DNA concentration and purity was assessed with a spectrophotometer, and PCR performance was assessed through the amplification of a fragment of the mitochondrial DNA control region or a nuclear marker of sex. Measurable DNA was recovered from 96 samples (98%), although DNA yield varied widely, both by sample (range: 0 to 4,406 ng, mean = 701.5, SD = 1,033.7) and by number of exhalations (ng DNA/exhale) (range: 0 to 3,723, mean = 427.1, SD = 721.8). The amount of DNA extracted per exhale was greater for aquarium samples than for wild samples, but total yield was not proportional with the number of exhales for either group. Successful beluga-specific PCR amplification occurred in 56/59 of the aquarium samples (23/25 single-exhale samples) and 28/39 of the wild samples tested (7/10 of the single-exhale samples). The forcefulness of the breath and chance collection of large pieces of cellular debris likely shaped the relationship between the number of exhales and the DNA yield. Using these methods, a single, forceful exhale should yield enough DNA to perform multiple experiments. This technique is immediately applicable to live-stranded belugas such as the temporary mass strandings that occasionally occur in Cook Inlet, Alaska, and has the potential to increase genetic sampling in protected populations with less disturbance than direct tissue sampling.

Parsons K. M., Everett M., Dahlheim M., Park L. (2018): Water, water everywhere: Environmental DNA can unlock population structure in elusive marine species. Royal Society Open Science 5: 180537.
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Determining management units for natural populations is critical for effective conservation and management. However, collecting the requisite tissue samples for population genetic analyses remains the primary limiting factor for a number of marine species. The harbour porpoise (Phocoena phocoena), one of the smallest cetaceans in the Northern Hemisphere, is a primary example. These elusive, highly mobile small animals confound traditional approaches of collecting tissue samples for genetic analyses, yet their nearshore habitat makes them highly vulnerable to fisheries by-catch and the effects of habitat degradation. By exploiting the naturally shed cellular material in seawater and the power of next-generation sequencing, we develop a novel approach for generating population-specific mitochondrial sequence data from environmental DNA (eDNA) using surface seawater samples. Indications of significant genetic differentiation within a currently recognized management stock highlights the need for dedicated eDNA sampling throughout the population’s range in southeast Alaska. This indirect sampling tactic for characterizing stock structure of small and endangered marine mammals has the potential to revolutionize population assessment for otherwise inaccessible marine taxa.

Raudino H. C., Tyne J. A., Smith A., Ottewell K., McArthur S., Kopps A. M., Chabanne D., Harcourt R. G., Pirotta V., Waples K. (2019): Challenges of collecting blow from small cetaceans. Ecosphere 10: e02901. 
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We trialed the collection of blow samples using a waterproof electric multirotor (quadcopter) drone from two free-ranging dolphin species, the abundant and approachable bottlenose dolphin (Tursiops aduncus) and the less common and boat shy humpback dolphin (Sousa sahulensis). This drone was fast, maneuverable, and quiet compared to other drones commonly used in studies of cetaceans and relative to their hearing thresholds. We were successful in collecting blow samples from four individual dolphins (three bottlenose dolphins and one humpback dolphin) in two groups. The success of obtaining samples was dependent on the individual dolphin’s activity. We were successful in sampling when dolphins were resting and socializing but found that socializing dolphins were not predictable in their surfacing and direction and therefore do not recommend drone sampling socializing dolphins. The suitability and preference of the sampling technique over biopsy sampling is highly dependent on the dolphin activity. We also attempted to extract DNA from the blow samples with the aim of assessing the feasibility of using blow sampling by drone for population genetic studies. We were unsuccessful in extracting DNA and recommend that others attempting to sample dolphin blow with a drone should prioritize collecting a larger volume of blow that may yield adequate concentrations of DNA to be amplified. Blow sample volume could potentially be increased by sampling with more absorbent materials.

Székely D., Corfixen N. L., Mørch L. L., Knudsen S. W., McCarthy M. L., Teilmann J., Heide‐Jørgensen M. P., Olsen M. T. (2021): Environmental DNA captures the genetic diversity of bowhead whales (Balaena mysticetus) in West Greenland. Environmental DNA 3: 248-260.
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Environmental changes are prominent in Arctic ecosystems, where the distribution, abundance, life history, and health of marine organisms such as the bowhead whale (Balaena mysticetus) are tightly connected to sea ice and sea temperature. However, due to logistical and other challenges of data collection in the Arctic, appropriate assessments of past, present and future effects of climate change and human activities are lacking for many Arctic species. Environmental DNA (eDNA) is emerging as a noninvasive and cost-effective way of obtaining genetic material from the environment and has the potential to complement traditional methods for biodiversity and genetic monitoring. In this study, we investigate whether eDNA isolated from seawater samples has the capacity to capture the genetic diversity of bowhead whales in Disko Bay, West Greenland, for the implementation of long-term genetic monitoring programs of key Arctic marine species. A total of 41 eDNA “footprint” samples were obtained from the water surface after a whale had dived and an additional 54 eDNA samples were collected along transect lines. Samples were screened for bowhead DNA using a species-specific qPCR primer and probe assay, and a subset of 30 samples were successfully Sanger-sequenced to generate individual mitochondrial control region haplotypes. Moreover, by shotgun sequencing ten footprint samples on an Illumina NovaSeq platform we show that footprints generally contain less than 1% endogenous DNA, resulting in partial mitochondrial genomes in four samples out of ten samples. Our findings suggest that sampling in the footprint or wake of traveling animals is a promising method for capturing the genetic diversity of bowhead whales and other marine megafauna. With optimization of sampling and target DNA sequencing for higher endogenous DNA yield, seawater eDNA samples have a large potential for implementation in the long-term population genetic monitoring of marine megafauna in the Arctic and elsewhere.

Neveceralova P., Carroll E. L., Steel D., Vermeulen E., Elwen S., Zidek J., Stafford J. K., Chivell W., Hulva P. (2022): Population changes in a whale breeding ground revealed by citizen science noninvasive genetics. Global Ecology and Conservation 37: e02141.
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Historical exploitation, and a combination of current anthropogenic impacts, such as climate change and habitat degradation, impact the population dynamics of marine mammalian megafauna. Right whales (Eubalaena spp.) are large cetaceans recovering from hunting, whose reproductive and population growth rate appear to be impacted by climate change. We apply noninvasive genetic methods to monitor southern right whale (E. australis, SRW) and test the application of noninvasive genetics to minimise the observer effects on the population. Our aim is to describe population structure, and interdecadal and interannual changes to assess species status in the Great Acceleration period of Anthropocene. As a basis for population genetic analyses, we collected samples from sloughed skin during post-migration epidermal moult. Considering the exploration-exploitation dilemma, we collaborated with whale watching companies, as part of a citizen science approach and to reduce ad hoc logistic operations and biopsy equipment. We used mitochondrial and microsatellite data and population genetic tools. We report for the first time the genetic composition and differentiation of the Namibian portion of the range. Population genetic parameters suggest that South Africa hosts the largest population. This corresponds with higher estimates of current gene flow from Africa compared to older samples. We have observed considerable interannual variation in population density at the breeding ground and an interdecadal shift in genetic variability, evidenced by an increase in the point estimate inbreeding. Clustering analyses confirmed differentiation between the Atlantic and Indo-Pacific, presumably originating during the ice ages. We show that population monitoring of large whales, essential for their conservation management, is feasible using noninvasive sampling within non-scientific platforms. Observed patterns are concurrent to changes of movement ecology and decline in reproductive success of the South African population, probably reflecting a large-scale restructuring of pelagic marine food webs.

ELEPHANTS

Flagstad Ø., Pradhan N. M., Kvernstuen L. G., Wegge P. (2012): Conserving small and fragmented populations of large mammals: non-invasive genetic sampling in an isolated population of Asian elephants in Nepal. Journal for Nature Conservation 20: 181-190.
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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.

Gray T. N., Vidya T. N. C., Potdar S., Bharti D. K., Sovanna P. (2014): Population size estimation of an Asian elephant population in eastern Cambodia through non-invasive mark-recapture sampling. Conservation Genetics 15: 803-810.
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The Asian elephant is a flagship species for conservation in tropical Asia, but reliable population estimates are available only from a few populations. This is because the species can be elusive and occurs at low densities in dense habitat over a large part of its range. Phnom Prich Wildlife Sanctuary in the Eastern Plains, Cambodia, which is part of one of the largest protected area complexes in South-East Asia, is one such habitat that had not been systematically censused for elephants. We, therefore, used fecal-DNA based capture-mark-recapture sampling to estimate the population size for establishing a monitoring baseline. Five sampling sessions targeted all areas in and adjacent to Phnom Prich Wildlife Sanctuary believed to be used by elephants. Fresh dung was collected as the source of DNA and genotyping was carried out based on nine microsatellite loci. The 224 samples collected yielded 78 unique genotypes. Using model averaging of closed population capture-mark-recapture models, the elephant population in Phnom Prich Wildlife Sanctuary was estimated to number 136 ± 18 (SE) individuals. Our results suggest that eastern Cambodia supports a regionally important Asian elephant population.

Bourgeois S., Kaden J., Senn H., Bunnefeld N., Jeffery K. J., Akomo-Okoue E. F., Ogden R., McEwing R. (2019): Improving cost-efficiency of faecal genotyping: New tools for elephant species. Plos One 14: e0210811.
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Despite the critical need for non-invasive tools to improve monitoring of wildlife populations, especially for endangered and elusive species, faecal genetic sampling has not been adopted as regular practice, largely because of the associated technical challenges and cost. Substantial work needs to be undertaken to refine sample collection and preparation methods in order to improve sample set quality and provide cost-efficient tools that can effectively support wildlife management. In this study, we collected an extensive set of forest elephant (Loxodonta cyclotis) faecal samples throughout Gabon, Central Africa, and prepared them for genotyping using 107 single-nucleotide polymorphism assays. We developed a new quantitative polymerase chain reaction (PCR) assay targeting a 130-bp nuclear DNA fragment and demonstrated its suitability for degraded samples in all three elephant species. Using this assay to compare the efficacy of two sampling methods for faecal DNA recovery, we found that sampling the whole surface of a dung pile with a swab stored in a small tube of lysis buffer was a convenient method producing high extraction success and DNA yield. We modelled the influence of faecal quality and storage time on DNA concentration in order to provide recommendations for optimized collection and storage. The maximum storage time to ensure 75% success was two months for samples collected within 24 hours after defecation and extended to four months for samples collected within one hour. Lastly, the real-time quantitative PCR assay allowed us to predict genotyping success and pre-screen DNA samples, thus further increasing the cost-efficiency of our approach. We recommend combining the validation of an efficient sampling method, the build of in-country DNA extraction capacity for reduced storage time and the development of species-specific quantitative PCR assays in order to increase the cost-efficiency of routine non-invasive DNA analyses and expand the use of next-generation markers to non-invasive samples.

Lohay G. G. (2020): An accurate molecular method to sex elephants using PCR amplification of Amelogenin gene. Pachyderm 61: 90-96.
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The use of molecular methods to identify the sex of elephants from non-invasive samples is essential for studies of population dynamics and population genetics. We designed a new technique for sex identification in savannah elephants using Amelogenin (AMEL) genes. The X-Y homologs of AMEL genes is known to be suitable for sex determination in pigs and some bovids. In this study on savannah elephants, the use of AMEL genes was more successful than previous methods that relied on genes found exclusively on Y-chromosomes, such as SRY, to distinguish males from females. We designed a common forward primer and two reverse primers for X- and Y-specific AMEL genes to obtain 262 bp and 196 bp PCR amplicons from X and Y genes, respectively. We tested the primers for the identification of the sex of 132 savannah elephants from fecal samples. The sex of 126 individuals (95.45%) matched observational data, while 6 (4.54%) did not match. This discrepancy observed was likely due to observational errors in the field, where high grass reduces the ability to accurately sex young individuals. Through our stool sample results, we have shown that the use of only three primers for AMELX/Y provides a highly accurate PCR-based method for sex identification in savannah elephants. The method is fast and shows more success than the SRY system by avoiding the inherent ambiguities of the previous PCR-based methods that made it difficult to distinguish between female samples and failed amplification reactions. Our sex identification method is non-invasive and can potentially be applied in population genetic studies and forensics tests on both savannah as well as forest elephants.

Bourgeois S., Ouitavon K., Kongmee P., Veeramaethaphan T., Kaden J., McEwing R. (2021): A simple sexing test for elephant species and its application to faecal DNA. Journal of Applied Genetics 62: 507-509.
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We developed a novel real-time PCR assay for rapid sexing in all three elephant species, which amplifies small fragments of the orthologous sexual chromosome zinc finger protein genes ZFX/ZFY (65 bp). This assay is a simple, inexpensive and reliable tool that is suitable for non-invasive DNA samples and can be incorporated into larger SNP panels for individual identification and population genetic studies.

Laguardia A., Bourgeois S., Strindberg S., Gobush K. S., Abitsi G., Ateme H. B. B., Ebouta F., Fay J. M., Gopalaswamy A. M., Maisels F., Daouda E. S. B. (2021): Nationwide abundance and distribution of African forest elephants across Gabon using non-invasive SNP genotyping. Global Ecology and Conservation 32: e01894.
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Robust monitoring programs are essential for understanding changes in wildlife population dynamics and distribution over time, especially for species of conservation concern. In this study, we applied a rapid non-invasive sampling approach to the Critically Endangered African forest elephant (Loxodonta cyclotis), at nationwide scale in its principal remaining population strongholds in Gabon. We used a species-specific customized genetic panel and spatial capture-recapture (SCR) approach, which gave a snapshot of current abundance and density distribution of forest elephants across the country. We estimated mean forest elephant density at 0.38 (95% Confidence Interval 0.24–0.52) per km2 from 18 surveyed sites. We confirm that Gabon is the main forest elephant stronghold, both in terms of estimated population size: 95,110 (95% CI 58,872–131,349) and spatial distribution (250,782 km2). Predicted elephant densities were highest in relatively flat areas with a high proportion of suitable habitat not in proximity to the national border. Protected areas and human pressure were not strong predictors of elephant densities in this study. Our nationwide systematic survey of forest elephants of Gabon serves as a proof-of-concept of application of noninvasive genetic sampling for rigorous population monitoring at large spatial scales. To our knowledge, it is the first nationwide DNA-based assessment of a free-ranging large mammal in Africa. Our findings offer a useful national baseline and status update for forest elephants in Gabon. It will inform adaptive management and stewardship of elephants and forests in the most important national forest elephant stronghold in Africa.

Laguardia A., Gobush K. S., Bourgeois S., Strindberg S., Abitsi G., Ebouta F., Fay J. M., Gopalaswamy A. M., Maisels F., Ogden R., White L. J. (2021): Assessing the feasibility of density estimation methodologies for African forest elephant at large spatial scales. Global Ecology and Conservation 27: e01550.
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Effective wildlife management requires information on population status and distribution. Survey methods that provide estimates of these population parameters can vary greatly in effort required, area covered, precision of estimates, and cost. Trade-offs are required, because increasing precision and area coverage generally requires increasing field effort and incurs a higher cost. We compare DNA- and camera trap based-spatial capture-recapture approaches (DNA-SCR and CT-SCR) to the widely-used, dung-based line transect distance sampling (LTDS) method to assess their performance when applied to three relatively large populations of forest elephant Loxodonta cyclotis (>500 individuals), in order to evaluate their feasibility for future use at national and regional scales. Six of the nine surveys had a coefficient of variation below 20%; area coverage via DNA-SCR and LTDS was comparable and greatly exceeded that of the CT-SCR as applied; overall cost was highest for the LTDS surveys compared to the other two methods. We designed a new metric with which to compare survey methods: an integrated feasibility index (IFI). This combines three typical survey components: total area covered, level of precision achieved, and cost. The IFI suggests that DNA-SCR and LTDS are equally acceptable in terms of the combination of the three survey components, and that either survey method is suitable for large (national or regional) spatial scales for forest elephant density estimation. CT-SCR provides more precise estimates, but has double the IFI, due to the high cost per km2. DNA-SCR in particular, given the improvements highlighted in this study, is now being used at a national scale in Gabon. In conclusion, we recommend that the use of these spatial capture-recapture (SCR) methods, and their development, continue. Future findings and improvements should be compiled across studies to ensure their robust evolution as an option for monitoring the African forest elephant across its range and inform strategies and action for its conservation.

Chen Y., Sun Y., Hua M., Shi K., Dudgeon D. (2022): Using genetic tools to inform conservation of fragmented populations of Asian elephants (Elephas maximus) across their range in China. Integrative Zoology 18: 453-468.
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A herd of 15 Chinese elephants attracted international attention during their 2021 northward trek, motivating the government to propose establishment of an Asian elephant national park. However, planning is hampered by a lack of genetic information on the remaining populations in China. We collected DNA from 497 dung samples from all 5 populations encompassing the entire range of elephants in China and used mitochondrial and microsatellite markers to investigate their genetic and demographic structure. We identified 237 unique genotypes (153 females, 84 males), representing 81% of the known population. However, the effective population size was small (28, range 25–32). Historic demographic contraction appeared to account for low haplotype diversity (Hd = 0.235), but moderate nucleotide and nuclear diversity (π = 0.6%, He = 0.55) was attributable to post-bottleneck recovery involving recent population expansion plus historical gene exchange with elephants in Myanmar, Lao PDR, and Vietnam. The 5 populations fell into 3 clusters, with Nangunhe elephants differing consistently from the other 4 populations (FST = 0.23); elephants from Mengyang, Simao, and Jiangcheng belonged to a single population (henceforth, MSJ), and differed from the Shangyong population (FST = 0.11). Interpopulation genetic variation reflected isolation by distance and female-biased dispersal. Chinese elephants should be managed as 2 distinct units: Nangunhe and another combining Shangyong and MSJ; their long-term viability will require restoring gene flow between Shangyong and MSJ, and between elephants in China and neighboring countries. Our results have the potential to inform conservation planning for an iconic megafaunal species.

HEDGEHOGS, SHREWS, AND DESMANS

Querejeta M., Castresana J. (2018): Evolutionary history of the endemic water shrew Neomys anomalus: recurrent phylogeographic patterns in semi‐aquatic mammals of the Iberian Peninsula. Ecology and Evolution 8: 10138-10146.
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The Cabrera’s water shrew (Neomys anomalus) is a small semi‐aquatic mammal whose taxonomic status was recently elevated from subspecies to species; as a consequence of this change, this species is now endemic to the Iberian Peninsula. In this study, we looked at its evolutionary history by combining phylogeography, the spatial distribution of genetic diversity, and species distribution modeling. To perform these analyses, we used noninvasive samples collected across the species distribution range and sequenced partial mitochondrial cytochrome b and D‐loop genes. Maximum‐likelihood and Bayesian phylogenetic trees derived from these sequences indicated that N. anomalus is divided into two main phylogroups that correlate strongly with geography, with two contact zones between the groups that showed limited spatial mixing between them. River basins were responsible for only a small percentage of the structure of the genetic diversity of this species despite its riparian habitat. The nucleotide diversity variation map showed the highest genetic diversity to be in the north of the Iberian Peninsula. Finally, species distribution modeling allowed the inference of an optimal area during the Last Interglacial in the north of the Iberian Peninsula, and multiple glacial refugia during the Last Glacial Maximum. The phylogeographic pattern of N. anomalus is strikingly similar to that of another semi‐aquatic Iberian mammal, the Pyrenean desman (Galemys pyrenaicus), revealing how Pleistocene glaciations could have had equivalent effects on species of similar ecology and distribution. This phylogeographic structure is consistent with N. anomalus having been isolated for long periods in multiple glacial refugia within the Iberian Peninsula, in agreement with the “refugia‐within‐refugia” hypothesis, and further supporting its status as a distinct species.

Barthel L. M., Wehner D., Schmidt A., Berger A., Hofer H., Fickel J. (2020): Unexpected gene-flow in urban environments: the example of the European hedgehog. Animals 10: 2315.
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We use the European hedgehog (Erinaceus europaeus), a mammal with limited mobility, as a model species to study whether the structural matrix of the urban environment has an influence on population genetic structure of such species in the city of Berlin (Germany). Using ten established microsatellite loci we genotyped 143 hedgehogs from numerous sites throughout Berlin. Inclusion of all individuals in the cluster analysis yielded three genetic clusters, likely reflecting spatial associations of kin (larger family groups, known as gamodemes). To examine the potential bias in the cluster analysis caused by closely related individuals, we determined all pairwise relationships and excluded close relatives before repeating the cluster analysis. For this data subset (N = 65) both clustering algorithms applied (Structure, Baps) indicated the presence of a single genetic cluster. These results suggest that the high proportion of green patches in the city of Berlin provides numerous steppingstone habitats potentially linking local subpopulations. Alternatively, translocation of individuals across the city by hedgehog rescue facilities may also explain the existence of only a single cluster. We therefore propose that information about management activities such as releases by animal rescue centres should include location data (as exactly as possible) regarding both the collection and the release site, which can then be used in population genetic studies.

MARSUPIALS

Walker F. M., Sunnucks P., Taylor A. C. (2006): Genotyping of “captured” hairs reveals burrow-use and ranging behavior of southern hairy-nosed wombats. Journal of Mammalogy 87: 690-699.
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The southern hairy-nosed wombat (Lasiorhinus latifrons) is a cryptic, primarily nocturnal marsupial, for which behavioral information is notably lacking. Animals that are difficult to observe can be identified and tracked by individual-specific genotypes obtained from remotely collected DNA sources; wombats are excellent candidates for such an approach because freshly plucked hair is easily captured at burrow entrances. Twice a year during 1999–2001, we employed an intensive remote hair-collection regime coupled with individual-resolution genetic analysis to sample a South Australian population in a 63.3-km2 protected conservation reserve. Individuals were identified using 5 highly polymorphic microsatellite markers and further examined using another 10 markers to resolve ambiguities, enabling assessment of burrow- and space-use patterns. Analyses of rate of discovery of new individuals indicated that nearly all wombats inhabiting the study area were detected (102); there were > 1,000 “capture” events, and 90% of individuals were detected multiple times. Surprisingly, a female sex bias existed despite females being the dispersing sex. Space use was conservative and likely a reflection of adaptations enabling energy conservation. Transits between the northern and southern sections of the study area were nearly nonexistent. No sex or consistent seasonal differences were found in burrow, warren, or space use, other than in interaction with other factors. However, a strong increase in activity and space use in September 2001 relative to other sampling periods may be attributed to activities associated with mating. The thoroughness and evenness of this study’s sampling strategy was markedly effective for this species, as, with appropriate modification and optimization, it will likely be for other shy and cryptic organisms.

Smith S., McRae P., Hughes J. (2009): Faecal DNA analysis enables genetic monitoring of the species recovery program for an arid-dwelling marsupial. Australian Journal of Zoology 57: 139-148. 
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The greater bilby, Macrotis lagotis, is a species of conservation significance in the arid and semiarid zones of Australia. A species recovery program has been underway since the mid-1990s but the incorporation of molecular genetic data within the program has been difficult due to the problems of obtaining regular, population-wide samples of this trap-shy and sparsely distributed species. In this study, we demonstrate that faecal pellets collected from around burrows in the dry, arid habitat of western Queensland provide a viable source for DNA extraction and analysis. Faecal DNA was used to generate population-level estimates of microsatellite and mtDNA diversity for comparison with previous estimates for the natural population derived from tissue samples. Data were used to assess both the reliability of faecal-derived genotypes and the extent of any diversity loss since the previous study. Microsatellite diversity recorded from eight polymorphic markers for the natural population (A = 4.31 ± 0.30, HE = 0.76 ± 0.03) was comparable with the previous study, indicating little change in genetic diversity for the natural population in the 10-year interim. Faecal genotypes generated for the recently reintroduced population matched the known number of founders as well as a known genotype, providing support for the reliability of the faecal DNA approach. The captive and reintroduced populations had significantly lower diversity levels than the natural population (A = 3.59 ± 0.28, HE = 0.68 ± 0.03; A = 3.57 ± 0.20, HE = 0.65 ± 0.03 respectively). Mitochondrial control region analysis, incorporating nested clade phylogeographic analysis (NCPA), agrees with earlier findings that populations of bilbies across the arid zone in Australia have only recently become fragmented, but the case for Queensland bilbies being strongly differentiated from other regions is diminished. Implications from this study include the need to further supplement the captive and reintroduced populations with additional out-bred individuals and that faecal DNA can be used effectively for ongoing monitoring and management of this species.

Wedrowicz F., Karsa M., Mosse J., Hogan F. E. (2013): Reliable genotyping of the koala (Phascolarctos cinereus) using DNA isolated from a single faecal pellet. Molecular Ecology Resources 13: 634-641.
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The koala, an Australian icon, has been added to the threatened species list. Rationale for the listing includes proposed declines in population size, threats to populations (e.g. disease) and loss and fragmentation of habitat. There is now an urgent need to obtain accurate data to assess the status of koala populations in Australia, to ensure the long‐term viability of this species. Advances in genetic techniques have enabled DNA analysis to study and inform the management of wild populations; however, sampling of individual koalas is difficult in tall, often remote, eucalypt forest. The collection of faecal pellets (scats) from the forest floor presents an opportunistic sampling strategy, where DNA can be collected without capturing or even sighting an individual. Obtaining DNA via noninvasive sampling can be used to rapidly sample a large proportion of a population; however, DNA from noninvasively collected samples is often degraded. Factors influencing DNA quality and quantity include environmental exposure, diet and methods of sample collection, storage and DNA isolation. Reduced DNA quality and quantity can introduce genotyping errors and provide inaccurate DNA profiles, reducing confidence in the ability of such data to inform management/conservation strategies. Here, we present a protocol that produces a reliable individual koala genotype from a single faecal pellet and highlight the importance of optimizing DNA isolation and analysis for the species of interest. This method could readily be adapted for genetic studies of mammals other than koalas, particularly those whose diet contains high proportions of volatile materials that are likely to induce DNA damage.

Schultz A. J., Cristescu R. H., Littleford‐Colquhoun B. L., Jaccoud D., Frère C. H. (2018): Fresh is best: Accurate SNP genotyping from koala scats. Ecology and Evolution 8: 3139-3151.
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Maintaining genetic diversity is a crucial component in conserving threatened species. For the iconic Australian koala, there is little genetic information on wild populations that is not either skewed by biased sampling methods (e.g., sampling effort skewed toward urban areas) or of limited usefulness due to low numbers of microsatellites used. The ability to genotype DNA extracted from koala scats using next‐generation sequencing technology will not only help resolve location sample bias but also improve the accuracy and scope of genetic analyses (e.g., neutral vs. adaptive genetic diversity, inbreeding, and effective population size). Here, we present the successful SNP genotyping (1272 SNP loci) of koala DNA extracted from scat, using a proprietary DArTseq protocol. We compare genotype results from two‐day‐old scat DNA and 14‐day‐old scat DNA to a blood DNA template, to test accuracy of scat genotyping. We find that DNA from fresher scat results in fewer loci with missing information than DNA from older scat; however, 14‐day‐old scat can still provide useful genetic information, depending on the research question. We also find that a subset of 209 conserved loci can accurately identify individual koalas, even from older scat samples. In addition, we find that DNA sequences identified from scat samples through the DArTseq process can provide genetic identification of koala diet species, bacterial and viral pathogens, and parasitic organisms.

Zemanova M. A., Ramp D. (2021): Genetic structure and gene flow in eastern grey kangaroos in an isolated conservation reserve. Diversity 13: 570. 
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Dispersal is a key process for population persistence, particularly in fragmented landscapes. Connectivity between habitat fragments can be easily estimated by quantifying gene flow among subpopulations. However, the focus in ecological research has been on endangered species, typically excluding species that are not of current conservation concern. Consequently, our current understanding of the behaviour and persistence of many species is incomplete. A case in point is the eastern grey kangaroo (Macropus giganteus), an Australian herbivore that is subjected to considerable harvesting and population control efforts. In this study, we used non-invasive genetic sampling of eastern grey kangaroos within and outside of the Mourachan Conservation Property to assess functional connectivity. In total, we genotyped 232 samples collected from 17 locations at 20 microsatellite loci. The clustering algorithm indicated the presence of two clusters, with some overlap between the groups within and outside of the reserve. This genetic assessment should be repeated in 10–15 years to observe changes in population structure and gene flow over time, monitoring the potential impact of the planned exclusion fencing around the reserve.

Cowen S., Smith M., McArthur S., Rayner K., Jackson C., Anderson G., Ottewell K. (2022): Novel microsatellites and investigation of faecal DNA as a non-invasive population monitoring tool for the banded hare-wallaby (Lagostrophus fasciatus). Australian Journal of Zoology 69: 55-66.
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Monitoring programs for populations of small or medium-sized animals often use live-capture or photo-monitoring trapping methods to estimate population size. The banded hare-wallaby (Lagostrophus fasciatus), a small macropodiform marsupial, does not readily enter traps or have individually unique distinguishing physical features and is consequently difficult to monitor using these methods. Isolating DNA from faecal material to obtain individual genotypes is a promising monitoring technique and may present an alternative approach for this species. We developed novel species-specific microsatellite markers and undertook trials to assess faecal DNA degradation in ambient environmental conditions at two locations where this species has been translocated. The quality of DNA yielded from faecal pellets was evaluated through amplification failure and genotyping error rates of microsatellite markers. Error rates were compared for different treatments and exposure duration across multiple individuals. DNA was successfully obtained from all samples and error rates increased with exposure duration, peaking after 14–30 days depending on the site and treatment. The level of solar exposure was the most significant factor affecting degradation rate but both this and exposure duration had significant effects on amplification failure. Analysing DNA obtained from faecal pellets may represent a practical non-invasive method of deriving population estimates for this species and warrants further development.

Treloar S., Lohr C., Hopkins A. J., Ottewell K., McArthur S., Davis R. A. (2023): Scat DNA as a non-invasive method for estimating the abundance of the vulnerable mala (Lagorchestes hirsutus). Wildlife Research.
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Population-monitoring programs often use direct (e.g. live capture or spotlighting) or indirect (e.g. scats sightings) observations to estimate population abundance. Such methods, however, are often inadequate for rare, elusive, or cryptic species due to the difficulty in achieving sufficient encounters or detection rates. The mala (Lagorchestes hirsutus), a small native Australian macropod, listed as Vulnerable by the IUCN, is difficult to capture, susceptible to capture myopathy, and not easily sighted in their dense habitat; consequently, the population size cannot always be estimated. The use of molecular markers to identify individual genotypes from non-invasively collected samples is increasingly being used in wildlife conservation and may be an alternative approach for mala. The aim of this study was to evaluate the efficacy of non-invasive scat DNA sampling to estimate the population abundance of mala. A panel of microsatellite markers was developed for the identification of individual mala via profiling of their scats. Scats were systematically collected from a wild mala population located in an 1100-ha fenced reserve in Western Australia. Individual genotypes were determined using the microsatellite markers, and the abundance of mala was estimated using the genotypes with spatially explicit capture–recapture (SECR) and mark–resight analyses. The genetic markers proved variable and with sufficient exclusionary power to confidently identify unique individuals (mean locus genotyping error rate: 3.1%). Individual genetic identification from scat sampling, when used with traditional mark–recapture/resight analytical models, provides feasible estimates of population abundance. This is the first reliable abundance estimate of this mala population, suggesting a >70% increase in population size since the initial reintroduction of 64 individuals in 2011–13. Given the inherent difficulties in surveying mala, this approach would be valuable to ensure effective monitoring of the few remaining fenced and island mala populations to prevent further decline of this vulnerable species. This is the first study to identify species-specific microsatellite markers for mala and use genetic-capture sampling with scat DNA to estimate the abundance of a mala population. The study provides an evaluation of a valuable species monitoring technique that can be applied to other rare, elusive, or cryptic threatened species.

MONOTREMES

Perry T., Toledo-Flores D., Kang W. X., Ferguson A., Laming B., Tsend-Ayush E., Lim S. L., Grützner F. (2019): Non-invasive genetic sexing technique for analysis of short-beaked echidna (Tachyglossus aculeatus) populations. Reproduction, Fertility and Development 31: 1289-1295.
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Identifying male and female echidnas is challenging due to the lack of external genitalia or any other differing morphological features. This limits studies of wild populations and is a major problem for echidna captive management and breeding. Non-invasive genetic approaches to determine sex minimise the need for handling animals and are used extensively in other mammals. However, currently available approaches cannot be applied to monotremes because their sex chromosomes share no homology with sex chromosomes in other mammals. In this study we used recently identified X and Y chromosome-specific sequences to establish a non-invasive polymerase chain reaction-based technique to determine the sex of echidnas. Genomic DNA was extracted from echidna hair follicles followed by amplification of two Y chromosome (male-specific) genes (mediator complex subunit 26 Y-gametolog (CRSPY) and anti-Müllerian hormone Y-gametolog (AMHY)) and the X chromosome gene (anti-Müllerian hormone X-gametolog (AMHX)). Using this technique, we identified the sex of 10 juvenile echidnas born at Perth Zoo, revealing that eight of the 10 echidnas were female. Future use of the genetic sexing technique in echidnas will inform captive management, continue breeding success and can be used to investigate sex ratios and population dynamics in wild populations.

PRIMATES

Inoue E., Inoue-Murayama M., Takenaka O., Nishida T. (2007): Wild chimpanzee infant urine and saliva sampled noninvasively usable for DNA analyses. Primates 48: 156-159.
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In many genetic studies on the great apes, fecal or hair samples have been used as sources of DNA. However, feces and hairs are difficult to collect from chimpanzee infants under 3 years of age. As alternative DNA sources, we investigated the efficiency of collecting urine samples from infants compared with fecal samples, as well as the validity of the DNA extracted from urine and saliva samples of well-habituated M group chimpanzees (Pan troglodytes schweinfurthii) in the Mahale Mountains National Park, Tanzania. We collected 40 urine and 3 fecal samples from 10 infants under 3 years. Compared with feces, the urine samples were relatively easy to collect. The saliva of infants, which remained on the twigs sucked by them, was collected using cotton swabs. The average amounts of DNA extracted from the 40 urine and 6 saliva samples were 3,920 and 458 pg/μl, respectively. The rate of positive PCR was low and the allelic dropout rate was high when using less than 25 pg of template DNA in the PCR mixtures. Based on the amounts of DNA, 50% of the urine samples and 100% of the saliva samples were judged usable for accurate microsatellite genotyping. For infant chimpanzees in particular, collecting urine and saliva as an alternative to fecal and hair samples can reduce the effort invested in collection in the field.

Améndola‐Pimenta M., García‐Feria L., Serio‐Silva J. C., Rico‐Gray V. (2009): Noninvasive collection of fresh hairs from free‐ranging howler monkeys for DNA extraction. American Journal of Primatology 71: 359-363.
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The use of noninvasive collected samples as source of DNA in studies of wild primate populations has increased in recent years. Fresh‐plucked hairs represent an important source of DNA, with relatively high quality and concentration. In this study, we describe a low‐cost noninvasive technique for collecting fresh‐plucked hairs used to obtain DNA samples from free‐ranging black howler monkey populations (Alouatta pigra). We designed and manufactured darts made of wooden dowels, with the anterior part smeared with glue, which were projected with blowpipes to trap howler monkey hairs. All of the materials to make the darts are inexpensive and are available locally. We collected 89 samples from 76 individuals residing in 15 troops, and the total number of hairs obtained was 754. We found no differences in the number of hairs collected among sex–age classes or among localities but the percentage of darts recovered with sample varied among localities. Preliminary results indicate that over 96% of samples yielded DNA suitable for polymerase chain reaction‐based microsatellite marker analysis. The technique proved successful for collecting fresh‐plucked hairs of free‐ranging black howler monkeys without any trauma to the animals and can be easily adapted to obtain samples from other wild primate and mammal species.

Perry G. H., Marioni J. C., Melsted P., Gilad Y. (2010): Genomic‐scale capture and sequencing of endogenous DNA from feces. Molecular Ecology 19: 5332-5344.
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Genomic‐level analyses of DNA from non‐invasive sources would facilitate powerful conservation and evolutionary studies in natural populations of endangered and otherwise elusive species. However, the typical low quantity and poor quality of DNA that is extracted from non‐invasive samples have generally precluded such work. Here we apply a modified DNA capture protocol that, when used in combination with massively‐parallel sequencing technology, facilitates efficient and highly‐accurate resequencing of megabases of specified nuclear genomic regions from fecal DNA samples. We validated our approach by comparing genetic variants identified from corresponding fecal and blood DNA samples of six western chimpanzees (Pan troglodytes verus) across more than 1.5 megabases of chromosome 21, chromosome X, and the complete mitochondrial genome. Our results suggest that it is now feasible to conduct genomic studies in natural populations for which constraints on invasive sampling have otherwise long been a barrier. The data we collected also provided an opportunity to examine western chimpanzee genetic diversity at unprecedented scale. Despite high mitochondrial genome diversity (π = 0.585%), western chimpanzees have a low ratio (0.42) of X chromosomal (π = 0.034%) to autosomal (chromosome 21 π = 0.081%) sequence diversity, a pattern that may reflect an unusual demographic history of this subspecies.

Smiley T., Spelman L., Lukasik-Braum M., Mukherjee J., Kaufman G., Akiyoshi D. E., Cranfield M. (2010): Noninvasive saliva collection techniques for free-ranging mountain gorillas and captive eastern gorillas. Journal of Zoo and Wildlife Medicine 41: 201-209.
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This study was designed to develop a simple, noninvasive method for saliva collection: a first step toward developing new diagnostic tests to survey gorillas for infectious diseases. The subjects included free-ranging mountain gorillas (Gorilla beringei beringei) in the Parc National des Volcans, Rwanda, and a group of orphan mountain and Grauer’s gorillas (Gorilla heringei graueri) housed nearby in a temporary holding facility. Three collection methods were used to recover saliva from discarded forest food: swabbing, soaking, and washing. Saliva was also collected from orphan gorillas maintained in a captive setting by using dental ropes inside mesh bags. The presence of gorilla saliva in each sample was confirmed by using a salivary s-amylase assay and forensic press test paper. The recovery of gorilla DNA was verified by polymerase chain reaction by using primers specific to mountain and Grauer’s gorillas. Of the three collection techniques used to recover saliva from forest food, directly swabbing plant bite marks was the most effective. Wild celery (Peucedanum linderi) provided for the most consistent saliva recovery and is eaten year round by mountain gorillas in Rwanda. This study shows that gorilla saliva can be recovered easily and noninvasively from known individual free-ranging gorillas by collecting pieces of wild celery discarded as the gorillas forage and from captive gorillas by offering them juice-soaked dental ropes inside mesh bags. Both methods can be used to recover gorilla DNA for genetic studies. Saliva collected from free-ranging and captive gorillas may prove to be a useful biologic sample for the development of new diagnostic tests and hormonal analysis.

Chancellor R. L., Langergraber K., Ramirez S., Rundus A. S., Vigilant L. (2012): Genetic sampling of unhabituated chimpanzees (Pan troglodytes schweinfurthii) in Gishwati Forest Reserve, an isolated forest fragment in western Rwanda. International Journal of Primatology 33: 479-488.
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Many primate populations currently live in forest fragments. These populations are often unhabituated, elusive, and contain few individuals, making them difficult to study through direct observation. Noninvasive genetic methods are useful for surveying these unhabituated populations to infer the number and sex of individuals and the genetic diversity of the population. We conducted genetic analysis on 70 fecal samples from eastern chimpanzees (Pan troglodytes schweinfurthii) in Gishwati Forest Reserve, a forest fragment in western Rwanda. We genotyped all but two of these samples using 12 autosomal and 13 Y-chromosome microsatellite markers previously used in analyses of other chimpanzee populations. The genetic data show that these samples represent a minimum of 19 individuals (7 females, 12 males). However, because we may not have sampled all individuals in the population, we also performed mark-recapture analysis with the genetic data and found that the entire population likely numbers between 19 and 29 individuals. These results are consistent with opportunistic observations of at least 19 individual chimpanzees. Levels of variation at the Y-chromosome microsatellites were similar to those observed in other chimpanzee communities, suggesting that the chimpanzees in this forest are members of a single community. These results provide a baseline count of the number of male and female chimpanzees in the Gishwati Forest Reserve, and the data provide the potential for follow-up studies aimed at tracking individuals over time, thus aiding conservation management of this unhabituated population.

Simons N. D., Lorenz J. G., Sheeran L. K., Li J. H., Xia D. P., Wagner R. S. (2012): Noninvasive saliva collection for DNA analyses from free‐ranging Tibetan macaques (Macaca thibetana). American Journal of Primatology 74: 1064-1070.
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Cryptic and endangered fauna, including many primate taxa, pose challenges for noninvasive collection of biomaterials. As a result, application of noninvasive genotyping to primates has been limited to the use of samples such as feces and hair for the extraction of PCR ‐amplifiable DNA. We present a method for noninvasive collection of saliva from habituated, free‐ranging monkeys. The method utilizes a low‐cost apparatus that controls for contamination and is usable with individual, free‐ranging primates. Saliva samples were collected from 18 individuals in a population of T ibetan macaques (Macaca thibetana) in the Valley of Wild Monkeys in Huangshan, People’s Republic of China. DNA was extracted from these samples and PCR ‐amplified for both mitochondrial and nuclear genes, Cytochrome B and MHC ‐DR B eta 1, respectively. These results indicate this is an effective technique for the noninvasive collection of saliva across age and sex class, and dominance rank in a free‐ranging, terrestrial primate species. This device could have wide application for obtaining high‐quality saliva samples from free‐ranging primate populations for use in epidemiological studies, hormonal analyses of HPA axis function, pathogen screening, noninvasive genotyping, and behavioral genetics.

Inoue E., Akomo‐Okoue E. F., Ando C., Iwata Y., Judai M., Fujita S., Hongo S., Nze‐Nkogue C., Inoue‐Murayama M., Yamagiwa J. (2013): Male genetic structure and paternity in western lowland gorillas (Gorilla gorilla gorilla). American Journal of Physical Anthropology 151: 583-588.
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The male dispersal patterns of western lowland gorillas (WLGs, Gorilla gorilla gorilla) are not well understood. To determine whether most silverbacks stay close to their relatives, we analyzed autosomal and Y‐chromosomal microsatellites (STRs) in wild WLGs at Moukalaba, Gabon. We obtained STR genotypes for 38 individuals, including eight silverbacks and 12 adult females in an approximately 40 km2 area. Among them, 20 individuals were members of one identified group (Group Gentil; GG), including one silverback and six adult females. The silverback sired all 13 of the offspring in GG and no Y‐STR polymorphism within GG was found, as expected in a one‐male group structure. Over all silverbacks sampled, Y‐STR diversity was high considering the limited sampling area, and silverbacks with similar Y‐STR haplotypes were not always located in nearby areas. Although the misclassification rate of kinship estimates in this study was not negligible, there were no kin dyads among all silverbacks sampled. These results suggest that silverbacks born in the same group do not stay close to each other after maturation. The Y‐STR diversity in this study was similar to that of a previous study conducted in an area that was approximately 150 times larger than our study area. Similarity of WLG Y‐STR diversity between studies at different sampling scales suggests that male gene flow may not be geographically limited. These results suggest that WLG males normally disperse from their natal areas after maturation, at least, in Moukalaba.

Basabose A. K., Inoue E., Kamungu S., Murhabale B., Akomo‐Okoue E. F., Yamagiwa J. (2015): Estimation of chimpanzee community size and genetic diversity in Kahuzi‐Biega National Park, Democratic Republic of Congo. American Journal of Primatology 77: 1015-1025.
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A small chimpanzee habitat in the montane forest of Kahuzi‐Biega National Park, Democratic Republic of Congo, is connected with the lowland forest of this park through a corridor, which is affected by human encroachment. To assess the conservation status of the chimpanzee population in this small habitat, we estimated the size of the community and evaluated its genetic diversity by using 279 fecal samples collected in the montane forest of Kahuzi. Using autosomal microsatellite (or short tandem repeat, STR) loci, we identified 32 individuals, comprising 19 females and 13 males. Samples from 24 individuals were collected at least twice and a genetic mark‐recapture analysis estimated that the community size was 36 (range: 32–42). Data on nest site sharing confirmed that all the samples belonged to the same community. Nest site sharing information may be useful in population studies of unhabituated chimpanzees. The genetic structure and diversity of the 32 genotyped individuals was assessed using Y‐chromosomal short tandem repeat (Y‐STR) loci and mitochondrial D‐loops. One dominant Y‐STR haplotype was found, whereas there was no dominant haplotype in the mitochondrial region, reflecting a female‐biased dispersal pattern, which is typical of chimpanzees. The genetic diversity for three markers in Kahuzi chimpanzees was comparable to that in other eastern chimpanzee populations. A relatively high heterozygosity and negative inbreeding coefficient (FIS) for STR loci suggests that the study community belongs to an outbreeding chimpanzee population. These findings suggest that individuals of the study community may have reproductive contact with other chimpanzee individuals from neighboring communities in Kahuzi‐Biega National Park, at least in the recent past.

Aylward M. L., Sullivan A. P., Perry G. H., Johnson S. E., Louis Jr, E. E. (2018): An environmental DNA sampling method for aye‐ayes from their feeding traces. Ecology and Evolution 8: 9229-9240.
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Noninvasive sampling is an important development in population genetic monitoring of wild animals. Particularly, the collection of environmental DNA (eDNA) which can be collected without needing to encounter the target animal facilitates the genetic analysis of endangered species. One method that has been applied to these sample types is target capture and enrichment which overcomes the issue of high proportions of exogenous (nonhost) DNA from these lower quality samples. We tested whether target capture of mitochondrial DNA from sampled feeding traces of the aye‐aye, an endangered lemur species would yield mitochondrial DNA sequences for population genetic monitoring. We sampled gnawed wood where aye‐ayes excavate wood‐boring insect larvae from trees. We designed RNA probes complementary to the aye‐aye’s mitochondrial genome and used these to isolate aye‐aye DNA from other nontarget DNA in these samples. We successfully retrieved six near‐complete mitochondrial genomes from two sites within the aye‐aye’s geographic range that had not been sampled previously. Our method demonstrates the application of next‐generation molecular techniques to species of conservation concern. This method can likely be applied to alternative foraged remains to sample endangered species other than aye‐ayes.

Guevara E. E., Frankel D. C., Ranaivonasy J., Richard A. F., Ratsirarson J., Lawler R. R., Bradley B. J. (2018): A simple, economical protocol for DNA extraction and amplification where there is no lab. Conservation Genetics Resources 10: 119-125. 
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Genetic analyses are well suited to address many research questions in the study of wild populations, yet species of interest often have distributions that are geographically distant from molecular laboratories, necessitating potentially lengthy transport of biological specimens. Performing basic genetic analyses on site would avoid the project delays and risks of sample quality decline associated with transport, as well as allow original specimens to remain in the country of origin. Further, diagnostic genetic assays performed in the field could provide real-time information allowing for more nimble adjustments to research plans and use of resources. To this end, we developed protocols for reliably performing front-end genetics bench work in the field, without the requirements of electricity or permanent shelter. We validated these protocols on buccal swabs collected during routine capturing of sifaka lemurs (Propithecus verreauxi) at Bezà Mahafaly Special Reserve in Southwest Madagascar and faecal samples collected from captive sifakas (P. coquereli) at the Duke Lemur Center. Our basic protocol pipeline involves a chelating resin based DNA extraction followed by whole genome amplification or polymerase chain reaction using reagents stored at ambient temperature and portable, compact equipment powered by a lightweight solar panel. We achieved a high success rate (>80%) in downstream procedures, demonstrating the promise of such protocols for performing basic genetic analyses in a broad range of field situations.

Ishizuka S., Kawamoto Y., Sakamaki T., Tokuyama N., Toda K., Okamura H., Furuichi T. (2018): Paternity and kin structure among neighbouring groups in wild bonobos at Wamba. Royal Society Open Science 5: 171006.
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Although both bonobos and chimpanzees are male-philopatric species, outcomes of male–male reproductive competition seem to be more closely associated with mating success in chimpanzees. This suggests that the extent of male reproductive skew is lower in bonobos. In addition, between-group male–male reproductive competition is more lethal in chimpanzees. This suggests that between-group differentiation in male kinship is lower in bonobos. We analysed the paternity of 17 offspring in two bonobo groups and estimated the relatedness of individuals among three neighbouring groups by using DNA extracted from non-invasive samples at Wamba, in the Democratic Republic of the Congo. The alpha males sired at least nine of 17 offspring. This supports a previous finding that the male reproductive skew is higher in bonobos than that in chimpanzees. Average relatedness among males within groups was significantly higher than that among males across groups, whereas there was no significant difference among females between within and across groups. These results are consistent with male philopatry, highly skewed reproductive success of males and female dispersal. Higher average relatedness among males within groups suggest that the differences in hostility towards males of different groups between bonobos and chimpanzees may be explained by factors other than kinship.

Stewart F. A., Piel A. K., Luncz L., Osborn J., Li Y., Hahn B. H., Haslam M. (2018): DNA recovery from wild chimpanzee tools. Plos One 13: e0189657.
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Most of our knowledge of wild chimpanzee behaviour stems from fewer than 10 long-term field sites. This bias limits studies to a potentially unrepresentative set of communities known to show great behavioural diversity on small geographic scales. Here, we introduce a new genetic approach to bridge the gap between behavioural material evidence in unhabituated chimpanzees and genetic advances in the field of primatology. The use of DNA analyses has revolutionised archaeological and primatological fields, whereby extraction of DNA from non-invasively collected samples allows researchers to reconstruct behaviour without ever directly observing individuals. We used commercially available forensic DNA kits to show that termite-fishing by wild chimpanzees (Pan troglodytes schweinfurthii) leaves behind detectable chimpanzee DNA evidence on tools. We then quantified the recovered DNA, compared the yield to that from faecal samples, and performed an initial assessment of mitochondrial and microsatellite markers to identify individuals. From 49 termite-fishing tools from the Issa Valley research site in western Tanzania, we recovered an average of 52 pg/μl chimpanzee DNA, compared to 376.2 pg/μl in faecal DNA extracts. Mitochondrial DNA haplotypes could be assigned to 41 of 49 tools (84%). Twenty-six tool DNA extracts yielded >25 pg/μl DNA and were selected for microsatellite analyses; genotypes were determined with confidence for 18 tools. These tools were used by a minimum of 11 individuals across the study period and termite mounds. These results demonstrate the utility of bio-molecular techniques and a primate archaeology approach in non-invasive monitoring and behavioural reconstruction of unhabituated primate populations.

Hagemann L., Arandjelovic M., Robbins M. M., Deschner T., Lewis M., Froese G., Boesch C., Vigilant L. (2019): Long-term inference of population size and habitat use in a socially dynamic population of wild western lowland gorillas. Conservation Genetics 20: 1303-1314.
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Genetic estimation of population sizes has been critical for monitoring cryptic and rare species; however, population estimates do not inherently reveal the permanence or stability of the population under study. Thus, it is important to monitor not only the number of individuals in a population, but also how they are associated in groups and how those groups are distributed across the landscape. Adding to the challenge of obtaining such information with high precision for endangered and elusive species is the need for long-term collection of such data. In this study we compare sampling approaches and genotype non-invasive genetic samples to estimate the number and distribution of wild western lowland gorillas occupying a ~ 100 km2 area in Loango National Park, Gabon, for the periods 2005–2007 and 2014–2017. Based on the number of genotyped individuals we inferred a minimum of 83 gorillas during the first and 81 gorillas during the second study period. We also obtained similar capture–recapture population size estimates for the two periods despite variance in social dynamics like group formations, group dissolutions and individual dispersal. We furthermore found area fidelity for two groups that were sampled for 10–12 years, despite variation in group membership. Our results revealed how individual movements link groups in a ‘network’ and show that western lowland gorilla populations can show a high degree of temporal and geographic stability concurrent with substantial social dynamics.

Ishizuka S., Kawamoto Y., Toda K., Furuichi T. (2019): Bonobos’ saliva remaining on the pith of terrestrial herbaceous vegetation can serve as non-invasive wild genetic resources. Primates 60: 7-13.
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Evaluating the genetic diversity of natural populations of endangered species is important for conservation. Although the genetic analysis of wildlife usually requires collecting DNA non-invasively, the variety of non-invasive DNA sampling methods is limited for each species. We present a method to obtain DNA of an endangered species, the bonobo (Pan paniscus), in which the pith of the terrestrial herbaceous vegetation (THV) that they consumed was newly utilized. We investigated the (1) frequency of encountering remnant saliva on three types of THV pith; (2) concentrations of DNA in the saliva samples by the real-time quantitative PCR; and (3) rates of positive PCR, accurate genotyping, and allelic drop out by analyzing two autosomal microsatellite loci (D7s817 and D9s910). The number of remnant saliva samples was recorded by following the bonobo groups on a daily basis. The frequency of encountering DNA samples was higher in saliva samples than in fecal samples. More than half of the saliva samples remaining on two types of THV pith provided sufficient concentrations of bonobo DNA (> 200 pg/μl). Rates of positive PCR and accurate genotyping were high, and allelic dropout rate was low when the amount of template DNA was above 200 pg per reaction. Our results suggest that the remnants of bonobo saliva on the pith of THV are a potential resource for obtaining DNA, and better than other kinds of samples from the perspective of the abundant sampling opportunities.

Ang A., Roesma D. I., Nijman V., Meier R., Srivathsan A. (2020): Faecal DNA to the rescue: Shotgun sequencing of non-invasive samples reveals two subspecies of Southeast Asian primates to be Critically Endangered species. Scientific Reports 10: 9396.
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A significant number of Southeast Asian mammal species described in the 19th and 20th century were subsequently synonymized and are now considered subspecies. Many are affected by rapid habitat loss which creates an urgent need to re-assess the conservation status based on species boundaries established with molecular data. However, such data are lacking and difficult to obtain for many populations and subspecies. We document via a literature survey and empirical study how shotgun sequencing of faecal DNA is a still underutilized but powerful tool for accelerating such evaluations. We obtain 11 mitochondrial genomes for three subspecies in the langur genus Presbytis through shotgun sequencing of faecal DNA (P. femoralis femoralis, P. f. percura, P. siamensis cf. cana). The genomes support the resurrection of all three subspecies to species based on multiple species delimitation algorithms (PTP, ABGD, Objective Clustering) applied to a dataset covering 40 species and 43 subspecies of Asian colobines. For two of the newly recognized species (P. femoralis, P. percura), the results lead to an immediate change in IUCN status to Critically Endangered due to small population sizes and fragmented habitats. We conclude that faecal DNA should be more widely used for clarifying species boundaries in endangered mammals.

Lester J. D., Vigilant L., Gratton P., McCarthy M. S., Barratt C. D., Dieguez P., Agbor A., Álvarez-Varona P., Angedakin S., Ayimisin E. A., et al. (2021): Recent genetic connectivity and clinal variation in chimpanzees. Communications Biology 4: 283.
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Much like humans, chimpanzees occupy diverse habitats and exhibit extensive behavioural variability. However, chimpanzees are recognized as a discontinuous species, with four subspecies separated by historical geographic barriers. Nevertheless, their range-wide degree of genetic connectivity remains poorly resolved, mainly due to sampling limitations. By analyzing a geographically comprehensive sample set amplified at microsatellite markers that inform recent population history, we found that isolation by distance explains most of the range-wide genetic structure of chimpanzees. Furthermore, we did not identify spatial discontinuities corresponding with the recognized subspecies, suggesting that some of the subspecies-delineating geographic barriers were recently permeable to gene flow. Substantial range-wide genetic connectivity is consistent with the hypothesis that behavioural flexibility is a salient driver of chimpanzee responses to changing environmental conditions. Finally, our observation of strong local differentiation associated with recent anthropogenic pressures portends future loss of critical genetic diversity if habitat fragmentation and population isolation continue unabated.

Ozga A. T., Webster T. H., Gilby I. C., Wilson M. A., Nockerts R. S., Wilson M. L., Pusey A. E., Li Y., Hahn B. H., Stone A. C. (2021): Urine as a high-quality source of host genomic DNA from wild populations. Molecular Ecology Resources 21: 170-182.
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The ability to generate genomic data from wild animal populations has the potential to give unprecedented insight into the population history and dynamics of species in their natural habitats. However, in the case of many species, it is impossible legally, ethically, or logistically to obtain tissues samples of high-quality necessary for genomic analyses. In this study we evaluate the success of multiple sources of genetic material (feces, urine, dentin, and dental calculus) and several capture methods (shotgun, whole-genome, exome) in generating genome-scale data in wild eastern chimpanzees (Pan troglodytes schweinfurthii) from Gombe National Park, Tanzania. We found that urine harbors significantly more host DNA than other sources, leading to broader and deeper coverage across the genome. Urine also exhibited a lower rate of allelic dropout. We found exome sequencing to be far more successful than both shotgun sequencing and whole-genome capture at generating usable data from low-quality samples such as feces and dental calculus. These results highlight urine as a promising and untapped source of DNA that can be noninvasively collected from wild populations of many species.

Toyoda A., Matsudaira K., Maruhashi T., Malaivijitnond S., Kawamoto Y. (2021): Highly versatile, non-invasive method for collecting buccal DNA from free-ranging non-human primates. Journal of Tropical Biology and Conservation 18: 251-267.
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Non-invasive techniques for collection of DNA samples of suitable quality and quantity are important for improving the efficiency of genetic wildlife research. The development of a non-invasive method for collection of DNA samples from wild stump-tailed macaques (Macaca arctoides) is described herein. Sterilized polyester rope was cut into 10 cm pieces, which were then soaked in a 20% sugar solution to bait individuals. Rope swabs were immediately collected and transferred to a lysis buffer solution after subjects had picked up, chewed and discarded them. DNA was later extracted from the buffer. Quantitative real-time PCR and both allelic dropout and genotype failure rates were used to compare the quantity and quality of the buccal DNA samples to those of intestinal slough cell DNA samples collected from freshly dropped feces. The buccal samples yielded significantly more DNA (27.1 ± 33.8 ng/μL) than did the fecal samples (11.4 ± 15.4 ng/μL) and exhibited lower allelic dropout and genotyping failure rates for the 10 autosomal microsatellites investigated. Buccal cell collection was also simple, inexpensive, reliable and less time-consuming compared to fecal sampling. Thus, this method should facilitate genome-wide studies of non-human primates and other wildlife species.

RABBITS, HARES, AND PIKAS

Henry P., Russello M. A. (2011): Obtaining high-quality DNA from elusive small mammals using low-tech hair snares. European Journal of Wildlife Research 57: 429-435.
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Noninvasive sampling approaches are becoming increasingly important for enabling genetic studies of wildlife populations. While a number of methods have been described to noninvasively sample hair from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. Here we describe a novel and inexpensive noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We explore the quality of the sample by assessing PCR amplification success of mitochondrial and nuclear DNA fragments across four commercially available DNA isolation kits and two different quantities of hair in a factorial design. Additionally, we determined the sex of the individual samples using PCR–RFLP of ZFX/ZFY loci. We found that our snare is effective in obtaining hair that yield DNA of sufficient quality and quantity to successfully amplify a range of mitochondrial and nuclear fragment sizes. Specifically, we found the greatest success in amplifying mitochondrial DNA, nuclear microsatellites and ZFX/ZFY loci using at least 25 hairs as starting material and the DNA IQ™ system. The hair snares thus provide a cost-effective and minimally intrusive approach to sample elusive or rare small mammals. We anticipate that this approach will be useful to obtain samples for molecular studies of the ecology, evolution and conservation of small, elusive mammals.

Russello M. A., Waterhouse M. D., Etter P. D., Johnson E. A. (2015): From promise to practice: pairing non-invasive sampling with genomics in conservation. PeerJ 3: e1106.
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Conservation genomics has become an increasingly popular term, yet it remains unclear whether the non-invasive sampling that is essential for many conservation-related studies is compatible with the minimum requirements for harnessing next-generation sequencing technologies. Here, we evaluated the feasibility of using genotyping-by-sequencing of non-invasively collected hair samples to simultaneously identify and genotype single nucleotide polymorphisms (SNPs) in a climate-sensitive mammal, the American pika (Ochotona princeps). We identified and genotyped 3,803 high-confidence SNPs across eight sites distributed along two elevational transects using starting DNA amounts as low as 1 ng. Fifty-five outlier loci were detected as candidate gene regions under divergent selection, constituting potential targets for future validation. Genome-wide estimates of gene diversity significantly and positively correlated with elevation across both transects, with all low elevation sites exhibiting significant heterozygote deficit likely due to inbreeding. More broadly, our results highlight a range of issues that must be considered when pairing genomic data collection with non-invasive sampling, particularly related to field sampling protocols for minimizing exogenous DNA, data collection strategies and quality control steps for enhancing target organism yield, and analytical approaches for maximizing cost-effectiveness and information content of recovered genomic data.

Rehnus M., Bollmann K. (2016): Non-invasive genetic population density estimation of mountain hares (Lepus timidus) in the Alps: systematic or opportunistic sampling? European Journal of Wildlife Research 62: 737-747.
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The development and evaluation of a reliable non-invasive genetic sampling (NIGS) is a crucial step towards accurately and reliably estimating population size for the long-term monitoring of wildlife species. We used NIGS data to obtain population density estimates of a mountain hare (Lepus timidus) population in the Swiss Alps. We evaluated and compared the effectiveness of systematic and opportunistic NIGS and their combination in spring 2014. Extraction success rate of DNA from faeces, hair and urine samples, their age-dependent variation as well as the completeness of microsatellite genotyping data were used as measures of effectiveness. We applied a spatially explicit capture-recapture (SECR) approach to estimate the minimum population size. We found that the extraction success of faecal samples decreased with the time since excretion and that urine and hair samples mostly yielded insufficient DNA for the successful genotyping of individuals. Mountain hare faeces up to 5 days old are most appropriate for NIGS because the risk of unsuccessful DNA extraction or genotyping errors/failure is considerably lower in these samples. Systematic sampling revealed more genotypes than opportunistic sampling, but the latter resulted in higher numbers of recapture and thus, increased the spatial resolution of the data. Depending on the sampling design, the population density estimates ranged from 3.2 to 3.6 mountain hares per 100 ha. This study informs ecologists and wildlife managers about suitable survey techniques for the monitoring of free-ranging lagomorph populations and addresses important principles for the development of accurate survey methods for other elusive wildlife species that inhabit difficult, mountainous terrain.

DeMay S. M., Becker P. A., Rachlow J. L., Waits L. P. (2017): Genetic monitoring of an endangered species recovery: demographic and genetic trends for reintroduced pygmy rabbits (Brachylagus idahoensis). Journal of Mammalogy 98: 350-364.
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Monitoring demographic and genetic parameters of reintroduced populations of endangered species is essential for evaluating and informing conservation strategies to maximize the chances of a successful recovery. We applied noninvasive genetic monitoring using fecal DNA to evaluate demographic and genetic parameters as they relate to the recovery of the endangered Columbia Basin pygmy rabbit (Brachylagus idahoensis) in central Washington, United States, during the initial 3 years of a renewed reintroduction effort. We quantified post-release dispersal, apparent survival, and reproduction in the wild, and monitored the genetic diversity and composition of the released cohorts, wild surviving population, and breeding population held in large enclosures. During this study, we reintroduced 1,206 pygmy rabbits into the wild, and detected a total of 176 individuals living on or near the release area during surveys the following winters. Median dispersal distance of juveniles was 776 m, compared to 471 m for adults, and dispersal distances for juveniles decreased for rabbits released later in the release season. Survival of juveniles differed across years and was positively associated with release date, release weight (a surrogate for age), and heterozygosity. Survival of adults was similarly influenced by release day, with some evidence for a positive effect of heterozygosity. Only 14 wild-born individuals were detected during the study. Genetic monitoring was an effective way to evaluate the demographic and genetic status of the reintroduced population within a limited study area, to inform changes to the conservation strategy, and to generate a data set to address long-term research and recovery goals.

Buglione M., Petrelli S., Notomista T., de Filippo G., Gregorio R., Fulgione D. (2020): Who is who? High Resolution Melting analysis to discern between hare species using non-invasive sampling. Conservation Genetics Resources 12: 727-732.
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Identification of the species is a crucial step in many ecological studies. Sometimes, this could become a challenge, because of animal elusive behavior, low density population or for sympatric species leaving similar signs that are impossible to discriminate based only on their morphology. Here, we set up non-invasive molecular method to discern between the Italian hare (Lepus corsicanus) and the European hare (Lepus europaeus) using High Resolution Melting assay on fecal DNA, for the first time on these species. The Italian hare is endemic of the Central-Southern Italy and Sicily and it is classified vulnerable by the International Union for Conservation of Nature. Our procedure could be a useful tool to help conservation and management strategies, mainly in areas where the Italian hare and the European hare live in sympatry. The 9.5% of the peninsular range distribution of the European hare is took up by the range of the Italian hare. Our workflow allows sure species discrimination, rapidly and inexpensively (in one day at least 36 samples could be processed at costs of about 259 euros, including both DNA extractions and HRM run), also when large numbers of samples have to be processed. Moreover, our method could be widely applicable to other Lepus and/or mammalian species with similar concerns, by small adjustments to the protocol and its further validation, focusing on primes and corresponding HRM annealing temperature.

Schenker L., Bollmann K., Rehnus M., Brodbeck S., Gugerli F. (2020): Hare’s affairs: Lessons learnt from a noninvasive genetic monitoring for tracking mountain hare individuals. Ecology and Evolution 10: 10150-10166.
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Systematic monitoring of individuals and their abundance over time has become an important tool to provide information for conservation. For genetic monitoring studies, noninvasive sampling has emerged as a valuable approach, particularly so for elusive or rare animals. Here, we present the 5‐year results of an ongoing noninvasive genetic monitoring of mountain hares (Lepus timidus) in a protected area in the Swiss Alps. We used nuclear microsatellites and a sex marker to identify individuals and assign species to noninvasively collected feces samples. Through including a marker for sex identification, we were able to assess sex ratio changes and sex‐specific demographic parameters over time. Male abundance in the area showed high fluctuations and apparent survival for males was lower than for females. Generally, males and females showed only little temporary migration into and out of the study area. Additionally, using genotyped tissue samples from mountain hares, European hares (Lepus europaeus) and their hybrids, we were able to provide evidence for the first occurrence of a European hare in the study area at an elevation of 2,300 m a.s.l. in spring 2016. For future monitoring studies, we suggest to include complementary analysis methods to reliably infer species identities of the individuals analyzed and, thus, not only monitor mountain hare individual abundance, but also assess the potential threats given through competitive exclusion by and hybridization with the European hare.

Alasaad S., Soriguer R. C., Jowers M. J., Marchal J. A., Romero I., Sanchez A. (2011): Applicability of mitochondrial DNA for the identification of Arvicolid species from faecal samples: a case study from the threatened Cabrera’s vole. Molecular Ecology Resources 11: 409-414.
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Arvicolid mitochondrial genomes evolve faster than in any other mammalian lineage. The genetic diversity exhibited by these rodents contrasts sharply with their phenotypic homogeneity. Furthermore, faecal droppings from Arvicolid rodents of similar body size are almost undistinguishable on the basis of pellet morphology and content. In this study, we advantaged from their high genetic diversity vs. phenotypic homogeneity to document the applicability of mtDNA extraction from vole droppings for latter identification of such via a rapid and efficient nested PCR‐based technique using the threatened Microtus cabrerae as a model species. We sequenced the mitochondrial control region from 75 individuals belonging to 11 species of Arvicolinae from Spain, Portugal, Greece and Italy, and an additional 19 sequences from ten Microtus species from other countries were downloaded from Genbank. Based on these control region sequences, we successfully designed and applied a nested PCR for M. cabrerae‐specific and arvicolid‐generic mtDNA markers to differentiate Cabrera’s vole faecal samples among other species of the Arvicolinae subfamily. Although this study used Cabrera’s vole as a model species, similar techniques based on mtDNA sequences may find a broader applicability for noninvasive genetic conservation of vole species and their populations.

Oliveira C. G., Martinez R. A., Giné G. A. F., Faria D. M., Gaiotto F. A. (2011): Genetic assessment of the Atlantic Forest bristle porcupine, Chaetomys subspinosus (Rodentia: Erethizontidae), an endemic species threatened with extinction. Genetics and Molecular Research 10: 923-931.
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The bristle-spined porcupine, Chaetomys subspinosus, an endemic rodent from Atlantic Forest, was considered to be abundant in the recent past, but population reductions due to habitat loss and expansion of human activities caused this species to be included in the “vulnerable” category of the World Conservation Union Red List. We performed the first genetic assessment in natural populations of this focal species along its geographical distribution. Thirty-five non-invasive samples (hair) were collected from three natural populations in the Brazilian States of Sergipe, Bahia and Espírito Santo. Genetic similarity obtained by Jaccard’s index, based on dominant RAPD and ISSR markers, varied between 25 and 100%. Four clusters, mainly coincident with the geographical distribution of the populations, were observed. Analysis of molecular variance based on 47 polymorphic loci showed that there was 15.99% genetic variability among populations and 84.01% within populations. The estimated genetic structure among populations (Φ(ST)) was 0.16. The populations may have formed a continuum along the past distribution of the Atlantic rainforest but historical events of human occupation resulted in recent divergence among sampled populations.

Reiners T. E., Encarnação J. A., Wolters V. (2011): An optimized hair trap for non-invasive genetic studies of small cryptic mammals. European Journal of Wildlife Research 57: 991-995.
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As sample quality and quantity is a crucial factor in non-invasive genetics, we focused on the improvement of sampling efficiency of glue hair traps. We invented an optimized hair trap with moveable parts which enhanced sampling of high-quality genetic material. With the aid of the optimized hair trap, we were able to remotely pluck a sufficient amount of hair bulbs from our study animal the common hamster (Cricetus cricetus) with a trapping success of 49.3% after one survey night. The number of collected hairs with bulbs ranged between 1 and 50, with an average of 20.7 ± 14.8. Subsequently, the use of the hair trap in combination with a simplified laboratory routine allowed us to amplify species-specific microsatellites with an amplification success of 96.2% and ADO of 4.6%. This optimized trap may find usage for species identification or could be used as an instrument for long-term genetic monitoring of mammal populations.

Barbosa S., Pauperio J., Searle J. B., Alves P. C. (2013): Genetic identification of Iberian rodent species using both mitochondrial and nuclear loci: application to noninvasive sampling. Molecular Ecology Resources 13: 43-56. 
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Species identification through noninvasive sampling is increasingly used in animal conservation genetics, given that it obviates the need to handle free-living individuals. Noninvasive sampling is particularly valuable for elusive and small species such as rodents. Although rodents are not usually assumed to be the most obvious target for conservation, of the 21 species or near-species present in Iberia, three are considered endangered and declining, while several others are poorly studied. Here, we develop a genetic tool for identifying all rodent species in Iberia by noninvasive genetic sampling. To achieve this purpose, we selected one mitochondrial gene [cytochrome b (cyt-b)] and one nuclear gene [interphotoreceptor retinoid-binding protein (IRBP)], which we first sequenced using tissue samples. Both genes allow for the phylogenetic distinction of all species except the sibling species Microtus lusitanicus and Microtus duodecimcostatus. Overall, cyt-b showed higher resolution than IRBP, revealing a clear barcoding gap. To allow these markers to be applied to noninvasive samples, we selected a short highly diagnostic fragment from each gene, which we used to obtain sequences from faeces and bones from owl pellets. Amplification success for the cyt-b and IRBP fragment was 85% and 43% in faecal and 88% and 64% in owl-pellet DNA extractions, respectively. The method allows the unambiguous identification of the great majority of Iberian rodent species from noninvasive samples, with application in studies of distribution, spatial ecology and population dynamics, and for conservation.

Buś M. M., Żmihorski M., Romanowski J., Balčiauskienė L., Cichocki J., Balčiauskas L. (2014): High efficiency protocol of DNA extraction from Micromys minutus mandibles from owl pellets: a tool for molecular research of cryptic mammal species. Acta Theriologica 59: 99-109.
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Owl pellets have high potential as a source of DNA. However, this noninvasive method of collecting DNA is rarely used, and its methodological aspects are poorly understood. We investigated the methodology for DNA extraction and amplification from owl pellets containing the smallest European rodent—the Harvest mouse Micromys minutus—as an example. We used mandibles identified in owl pellets for mitochondrial and nuclear DNA amplification. For DNA extraction, we tested two commercial protocols and utilized a protocol being a combination of two commercial kits which ensured high efficiency of DNA extraction. Additionally, we recorded that the amount of DNA was five times higher in extracts from teeth as compared to DNA extracts from jawbones derived from the same mandible. The quantity of DNA was significantly positively correlated with biological sample weight; however, the age of the pellet remains had an impact on the level of inhibition. We recorded inhibition in 40 % of mtDNA extracts derived from pellets older than 150 months, whereas in DNA extracts from pellets younger than 80 months, we did not observe a negative impact of inhibition on PCR efficiency. The amplification success rate was 89.9 % for the mitochondrial fragment and 39.4 % in the case of the nuclear fragment. We observed partial degradation of DNA evidenced by the fact that the longest fragments that we were able to amplify in the case of mtDNA were 450 and 200 bp for nuDNA. The study shows that pellets can be considered as a source of DNA and have high potential for molecular research in the case of threatened species and species that are difficult to study using standard field techniques.

Mestre F., Pita R., Paupério J., Martins F. M., Alves P. C., Mira A., Beja P. (2015): Combining distribution modelling and non-invasive genetics to improve range shift forecasting. Ecological Modelling 297: 171-179.
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Forecasting species range shifts under climate change is critical to adapt conservation strategies to future environmental conditions. Ecological niche models (ENMs) are often used to achieve this goal, but their accuracy is limited when species niches are inadequately sampled. This problem may be tackled by combining ENM with field validation to fine-tune current species distribution, though the traditional methods are often time-consuming and the species ID inaccurate. Here we combine ENM with novel field validation methods based on non-invasive genetic sampling to forecast range shifts in the globally near-threatened Cabrera vole (Microtus cabrerae). Using occurrence records mapped at 10 km × 10 km resolution, we built the first ENM (ENM1) to estimate the current species distribution. We then selected 40 grid squares with no previous data along the predicted range margins, and surveyed suitable habitats through presence-sign searches. Faecal samples visually assigned to the species were collected for genetic identification based on the mitochondrial cytochrome-b gene, which resulted in 19 new grid squares with confirmed presence records. The second model (ENM2) was built by adding the new data, and species distribution maps predicted by each model under current and future climate change scenarios were compared. Both models had high predictive ability, with strong influence of temperature and precipitation. Although current distribution ranges predicted by each model were quite similar, the range shifts predicted under climate change differed greatly when using additional field data. In particular, ENM1 overlooked areas identified as important by ENM2 for species conservation in the future. Overall, results suggest that combining ENM with non-invasive genetics may provide a cost-effective approach in studies regarding species conservation under environmental change.

Ferreira C. M., Sabino-Marques H., Barbosa S., Costa P., Encarnação C., Alpizar-Jara R., Pita R., Beja P., Mira A., Searle J. B., Paupério J. (2018): Genetic non-invasive sampling (gNIS) as a cost-effective tool for monitoring elusive small mammals. European Journal of Wildlife Research 64: 46.
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Genetic non-invasive sampling (gNIS) may provide valuable information for population monitoring, as it allows inferences of population density and key behavioural traits such as dispersal, kinship and reproduction. Despite its enormous potential, gNIS has rarely been applied to small mammals, for which live-trapping is still the most commonly used sampling method. Here we evaluated the applicability and cost-effectiveness of gNIS compared with live-trapping, to monitor a metapopulation of an Iberian endemic and elusive rodent: the Cabrera vole (Microtus cabrerae). We compared the genetic diversity, kinship and dispersal movements inferred using both methods. For that, we optimised microsatellite markers for individual identification of M. cabrerae, using both tissue (n = 31) and faecal samples (n = 323) collected from a metapopulation in south-western Iberia. An initial set of 20 loci was optimised for tissue samples, from which 11 were selected to amplify in faecal samples. Overall, gNIS revealed a higher number of identified individuals (65) than live-trapping (31), and the estimated genetic diversity was similar using data from tissues and gNIS. Kinship analysis showed a higher number of inferred relationships and dispersal events when including gNIS, and indicated absence of sex-biased dispersal. The total cost (fieldwork and genetic analysis) of each genotype obtained through live-trapping was three times greater than for gNIS. Our data strongly supports the high potential and cost-effectiveness of gNIS for monitoring populations of elusive and/or threatened small mammals. We also illustrate how this genetic tool can be logistically feasible in conservation.

UNGULATES

Hausknecht R., Salzer K., Filli F., Kuehn R. (2009): Techniques for noninvasive genetic monitoring of Alpine Ibex Capra ibex. Acta Theriologica 54: 277-287.
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Noninvasive sampling is of increasing importance for the molecular genetic monitoring of wild animal populations, although reduced quality and quantity of such samples’ DNA can affect genetic data and their subsequent interpretation. Consequently, we performed a pilot study to establish a feasible approach for the genetic investigation of free-ranging Alpine ibex Capra ibex Linnaeus, 1758 populations. Establishing an ibex-specific PCR-RFLP based on Cytochrome b gene differences allowed the discrimination of noninvasive ibex samples from those of other sympatric ungulates. In addition, we established a quantitative PCR for ibex samples. The quantification of 35 faecal samples clearly exhibited a strong variability of DNA contents among samples and individuals. Furthermore, we performed threefold genotyping experiments on six microsatellite loci to determine the extent of genotyping errors in reference to blood samples of the respective individuals. The analyses exhibited a strong dependence of erroneous microsatellite genotypes on the starting amount of template DNA. Variability in reliability was observed between individual loci, resulting in a mandatory high DNA concentration necessary for consistent genotyping. This study serves as basis for further ibex research and we propose the application of DNA quantification of faecal samples to focus genotyping efforts solely on suitable samples.

Yang J., Jiang Z. (2011): Genetic diversity, population genetic structure and demographic history of Przewalski’s gazelle (Procapra przewalskii): implications for conservation. Conservation Genetics 12: 1457-1468.
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The Przewalski’s gazelle (Procapra przewalskii) is one of the most endangered antelope species in the world. It is endemic to China and is a flagship species in the eastern part of the Qinghai–Tibet plateau. To establish effective conservation measures on this species, genetic information such as genetic structure is needed. However, there has not been a comprehensive genetic assessment on this gazelle using nuclear DNA markers yet. Here, we employed 13 microsatellite loci to investigate genetic diversity, population genetic structure and demographic history of Przewalski’s gazelle using noninvasive samples of 169 wild gazelles collected from nine populations. A total of 76 alleles were detected from the entire samples, mean allele number was 5.85, and overall H O and H E were 0.525 and 0.552, respectively. Structure and GENELAND analyses found six genetic groups in the nine populations. Between the inferred genetic groups, significant genetic differentiation and low migration rates were detected. Demographic analyses indicated that Przewalski’s gazelle experienced genetic bottleneck and severe population decline, with the ancestral effective population size reducing to less than one percent. Based on the results of this study, we provide several conservation recommendations for Przewalski’s gazelle, such as six management units, periodic monitoring and special conservation consideration on the Qiejitan population.

Ebert C., Knauer F., Spielberger B., Thiele B., Hohmann U. (2012): Estimating wild boar Sus scrofa population size using faecal DNA and capture-recapture modelling. Wildlife Biology 18: 142-152.
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Increasing populations of wild boar and feral domestic pigs Sus scrofa have evoked growing concern due to their potential as disease reservoir and as an origin of agricultural damages. Reliable population estimates are needed for effective management measures of this species. As an alternative to traditional methods, non-invasive genetic population estimation approaches based on hair or faeces sampling have yielded promising results for several species in terms of feasibility and precision. We developed and applied a non-invasive population estimation approach based on wild boar faeces in a study area situated in the Palatinate Forest, southwestern Germany. We collected 515 faeces samples along transects in January 2008. We carried out genotyping using six microsatellite markers to discriminate between individuals. During the trial, we identified 149 individual wild boar. Using multimodel inference and model averaging, we obtained relatively consistent estimates. Population densities calculated using the estimated population sizes ranged from 4.5 (2.9-7.8) to 5.0 (4.0-7.0) wild boar/km2. In the future, to further improve the precision of population estimates based on wild boar faeces, the detection probability should be increased. However, even when comparing a conservative population estimate to the hunting bag, our results show that the present hunting regime in our study area is not effective in regulating the wild boar population. The method which we present here offers a tool to calibrate hunting or other management measures for wild boar.

Hoffmann G. S., Griebeler E. M. (2013): An improved high yield method to obtain microsatellite genotypes from red deer antlers up to 200 years old. Molecular Ecology Resources 13: 440-446.
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Analysis of DNA from older samples, such as museum specimen, is a promising approach to studying genetics of populations and ecological processes across several generations. Here, we present a method for extracting high quality nuclear DNA for microsatellite analysis from antlers of red deer (Cervus elaphus). The genotyping of individuals was based on nine microsatellite loci. Because the amount of DNA found in antlers was high, we could reduce the amount of sample and chemicals used and shorten the decalcification time in comparison to other methods. Using these methods, we obtained genotypes from antlers up to 200 years old.

Akomo-Okoue E. F., Inoue E., Atteke C., Nakashima Y., Hongo S., Inoue-Murayama M., Yamagiwa J. (2015): Noninvasive genetic analysis for assessing the abundance of duiker species among habitats in the tropical forest of Moukalaba, Gabon. Mammal Research 60: 375-384.
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We investigated the relative abundance of four duiker species and the environmental effect including human impacts on their abundance in the forest of Moukalaba, Gabon. We counted and collected fresh faeces from duikers in thirteen 2-km line transects established in different types of vegetation. Genetic species identification showed that blue duiker (Philantomba monticola), Ogilby’s duiker (Cephalophus ogilbyi, which might include the sister species Peter’s duiker Cephalophus callipygus), bay duiker (Cephalophus dorsalis), and yellow-backed duiker (Cephalophus silvicultor) coexisted. Genetic analyses successfully identified Ogilby’s and bay duiker, which are difficult to discriminate using other methods. The blue, Ogilby’s, and yellow-backed duikers were found in all transects. The dung abundance of bay duiker was quite low compared to other duiker species. This result suggests the importance of accumulating information regarding the abundance of each duiker species for updating their conservation status. The dung count method with genetic species identification may be useful for this purpose. The dung abundance of Ogilby’s duikers and blue duikers was affected by human impacts, while that of yellow-backed duikers was not affected by human impacts. The dung abundance of Ogilby’s duikers was lower in young secondary forests, which are regenerating after significant human disturbance. In contrast, generalized liner mixed model showed that the dung abundance of blue duikers was not affected by vegetation type and was lower in transects nearer to the villages, which might be due to past hunting activity. The difference in human impacts among duiker species should be considered for their conservation management.

Das P. K., Borthakur U., Sarma H. K., Talukdar B. K. (2015): Population genetic assessment of extant populations of greater one-horned rhinoceros (Rhinoceros unicornis) in India. European Journal of Wildlife Research 61: 841-851.
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The greater one-horned rhino has a wide range of historical distribution across the northern Indian subcontinent and now being confined to restricted patches in India and Nepal. Development of effective conservation strategy for rhino requires a clear understanding of the extant and spatial distribution of genetic diversity. In the present study, we employed nine microsatellite markers to analyze 238 noninvasively sampled individual rhinos from five protected areas in India, in order to assess genetic diversity and population genetic structure in the wild. We observed a moderate to high level of genetic diversity with allelic richness (Ar) ranging from 2.589 (±0.88) to 3.635 (±0.93) and expected heterozygosity (He) ranging from 0.352 (±0.20) to 0.59 (±0.13) in the area. Significant level of genetic differentiation was observed between the Protected Areas of Assam and West Bengal, especially, Gorumara National Park showing a unique genetic signature (F ST ≥ 0.25; p < 0.001, with all other protected areas). Given the degree of population genetic structure observed, prolonged separation of these protected areas is unwanted as this could lead to further loss of genetic diversity, consequently, affecting long-term viability of the species. The results presented here will be crucial in designing in situ conservation and management strategies of the species.

Kim Y. H., Lee J. W., Chae S., Moon S. H., Do E. J., Oh S. E., Zhang G. J., Lee M. Y. (2015): Development of a PCR-based assay to differentiate Cervus elaphus sibiricus from Cervus antlers. Journal of the Korean Society for Applied Biological Chemistry 58: 61-66.
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Deer antlers (Cervi Parvum Cornu) are reported to possess various pharmacological properties, including anti-infective, anti-arthritic, anti-allergic, and anti-endometriotic properties, and are credited with reversing memory impairment. To determine the global distribution of Cervus subspecies by analyzing antlers, 166 samples of Cervus subspecies antlers were collected from Russia, Canada, China, New Zealand, and Korea. The respective deer subspecies were identified by amplifying the D-loop region of mitochondrial DNA isolated from the antler samples. On the basis of the mitochondrial DNA sequence, a C. elaphus sibiricus-specific primer was developed that differentiates C. e. sibiricus from four C. e. subspecies (C. e. manitobensis, C. e. nelsoni, C. e. canadensis, and C. e. bactrianus), C. elaphus, and C. nippon. This was confirmed using agarose gel electrophoresis. This primer set produced a specific 396-bp fragment that amplified only C. e. sibiricus in antler samples. A 468-bp fragment derived from the cytochrome b gene was used as the internal control to verify the success of amplification in all samples. This method may assist in rapid and effective differentiation between products from Cervus species and other deer products.

Schoenecker K. A., Watry M. K., Ellison L. E., Schwartz M. K., Luikart G. (2015): Estimating bighorn sheep (Ovis canadensis) abundance using noninvasive sampling at a mineral lick within a national park wilderness area. Western North American Naturalist 75: 181-191.
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Conservation of species requires accurate population estimates. We used genetic markers from feces to determine bighorn sheep abundance for a herd that was hypothesized to be declining and in need of population status monitoring. We sampled from a small but accessible portion of the population’s range where animals naturally congregate at a natural mineral lick to test whether we could accurately estimate population size by sampling from an area where animals concentrate. We used mark-recapture analysis to derive population estimates, and compared estimates from this smaller spatial sampling to estimates from sampling of the entire bighorn sheep range. We found that estimates were somewhat comparable; in 2009, the mineral lick sample and entire range sample differed by 20 individuals, and in 2010 they differed by only one individual. However, we captured 13 individuals in the entire range sample that were not captured at the mineral lick, and thus violated a model assumption that all individuals had an equal opportunity of being captured. This eliminated the possibility of inferring a total population estimate from just animals visiting the mineral lick, but because estimates were relatively similar, monitoring at the mineral lick can provide a useful index for management and conservation. We compared our results to a radio-collar study conducted in 2003–2004 and confirmed that the population remained stable since 2004. Our population estimates were 78 (CI 62–114) in 2009 and 95 (CI 77–131) in 2010. Between 7 and 11 sampling dates were needed to achieve a CV of 20% for population estimates, assuming a capture probability between 0.09 and 0.13. We relied on citizen science volunteers to maximize data collection and reduce costs; 71% of all fecal samples were collected by volunteers, compared to 29% collected by paid staff. We conclude that our technique provides a useful monitoring tool for managers. The technique could be tested and applied in similar populations where animals congregate with high fidelity at a mineral lick or other area.

Woodruff S. P., Johnson T. R., Waits L. P. (2015): Evaluating the interaction of faecal pellet deposition rates and DNA degradation rates to optimize sampling design for DNA‐based mark-recapture analysis of Sonoran pronghorn. Molecular Ecology Resources 15: 843-854.
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Knowledge of population demographics is important for species management but can be challenging in low‐density, wide‐ranging species. Population monitoring of the endangered Sonoran pronghorn (Antilocapra americana sonoriensis) is critical for assessing the success of recovery efforts, and noninvasive DNA sampling (NDS) could be more cost‐effective and less intrusive than traditional methods. We evaluated faecal pellet deposition rates and faecal DNA degradation rates to maximize sampling efficiency for DNA ‐based mark–recapture analyses. Deposition data were collected at five watering holes using sampling intervals of 1–7 days and averaged one pellet pile per pronghorn per day. To evaluate nuclear DNA (nDNA) degradation, 20 faecal samples were exposed to local environmental conditions and sampled at eight time points from one to 124 days. Average amplification success rates for six nDNA microsatellite loci were 81% for samples on day one, 63% by day seven, 2% by day 14 and 0% by day 60. We evaluated the efficiency of different sampling intervals (1–10 days) by estimating the number of successful samples, success rate of individual identification and laboratory costs per successful sample. Cost per successful sample increased and success and efficiency declined as the sampling interval increased. Results indicate NDS of faecal pellets is a feasible method for individual identification, population estimation and demographic monitoring of Sonoran pronghorn. We recommend collecting samples >7 days old and estimate that a sampling interval of 4–7 days in summer conditions (i.e. extreme heat and exposure to UV light) will achieve desired sample sizes for mark–recapture analysis while also maximizing efficiency.

Borthakur U., Das P. K., Talukdar A., Talukdar B. K. (2016): Noninvasive genetic census of greater one-horned rhinoceros Rhinoceros unicornis in Gorumara National Park, India: a pilot study for population estimation. Oryx 50: 489-494.
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The greater one-horned rhinoceros Rhinoceros unicornis is a flagship species for conservation in protected areas in India and Nepal. In India the species is afforded the highest level of legal protection under Schedule I of the Wildlife (Protection) Act 1972. Although censuses of greater one-horned rhinoceros have been carried out for decades using the traditional total count method, no advanced scientific approach has been adopted for population estimation of the species in India or elsewhere. We optimized noninvasive genetic techniques for identification of greater one-horned rhinoceros from dung samples, and applied these to estimate the number of rhinoceros in Gorumara National Park, in West Bengal, India. Our results confirmed the presence of 43 individuals from 60 dung samples collected throughout the Park in 2011. We confirmed a male-to-female sex ratio of 3.8:1, based on analysis of DNA from dung samples, using a y-chromosome linked marker. Our results are in concordance with a census carried out by the West Bengal Forest Department that found 42 rhinoceros in the Park, with a male-to-female sex ratio of 3.5:1. Our study thus demonstrates the feasibility of using a noninvasive genetic approach for population estimation of greater one-horned rhinoceros in the wild.

Rea R. V., Johnson C. J., Murray B. W., Hodder D. P., Crowley S. M. (2016): Timing moose pellet collections to increase genotyping success of fecal DNA. Journal of Fish and Wildlife Management 7: 461-466. 
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Fecal pellets can serve as a noninvasive source of DNA for identifying the distribution of individual animals when conducting population estimates. The quality of fecal DNA, however, can be degraded by wet or warm environmental conditions. We tested the effect of time of pellet collection (mid-March–mid-June) and temperature on the success of genotyping microsatellite loci in moose Alces alces by using DNA extracted from moose fecal pellets collected from north central British Columbia, Canada. Using the number of microsatellite loci genotyped as a measure of genotyping success clearly indicated that fecal pellets collected in March and April contained DNA that yielded high-confidence genotypes, whereas those collected in May and June did not. Pellets collected in March and April were more likely to be collected in cooler (often subzero) temperatures than those collected in May and June. Pellets collected later in the year were also more likely to be exposed to rain and increased solar radiation, all of which are likely to contribute to degradation of fecal DNA. Our findings suggest that pellets collected in late winter in the Northern Hemisphere have sufficient DNA to permit genotyping of moose.

Goodman I., Shahar N., Bar-Gal G. K., Saltz D., Bar-David S. (2017): Evaluation of noninvasive genetic methods for Nubian ibex. Conservation Genetics Resources 9: 181-183.
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Noninvasive genetic methods enable the sampling of natural populations while minimizing detrimental effects on them. However, noninvasive methods are marred by DNA extraction and amplification difficulties that can be mitigated by improved methodology. Past studies have shown that noninvasive genetic protocols are system specific and highlighted the importance of pilot studies in the establishment of genetic monitoring programs. We tested, using a factorial design experiment, the effect of different methods for the collection-preservation and extraction on the amplification of DNA from Nubian ibex (Capra nubiana) fecal samples. We found collection-preservation using paper bags and extraction with QIAamp® fast DNA stool mini kit to significantly enhance success rate compared to collection-preservation in ethanol and extraction with QIAamp® DNA mini kit. Our results will contribute to the studies of natural populations of the vulnerable C. nubiana.

King S. R., Schoenecker K. A., Fike J. A., Oyler‐McCance S. J. (2018): Long‐term persistence of horse fecal DNA in the environment makes equids particularly good candidates for noninvasive sampling. Ecology and Evolution 8: 4053-4064. 
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Fecal DNA collected noninvasively can provide valuable information about genetic and ecological characteristics. This approach has rarely been used for equids, despite the need for conservation of endangered species and management of abundant feral populations. We examined factors affecting the efficacy of using equid fecal samples for conservation genetics. First, we evaluated two fecal collection methods (paper bag vs. ethanol). Then, we investigated how time since deposition and month of collection impacted microsatellite amplification success and genotyping errors. Between May and November 2014, we collected feral horse fecal samples of known age each month in a feral horse Herd Management Area in western Colorado and documented deterioration in the field with photographs. Samples collected and dried in paper bags had significantly higher amplification rates than those collected and stored in ethanol. There was little difference in the number of loci that amplified per sample between fresh fecal piles and those that had been exposed to the environment for up to 2 months (in samples collected in paper bags). After 2 months of exposure, amplification success declined. When comparing fresh (0–2 months) and old (3–6 months) fecal piles, samples from fresh piles had more matching genotypes across samples, better amplification success and less allelic dropout. Samples defecated during the summer and collected within 2 months of deposition had highest number of genotypes matching among samples, and lowest rates of amplification failure and allelic dropout. Due to the digestive system and amount of fecal material produced by equids, as well as their occurrence in arid ecosystems, we suggest that they are particularly good candidates for noninvasive sampling using fecal DNA.

le Roex N., Paxton M., Adendorff J., Ferreira S., O’Riain M. J. (2018): Starting small: long‐term consequences in a managed large‐mammal population. Journal of Zoology 306: 95-100.
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Small populations are vulnerable to founder effects, stochastic demographic events and inbreeding depression. These factors raise the extinction probability of small populations, and thus effective management plans for endangered species have become essential. The black rhinoceros (Diceros bicornis) survived a historic global population crash and is currently facing renewed threat through poaching, population fragmentation and isolation. South Africa is home to approximately 36% of the world’s remaining black rhinos; however, this population is fragmented across parks and reserves, and many of these small populations remain geographically and genetically isolated. The historic population crash, subsequent fragmentation and relentless poaching pressure together threaten the viability of the black rhino population within South Africa. This study employed a practical, non‐invasive sampling protocol and genetic analyses to assess the growth and performance of a small founder population in a fenced reserve. We used these data and recorded life‐history data to compare the population growth and vital rates between the first and second 10‐year periods since founding. We report a decrease in population growth rate and male survival in the second 10‐year period (0.06; 0.89) compared to the first (0.09; 0.97). Genetic diversity (HEXP = 0.405) was low when compared to larger black rhino populations, and average pairwise relatedness was high (0.193). These results suggest that negative genetic consequences and the beginning of density‐dependant growth regulation may be present in this population. This highlights the importance of establishing an evaluation framework for the assessment of small populations of long‐lived mammals in order to inform metapopulation management.

Park H. B., Park H. C., Cowan P. E., Hong S. (2018): Comparison of hair‐trapping methods for the long‐tailed goral. Wildlife Society Bulletin 42: 310-313.
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In South Korea, there is a need for more ecological information about endangered herbivores such as the long‐tailed goral (Naemorhedus caudatus). This species lives in steep, rocky valleys and is difficult to trap and study. Noninvasive hair capture techniques have frequently been applied to carnivores to enable DNA or isotope analysis. Therefore, we assessed the effectiveness of hair‐trapping using a population of goral in the Wangpi River basin in South Korea. To compare efficiency of the different shapes of hair traps, we used barbed traps at 4 (2008) and 9 sites (2009), and a hook‐style trap (2010) with barbed traps at each of 15 sites. In 2008, we collected 15 hairs at all 4 sites with barbs. In 2009, we obtained 48 hairs at 8 of the 9 sites using barbs; and in 2010, we collected 99 hairs (barbed: 50 hairs, hook traps: 49 hairs) at 10 of the 15 sites using both traps. In 2010, the 2 shapes of hair traps were equally efficient at providing samples. We concluded that the hair‐trapping method allows for noninvasive monitoring of the endangered goral, and could be used to monitor other species in South Korea.

Blåhed I. M., Ericsson G., Spong G. (2019): Noninvasive population assessment of moose (Alces alces) by SNP genotyping of fecal pellets. European Journal of Wildlife Research 65: 96.
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Noninvasive genetic studies of wild animals enable the recovery of information infeasible to obtain using other means. However, the low quantity and quality of noninvasively collected DNA often challenge the retrieval of reliable genotypes, which may cause biases in downstream analyses. In this study, we optimized SNP (single nucleotide polymorphism) genotyping of fecal samples from moose (Alces alces) with the main purpose of exploring the potential of using noninvasively retrieved genotypes for individual- and sex identification. Fecal pellets were collected during the late winter of 2016 on the Swedish island of Öland in the Baltic Sea and DNA was extracted and genotyped using 86 autosomal, six sex-specific and five species diagnostic SNPs. The SNP error rate of the quality filtered dataset was 0.06 and the probability of identity for siblings below 0.001. Following a thorough quality filtering process, 182 reliable genotypes were obtained, corresponding to 100 unique individuals (37 males, 63 females), with an estimated male proportion of 37% (± 9%). The population size, estimated using two different capture-mark-recapture approaches, was found to be in the range of 115–156 individuals (95% CI). Furthermore, moose on Öland showed significantly lower heterozygosity levels (zHexp = −5.51, N = 69, p Hexp = 3.56·10−8, zHobs = −3.58, N = 69, pHobs = 3.38·10−4) and appeared genetically differentiated from moose on the Swedish mainland. Thus, we show that quality controlled noninvasively derived SNP genotypes can be highly informative for individual and population monitoring in a large ungulate.

Jang J. E., Kim N. H., Lim S., Kim K. Y., Lee H. J., Park Y. C. (2020): Genetic integrity and individual identification-based population size estimate of the endangered long-tailed goral, Naemorhedus caudatus from Seoraksan National Park in South Korea, based on a non-invasive genetic approach. Animal Cells and Systems 24: 171-179.
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The long-tailed goral (also called the Amur goral) Naemorhedus caudatus (subfamily Caprinae), a vulnerable and protected species designated by IUCN and CITES, has sharply been declining in the population size and is now becoming critically endangered in South Korea. This species has been conserved as a natural monument by the Korean Cultural Heritage Administration since 1968. In this study, using 78 fecal DNA samples with a non-invasive genetic approach, we assessed the genetic integrity and individual identification-based population size for the goral population from Seoraksan National Park representing the largest wild population in Korea. Using the successfully isolated 38 fecal DNA, phylogeographic and population genetic analyses were performed with mitochondrial DNA control region (CR) sequences and nine microsatellite loci. We found seven CR haplotypes, of which five were unique to the Seoraksan population, considering previously determined haplotypes in Korean populations. The Seoraksan population showed higher haplotype diversity (0.777 ± 0.062) and mean number of alleles (4.67 ± 1.563) relative to southern populations in Korea reported from previous studies, with no signal of a population bottleneck. Microsatellite-based individual identification estimate based on probability of identity (PID) indicated a population size of ≥30 in this population. Altogether, we suggest that for future management efforts of this species in the Seoraksan National Park, conserving its genetic integrity as an ‘endemic’ lineage, and curbing a decrease in its number through mitigating habitat destruction might be key to secure the population for the long term.

Venegas C., Varas V., Vásquez J. P., Marín J. C. (2020): Non-invasive genetic sampling of deer: a method for DNA extraction and genetic analysis from antlers. Gayana 84: 67-74.
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We describe a non-invasive technique to isolate genomic DNA from connective tissue present in the antlers of deer of the genus Hippocamelus. This method is simpler and more effective than conventional more-destructive procedures which damage the collection material. This method is applicable to all cervids that annually regenerate their antlers.

Brandt J. R., Saidah S. H., Zhao K., Ishida Y., Apriyana I., Ryder O. A., Ramono W., Sudoyo H., Suryadi H., Van Coeverden de Groot P. J., Roca A. L. (2021): Characterization of 29 polymorphic microsatellite markers developed by genomic screening of Sumatran rhinoceros (Dicerorhinus sumatrensis). BMC Research Notes 14: 119.
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The Sumatran rhinoceros is critically endangered, with fewer than 100 individuals surviving across its current range. Accurate census estimates of the remaining populations are essential for development and implementation of conservation plans. In order to enable molecular censusing, we here develop microsatellite markers with amplicon sizes of short length, appropriate for non-invasive fecal sampling. Due to limited sample quantity and potential lack of genome-wide diversity, Illumina sequence reads were generated from two Sumatran rhinoceros samples. Genomic screening identified reads with short tandem repeats and loci that were polymorphic within the dataset. Twenty-nine novel polymorphic microsatellite markers were characterized (A = 2.4; HO = 0.30). These were sufficient to distinguish among individuals (PID < 0.0001), and to distinguish among siblings (PID(sib) < 0.0001). Among rhinos in Indonesia, almost all markers were established as polymorphic and effective for genotyping DNA from fecal samples. Notably, the markers amplified and displayed microsatellite polymorphisms using DNA extracted from 11 fecal samples collected non-invasively from wild Sumatran rhinoceros. These microsatellite markers provide an important resource for a census and genetic studies of wild Sumatran rhinos.

Bach B. H., Quigley A. B., Gaynor K. M., McInturff A., Charles K. L., Dorcy J., Brashares J. S. (2022): Identifying individual ungulates from fecal DNA: a comparison of field collection methods to maximize efficiency, ease, and success. Mammalian Biology 102: 863-874.
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Non-invasive genetic sampling can facilitate the identification of individual animals across a landscape, with applications to management and conservation. Fecal material is a readily available source of DNA, and various methods exist for collecting fecal samples for DNA preservation. In particular, swab methods offer considerable promise, but their utility in real-world field contexts remains relatively untested. We systematically compared multiple genetic fecal sampling methods across all stages of data collection and analysis, including sampling in the field, DNA extraction in the lab, and identification of individuals using microsatellite genotyping. We collected 112 fecal samples from black-tailed deer (Odocoileus hemionus columbianus) in the field in Mendocino County, California, across a range of sample conditions of unknown age. We systematically compared the efficiency, ease, and genotyping success of three methods for field collection and storage of ungulate fecal samples: whole pellets in ethanol, whole dry pellets in paper envelopes, and cotton swabs in buffer. Storage method, sample condition, and their interaction predicted genotyping success in the top binomial GLMMs. We found that swabbing pellets resulted in the greatest percentage of individually identifiable genotypes (81%, compared to 60% for dry samples and 56% for ethanol), despite lower DNA concentrations. While swabbing pellets requires a greater time investment in the field, the samples are easier and safer to store and transport, and subsequent labwork is more efficient as compared to whole-pellet collection methods. We, therefore, recommend the swab method for most contexts. We provide additional recommendations and field protocols based on subsequent collection of 2284 swab samples for a larger monitoring study of the deer population, given that this large number of samples spanned a range of sample conditions and time spent in storage.

Gavriliuc S., Reza S., Jeong C., Getachew F., McLoughlin P. D., Poissant J. (2022): Targeted genome-wide SNP genotyping in feral horses using non-invasive fecal swabs. Conservation Genetics Resources 14: 203-213.
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The development of high-throughput sequencing has prompted a transition in wildlife genetics from using microsatellites toward sets of single nucleotide polymorphisms (SNPs). However, genotyping large numbers of targeted SNPs using non-invasive samples remains challenging due to relatively large DNA input requirements. Recently, target enrichment has emerged as a promising approach requiring little template DNA. We assessed the efficacy of Tecan Genomics’ Allegro Targeted Genotyping (ATG) for generating genome-wide SNP data in feral horses using DNA isolated from fecal swabs. Total and host-specific DNA were quantified for 989 samples collected as part of a long-term individual-based study of feral horses on Sable Island, Nova Scotia, Canada, using dsDNA fluorescence and a host-specific qPCR assay, respectively. Forty-eight samples representing 44 individuals containing at least 10 ng of host DNA (ATG’s recommended minimum input) were genotyped using a custom multiplex panel targeting 279 SNPs. Genotyping accuracy and consistency were assessed by contrasting ATG genotypes with those obtained from the same individuals with SNP microarrays, and from multiple samples from the same horse, respectively. 62% of swabs yielded the minimum recommended amount of host DNA for ATG. Ignoring samples that failed to amplify, ATG recovered an average of 88.8% targeted sites per sample, while genotype concordance between ATG and SNP microarrays was 98.5%. The repeatability of genotypes from the same individual approached unity with an average of 99.9%. This study demonstrates the suitability of ATG for genome-wide, non-invasive targeted SNP genotyping, and will facilitate further ecological and conservation genetics research in equids and related species.

Koitzsch K. B., Anton C. B., Koitzsch L. O., Tjepkes T. L., Schumann A. C., Strasburg J. L. (2022): A noninvasive and integrative approach for improving density and abundance estimates of moose. The Journal of Wildlife Management 86: e22200.
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Acquiring demographic data for moose (Alces alces) can be difficult because they are solitary in nature, they prefer densely vegetated and mountainous habitats, and they often occur at low density. Such data, however, are essential for long-term population monitoring, evaluating management practices, and effective conservation. Winter aerial surveys are the standard method for estimating moose population parameters, but they can be logistically challenging, expensive, and subject to sightability correction, which necessitates the capture of study animals for initial model development. Herein, we demonstrate a noninvasive alternative approach for estimating population parameters of moose in northern Yellowstone National Park, where aerial surveys were attempted but proved ineffective. We determined individual moose genotype and sex using microsatellite polymerase chain reaction amplification of DNA extracted from fecal pellets, integrated ancillary pellet sample data (i.e., metadata) in genotype analysis to aid in the identification of matching genotypes, and used spatially explicit capture-recapture (SECR) modeling to estimate sex-specific density and abundance. We collected 616 samples over 3 consecutive winters (Dec 2013–Apr 2016) and within 2 sampling occasions each winter. We recorded 514 captures of 142 individual moose (69 males, 73 females). Overall density ranged between 0.062 moose/km2 and 0.076 moose/km2 and averaged 0.034/km2 for females and 0.033/km2 for males. Abundance estimates were 150 moose in 2013 (female = 76, 95% CI = 55–105; male = 74, 95% CI = 54–103), 186 in 2014 (female = 95, 95% CI = 63–142; male = 91, 95% CI = 60–138), and 160 in 2015 (female = 79, 95% CI = 58–108; male = 81, 95% CI = 59–110). Average population sex ratio was 0.99 males/female. We demonstrate that SECR analysis of fecal DNA genotypes, using metadata in genotype analysis to help identify matching moose genotypes, is a promising alternative method for estimating sex-specific density and abundance of a low-density moose population in a mountainous and forested landscape.

MULTIPLE SPECIES

Ramón‐Laca A., Soriano L., Gleeson D., Godoy J. A. (2015): A simple and effective method for obtaining mammal DNA from faeces. Wildlife Biology 21: 195-203.
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The use of faecal DNA, although a promising tool for the population monitoring of mammals, has not yet become a fully exploited and standard practice, mainly because low target DNA concentration, DNA degradation, and co-purification of inhibitors demand extra laboratory procedures to improve success and reliability. Here we evaluate a simple method that enables sampling of DNA in the field through the collection of the intestinal cells present on the surface of a scat using a swab. The swab is immediately placed in a vial containing a lysis buffer that preserves the DNA for its later extraction. DNA extracts of three species of herbivores (goat, fallow deer and white-tailed deer), two carnivores (Iberian lynx and domestic dog) and one omnivore species (brushtail possum) were characterised in terms of target and total DNA quantity, PCR inhibition and genotyping success. Direct comparison was carried out with duplicate samples preserved in 96% ethanol and extracted via a commonly used commercial DNA extraction kit for faecal material. Results from these comparisons show that swabbing the samples in situ not only simplifies field collection and sample handling in the laboratory, but generally optimises target DNA recovery, minimises co-purification of PCR inhibitors and provides good quality DNA for the species tested, especially for herbivores. This method is also less time-consuming and more cost-effective, thus providing a more convenient and efficient alternative for non-invasive genetic studies.

Joshi B. D., Singh S. K., Singh V. K., Jabin G., Ghosh A., Dalui S., Singh A., Priyambada P., Dolker S., Mukherjee T., Sharief A., Kumar V., Singh H., Thapa A., Sharma C. M., Dutta R., Bhattacharjee S., Singh I., Mehar B. S., Chandra K., Thakur M. (2022): From poops to planning: A broad non-invasive genetic survey of large mammals from the Indian Himalayan Region. Science of The Total Environment 853: 158679.
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Large forested landscapes often harbour significant amount of biodiversity and support mankind by rendering various livelihood opportunities and ecosystem services. Their periodic assessment for health and ecological integrity is essential for timely mitigation of any negative impact of human use due to over harvesting of natural resources or unsustainable developmental activities. In this context, monitoring of mega fauna may provide reasonable insights about the connectivity and quality of forested habitats. In the present study, we conducted a largest non-invasive genetic survey to explore mammalian diversity and genetically characterized 13 mammals from the Indian Himalayan Region (IHR). We analyzed 4806 faecal samples using 103 autosomal microsatellites and with three mitochondrial genes, we identified 37 species of mammal. We observed low to moderate level of genetic variability and most species exhibited stable demographic history. We estimated an unbiased population genetic account (PGAunbias) for 13 species that may be monitored after a fixed time interval to understand species performance in response to the landscape changes. The present study has been evident to show pragmatic permeability with the representative sampling in the IHR in order to facilitate the development of species-oriented conservation and management programmes.