Evaluation of Noninvasive Genetic Sampling Methods for Felid and Canid Populations

Abstract: Noninvasive sampling methods provide a means for studying species such as large mammalian carnivores that are difficult to survey using traditional techniques. Focusing on bobcat (Lynx rufus), we compared the effectiveness of noninvasive hair and scat genetic sampling in terms of field sample collection, species identification, and individual identification. We describe a novel hair-snare design and sampling protocol that successfully sampled 4 sympatric carnivore species, bobcat, mountain lion (Felis concolor), coyote (Canis latrans), and gray fox (Urocyon cinereoargenteus), in 3 habitat blocks in coastal southern California, USA. Scat surveys were also successful at sampling bobcats and other carnivores in the area. Hair and scat sampling methods had similar species identification success (81% and 87%, respectively) using mitochondrial DNA amplification and restriction enzyme digestion patterns. Therefore, for studies focused on the distribution and activity of a suite of carnivore species, we recommend a combination of noninvasive methodologies, for example, targeting hair and scat surveys toward species and sites where they are most effective. Because of a higher success rate for scat (85%) than for hair samples (10%) when using 4 microsatellite loci and a multiple-tubes approach to verify individual genotypes, we suggest scat sampling is a better choice for studies that require individual identification of bobcats.     

Balancing sample accumulation and DNA degradation rates to optimize noninvasive genetic sampling of sympatric carnivores

Noninvasive genetic sampling, or noninvasive DNA sampling (NDS), can be an effective monitoring approach for elusive, wide‐ranging species at low densities. However, few studies have attempted to maximize sampling efficiency. We present a model for combining sample accumulation and DNA degradation to identify the most efficient (i.e. minimal cost per successful sample) NDS temporal design for capture–recapture analyses. We use scat accumulation and faecal DNA degradation rates for two sympatric carnivores, kit fox (Vulpes macrotis) and coyote (Canis latrans) across two seasons (summer and winter) in Utah, USA, to demonstrate implementation of this approach. We estimated scat accumulation rates by clearing and surveying transects for scats. We evaluated mitochondrial (mtDNA) and nuclear (nDNA) DNA amplification success for faecal DNA samples under natural field conditions for 20 fresh scats/species/season from <1–112 days. Mean accumulation rates were nearly three times greater for coyotes (0.076 scats/km/day) than foxes (0.029 scats/km/day) across seasons. Across species and seasons, mtDNA amplification success was ≥95% through day 21. Fox nDNA amplification success was ≥70% through day 21 across seasons. Coyote nDNA success was ≥70% through day 21 in winter, but declined to <50% by day 7 in summer. We identified a common temporal sampling frame of approximately 14 days that allowed species to be monitored simultaneously, further reducing time, survey effort and costs. Our results suggest that when conducting repeated surveys for capture–recapture analyses, overall cost‐efficiency for NDS may be improved with a temporal design that balances field and laboratory costs along with deposition and degradation rates.     

Implications of the placental structure compatibility for interspecies embryo transfer

Comparative histological features of the chorionic villi in placental cotyledons of the common eland (Taurotragus oryx ) and bongo (Boocercus euryceros ) antelopes and okapi (Okapia johnstoni ) and giraffe (Giraffa camelopardalis ) were examined. The chorionic villi in both antelope species showed only moderate branching and/or surface corrugation and their cross-sections were polygonal to oval. The close similarity in the structure of cotyledons has been apparently a contributing factor for success in mutual interspecies embryo transfer. The chorionic villi in okapi and giraffe had very different structures. In okapi the villi on cross-section were round and filled with thin connective tissue. They showed minimal branching and surface corrugation. In giraffe the villi showed extensive surface corrugation, had multiple fine branches, and were filled with a more dense connective tissue. Prospect for materno-fetal compatibility in mutual embryo transfer between these species is guarded.     

An Improved Field Method to Obtain DNA for Individual Identification From Wolf Scat

ABSTRACT Sampling of feces for genetic studies of wild populations can be problematic because of the low quality and quantity of template DNA obtained. We used cotton swabs in the field to isolate the mucous layer on the surface of fresh wolf (Canis lupus, C. lycaon, and their hybrids) scats followed by immediate preservation, and compared microsatellite genotyping of DNA from these fresh field swabs (FS) to that of previously frozen laboratory swabs (LS). In single polymerase chain reactions (PCRs) of 2 multiplexes, amplification at 8 loci was higher in the FS samples (FS = 50%, LS = 15%; P = 0.02) because proportion, quantity, and quality of large fragment wolf nuclear DNA from these samples was greater (2.5–25%, 6.25–62.5 ng/swab, 35% amplified at 1,000 base pairs [bp]) than from the LS samples (1.9%–10%, 4.7–25 ng/swab, 10% amplified at 1,000 bp). Paired blood and fresh field-swabbed samples had identical genotypes. In 84 multiplex PCRs we found no evidence of allelic dropout associated with low template quality or quantity. We conclude that field swabbing of fresh wolf scat facilitates field storage and reduces the need for multiple amplifications at single microsatellite loci, thereby reducing the genotyping costs for wildlife projects that use noninvasive samples.     

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

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

Skin papillomas in an impala (Aepyceros melampus) and a giraffe (Giraffa camelopardalis).

Viral particles, typical of the papovavirus family, were demonstrated by electronmicroscopy in small papillomas found on the feet of an impala (Aepyceros melampus) and on the face of a giraffe (Giraffa camelopardalis) in Kenya. Histologically the tissues proved to be typical papillomas. The viral particles measured 38 nm and 40 nm in diameter in all tissue sections from the impala and giraffe respectively.     

An efficient method for screening faecal DNA genotypes and detecting new individuals and hybrids in the red wolf (<Emphasis Type="Italic">Canis rufus</Emphasis>) experimental population area

Previously, sequencing of mitochondrial DNA (mtDNA) from non-invasively collected faecal material (scat) has been used to help manage hybridization in the wild red wolf (Canis rufus) population. This method is limited by the maternal inheritance of mtDNA and the inability to obtain individual identification. Here, we optimize the use of nuclear DNA microsatellite markers on red wolf scat DNA to distinguish between individuals and detect hybrids. We develop a data filtering method in which scat genotypes are compared to known blood genotypes to reduce the number of PCR amplifications needed. We apply our data filtering method and the more conservative maximum likelihood ratio method (MLR) of Miller et al. (2002 Genetics 160:357–366) to a scat dataset previously screened for hybrids by sequencing of mtDNA. Using seven microsatellite loci, we obtained genotypes for 105 scats, which were matched to 17 individuals. The PCR amplification success rate was 50% and genotyping error rates ranged from 6.6% to 52.1% per locus. Our data filtering method produced comparable results to the MLR method, and decreased the time and cost of analysis by 25%. Analysis of this dataset using our data filtering method verified that no hybrid individuals were present in the Alligator River National Wildlife Refuge, North Carolina in 2000. Our results demonstrate that nuclear DNA microsatellite analysis of red wolf scats provides an efficient and accurate approach to screen for new individuals and hybrids.     

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

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_E = 0.60 ± 0.18 (SD) for jaguars, H_E = 0.65 ± 0.21 (SD) for pumas and H_E = 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.     

DNA metabarcoding for diet analysis and biodiversity: A case study using the endangered Australian sea lion (Neophoca cinerea)

The analysis of apex predator diet has the ability to deliver valuable insights into ecosystem health, and the potential impacts a predator might have on commercially relevant species. The Australian sea lion (Neophoca cinerea) is an endemic apex predator and one of the world's most endangered pinnipeds. Given that prey availability is vital to the survival of top predators, this study set out to understand what dietary information DNA metabarcoding could yield from 36 sea lion scats collected across 1,500 km of its distribution in southwest Western Australia. A combination of PCR assays were designed to target a variety of potential sea lion prey, including mammals, fish, crustaceans, cephalopods, and birds. Over 1.2 million metabarcodes identified six classes from three phyla, together representing over 80 taxa. The results confirm that the Australian sea lion is a wide‐ranging opportunistic predator that consumes an array of mainly demersal fauna. Further, the important commercial species Sepioteuthis australis (southern calamari squid) and Panulirus cygnus (western rock lobster) were detected, but were present in <25% of samples. Some of the taxa identified, such as fish, sharks and rays, clarify previous knowledge of sea lion prey, and some, such as eel taxa and two gastropod species, represent new dietary insights. Even with modest sample sizes, a spatial analysis of taxa and operational taxonomic units found within the scat shows significant differences in diet between many of the sample locations and identifies the primary taxa that are driving this variance. This study provides new insights into the diet of this endangered predator and confirms the efficacy of DNA metabarcoding of scat as a noninvasive tool to more broadly define regional biodiversity.     

Successful carnivore identification with faecal DNA across a fragmented Amazonian landscape

The use of scat surveys to obtain DNA has been well documented in temperate areas, where DNA preservation may be more effective than in tropical forests. Samples obtained in the tropics are often exposed to high humidity, warm temperatures, frequent rain and intense sunlight, all of which can rapidly degrade DNA. Despite these potential problems, we demonstrate successful mtDNA amplification and sequencing for faeces of carnivores collected in tropical conditions and quantify how sample condition and environmental variables influence the success of PCR amplification and species identification. Additionally, the feasibility of genotyping nuclear microsatellites from jaguar (Panthera onca) faeces was investigated. From October 2007 to December 2008, 93 faecal samples were collected in the southern Brazilian Amazon. A total of eight carnivore species was successfully identified from 71% of all samples obtained. Information theoretic analysis revealed that the number of PCR attempts before a successful sequence was an important negative predictor across all three responses (success of species identification, success of species identification from the first sequence and PCR amplification success), whereas the relative importance of the other three predictors (sample condition, season and distance from forest edge) varied between the three responses. Nuclear microsatellite amplification from jaguar faeces had lower success rates (15-44%) compared with those of the mtDNA marker. Our results show that DNA obtained from faecal samples works efficiently for carnivore species identification in the Amazon forest and also shows potential for nuclear DNA analysis, thus providing a valuable tool for genetic, ecological and conservation studies.     

Community‐based wildlife management area supports similar mammal species richness and densities compared to a national park

Community‐based conservation models have been widely implemented across Africa to improve wildlife conservation and livelihoods of rural communities. In Tanzania, communities can set aside land and formally register it as Wildlife Management Area (WMA), which allows them to generate revenue via consumptive or nonconsumptive utilization of wildlife. The key, yet often untested, assumption of this model is that economic benefits accrued from wildlife motivate sustainable management of wildlife. To test the ecological effectiveness (here defined as persistence of wildlife populations) of Burunge Wildlife Management Area (BWMA), we employed a participatory monitoring approach involving WMA personnel. At intermittent intervals between 2011 and 2018, we estimated mammal species richness and population densities of ten mammal species (African elephant, giraffe, buffalo, zebra, wildebeest, waterbuck, warthog, impala, Kirk's dik‐dik, and vervet monkey) along line transects. We compared mammal species accumulation curves and density estimates with those of time‐matched road transect surveys conducted in adjacent Tarangire National Park (TNP). Mammal species richness estimates were similar in both areas, yet observed species richness per transect was greater in TNP compared to BWMA. Species‐specific density estimates of time‐matched surveys were mostly not significantly different between BWMA and TNP, but elephants occasionally reached greater densities in TNP compared to BWMA. In BWMA, elephant, wildebeest, and impala populations showed significant increases from 2011 to 2018. These results suggest that community‐based conservation models can support mammal communities and densities that are similar to national park baselines. In light of the ecological success of this case study, we emphasize the need for continued efforts to ensure that the BWMA is effective. This will require adaptive management to counteract potential negative repercussions of wildlife populations on peoples' livelihoods. This study can be used as a model to evaluate the effectiveness of wildlife management areas across Tanzania.     

Evaluation of fecal DNA preservation techniques and effects of sample age and diet on genotyping success

Optimal collection and preservation protocols for fecal DNA genotyping are not firmly established. We evaluated 3 factors that influence microsatellite genotyping success of fecal DNA extracted from coyote (Canis latrans) scats: 1) age of scat, 2) preservative, and 3) diet content. We quantified genotyping success by comparing rates of allelic dropout, false alleles, and failed amplifications among consensus genotypes. We used a panel of 6 microsatellite loci to genotype 20 scat samples, each of which was subjected to 3 age (1 day, 5 days, and 10 days post-deposition) and 3 preservation (DET buffer, 95% ethanol [EtOH], and lysis buffer) treatments. Both sample age and storage buffer had a significant effect on success and reliability. Ethanol and DET buffer preserved fecal samples with similar efficiency, and both were superior to lysis buffer. Our analysis of DNA degradation rates revealed that samples collected as early as 5 days of age yielded DNA that was highly degraded relative to samples collected on day 1. We tested the influence of dietary remains on microsatellite genotyping by using scat samples consisting predominantly of insect prey (n = 5), mammalian prey (n = 9), or the remains of juniper (Juniperus spp.) berries (n = 6) and compared EtOH and DET buffer preservation efficacy. We observed a significant interaction effect between storage buffer and diet for the probability of a false allele in a polymerase chain reaction (PCR), suggesting that the optimal preservation technique depended on the food remains comprising the scat. Scats comprised of juniper berry remains were more reliably genotyped when preserved in DET than EtOH. Mammalian prey-based scats were more reliable when stored in EtOH than DET buffer. Insect-predominant scats were preserved in EtOH and DET buffer with similar efficiency. Although accurate and reliable results can be obtained from scats collected at ≥5 days of age, we suggest sampling design to include collection of scats <5 days of age to minimize field and laboratory expenses. We suggest EtOH preservation for scats of obligate carnivores and of facultative carnivores with a diet consisting primarily of mammals. We suggest DET buffer preservation for animals with a diet consisting of plant-derived foods. Lysis buffer protocols that we employed should not be used for fecal DNA preservation. © 2011 The Wildlife Society.     

Changing vulnerability to predation related to season and sex in an African ungulate assemblage

The consequences of predation for prey population dynamics depend on the extent to which this mortality is predisposed by malnutrition or senescence, or additive in the sense that animals that would otherwise not have died at that time were killed. In places lacking effective predators, few adult ungulates die during the summer or wet season months when food is plentifully available. Hence the seasonal distribution of predator kills as well as the age and sex classes of the prey indicates the extent to which malnutrition contributes to mortality as well as other influences on vulnerability. Using records of animal deaths assembled over 35 years in South Africa's Kruger National Park, these patterns were investigated for 12 ungulate species forming the prey of lions, and for three other large predators with respect to one prey species. Buffalo, kudu and giraffe were more strongly represented in kills made during the late dry season, while wildebeest and zebra made relatively greater contributions during the wet season. Impala, waterbuck, warthog and rarer antelope species became more prominent in kills during transitional periods between seasons. Five prey species showed an elevation in representation of males in lion kills during the mating season, as well as impala for all predator species. Females were more prominently represented in kills during the time of late gestation and parturition for three prey species. Hence reproductive activities as well as changing vegetation cover and food resources affected vulnerability to predation. Shifts in susceptibility to predation over the seasonal cycle corresponded with rainfall‐related variation in the annual representation of these ungulate species in lion kills. The availability of vulnerable prey species, age and sex classes at different stages of the seasonal cycle helps maintain a high abundance of lions. These factors contribute to the strong additive impact that predation has had on the abundance of some of these ungulate populations.     

Enhanced understanding of predator–prey relationships using molecular methods to identify predator species,individual and sex

Predator species identification is an important step in understanding predator‐prey interactions, but predator identifications using kill site observations are often unreliable. We used molecular tools to analyse predator saliva, scat and hair from caribou calf kills in Newfoundland, Canada to identify the predator species, individual and sex. We sampled DNA from 32 carcasses using cotton swabs to collect predator saliva. We used fragment length analysis and sequencing of mitochondrial DNA to distinguish between coyote, black bear, Canada lynx and red fox and used nuclear DNA microsatellite analysis to identify individuals. We compared predator species detected using molecular tools to those assigned via field observations at each kill. We identified a predator species at 94% of carcasses using molecular methods, while observational methods assigned a predator species to 62.5% of kills. Molecular methods attributed 66.7% of kills to coyote and 33.3% to black bear, while observations assigned 40%, 45%, 10% and 5% to coyote, bear, lynx and fox, respectively. Individual identification was successful at 70% of kills where a predator species was identified. Only one individual was identified at each kill, but some individuals were found at multiple kills. Predator sex was predominantly male. We demonstrate the first large‐scale evaluation of predator species, individual and sex identification using molecular techniques to extract DNA from swabs of wild prey carcasses. Our results indicate that kill site swabs (i) can be highly successful in identifying the predator species and individual responsible; and (ii) serve to inform and complement traditional methods.     

Amplification of DNA markers from scat samples of the least weasel<Emphasis Type="Italic">Mustela nivalis nivalis</Emphasis>

To test the feasibility of using field-collected scats as a source of DNA in the study of the least weaselMustela nivalis nivalis Linnaeus, 1766, DNA was extracted from scat samples collected from captive weasels using a modified extraction protocol. Using universal primers, the control region of the mitochondrial genome was successfully amplified from scat-extracted DNA. This amplification resulted in two products; one equivalent in size and sequence to the product obtained from tissue-extracted weasel DNA, and the other slightly larger and equivalent in size and sequence to the domestic house mouseMus musculus, the food source of the captive weasels. This demonstrates the reliability of DNA extraction from scats, as well as the possibility, under favourable circumstances, of identifying the prey species from the same samples. In addition, we attempted to amplify microsatellite loci from both tissue and scat-extracted DNA using six primer pairs designed for other mustelids, the American minkMustela vison and the wolverineGulo gulo. While three loci, Mvi57 (American mink), Ggu216 and Ggu234 (wolverine), were found to be polymorphic in the least weasel, amplification of these loci from the scat extracted DNA was only successful for approximately half of the samples. Although further work is needed, the present results suggest that it is possible to use scats as a source of DNA in field studies of the least weasel.     

Migratory herds of wildebeests and zebras indirectly affect calf survival of giraffes

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Introduction of giraffe changes acacia distribution in a South African savanna

Large mammal herbivores can have significant effects on the structure and composition of plant communities. We studied the impacts of an introduced giraffe population on Acacia species at Ithala Game Reserve in South Africa. Browse intensity and Acacia mortality were assessed in field transects, and in road transects covering the reserve road network. Several Acacia species occurring in high‐density giraffe areas had high levels of mortality. Populations of Acacia davyi were extinct in areas accessible to giraffe. Most A. caffra trees within giraffe browse height were dead and A. karroo, the most common species, was also heavily affected. Some species, including A. tortilis, showed no or very low mortality attributable to giraffe browsing. Healthy populations of sensitive species occurred in areas within, and adjacent to, the reserve in areas with low or no giraffe browsing. Areas too steep for giraffe access formed spatial refuges for these trees. The differential mortality that is occurring as a consequence of giraffe browsing is altering species composition and species distribution in this savanna landscape.     

Preservation of corals in salt-saturated DMSO buffer is superior to ethanol for PCR experiments

Specimen collection is time consuming and expensive, yet few laboratories test preservation methods before setting out on field expeditions. The most common preservation buffer used for coral specimens is >70% EtOH. However, alternatives exist that are less flammable, easier to ship, and are widely used in other taxa. Here, we compare the effects of salt-saturated DMSO (SSD) and EtOH preservation buffers on post-extraction DNA quantity and quality. We found that soft tissue integrity was better maintained and higher quantities of DNA were extracted from EtOH-preserved specimens; however, by all other measures, SSD was a superior preservative to EtOH. Extractions of SSD-preserved specimens resulted in higher molecular weight DNA, higher PCR success, and more efficient amplification than specimens preserved in EtOH. Our results show that SSD is generally a superior preservative to EtOH for specimens destined for PCR studies, but species-specific differences indicate that preservation comparisons should be undertaken before collection and storage of samples.     

Antipredator behaviour of African ungulates around human settlements

As human populations grow and come into more frequent contact with wildlife, it is important to understand how anthropogenic disturbance alters wildlife behaviour. Using fine‐scale spatial analyses, we examined how proximity to human settlements affects antipredator responses of ungulates. We studied seven common ungulate species (Kirk's dik‐dik, Thomson's gazelle, impala, common warthog, common wildebeest, common zebra and Masai giraffe) in the Tarangire–Manyara ecosystem in northern Tanzania. In zebra and giraffe, flight responses to humans were significantly more likely when closer to settlements; however, there was a weak relationship between flight responses and distance to settlement in all other species. While there was largely a weak relationship between proximity to human settlements, the distribution of settlements in the landscape appears to affect wildlife behaviour, suggesting that animals perceive and respond to spatial variation in risk exerted by humans.