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

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.     

Evaluating DNA degradation rates in faecal pellets of the endangered pygmy rabbit

Noninvasive genetic sampling of faecal pellets can be a valuable method for monitoring rare and cryptic wildlife populations, like the pygmy rabbit (Brachylagus idahoensis). To investigate this method's efficiency for pygmy rabbit monitoring, we evaluated the effect of sample age on DNA degradation in faecal pellets under summer field conditions. We placed 275 samples from known individuals in natural field conditions for 1–60 days and assessed DNA quality by amplifying a 294‐base‐pair (bp) mitochondrial DNA (mtDNA) locus and five nuclear DNA (nDNA) microsatellite loci (111–221 bp). DNA degradation was influenced by sample age, DNA type, locus length and rabbit sex. Both mtDNA and nDNA exhibited high PCR success rates (94.4%) in samples <1 day old. Success rates for microsatellite loci declined rapidly from 80.0% to 42.7% between days 5 and 7, likely due to increased environmental temperature. Success rates for mtDNA amplification remained higher than nDNA over time, with moderate success (66.7%) at 21 days. Allelic dropout rates were relatively high (17.6% at <1 day) and increased to 100% at 60 days. False allele rates ranged from 0 to 30.0% and increased gradually over time. We recommend collecting samples as fresh as possible for individual identification during summer field conditions. Our study suggests that this method can be useful for future monitoring efforts, including occupancy surveys, individual identification, population estimation, parentage analysis and monitoring of genetic diversity both of a re‐introduced population in central Washington and across their range.     

Disappearance rate of chimpanzee scats: Implications for census work on Pan troglodytes

Scat (faeces) decay rate estimates are used to calculate animal species abundance and density. For African great apes, this has been measured only for Gorilla; chimpanzee scats are assumed to decay at a faster rate due to lower fibre content. We provide the first systematic measure of scat decay rate duration for Pan troglodytes schweinfurthii, in Kanyawara, Kibale National Park, Uganda. We used two methods: (1) multiple visits to obtain prospective decay rates (PDR) (N = 96 scats) and (2) a novel approach of time‐lapse photography (TLP) (N = 17 scats). Most of the visited scats (67%) decayed in ≤24 hr, and median decay rate duration from photographic documentation was 18 hr. Using regression analyses, we tested 11 covariables to determine predictors for decay rate duration. Greater volume of scat and reduced levels of diurnal dung beetle activity were positively associated with longer decay rate duration. Given a high prevalence of dung beetle activity (88% of scats), particularly within 3 hr post‐defaecation, we suggest the use of the alternative term, disappearance rate of scats. With a rapid disappearance rate, scat count surveys of unhabituated chimpanzees are challenging; further work is then needed for Pan spp. to determine spatial and temporal differences at intra‐ and inter‐species level.     

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.     

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.     

Endozoochorous dispersal of forest seeds by carnivorous mammals in Sierra Fría,Aguascalientes, Mexico

Some carnivorous mammals ingest fruit and disperse seeds of forest plant species capable of colonizing disturbed areas in ecosystems. The objective of the present study was to evaluate the dissemination of Arctostaphylos pungens and Juniperus deppeana seeds by the gray fox (Urocyon cinereoargenteus), coyote (Canis latrans), and other carnivores in the Protected Natural Area Sierra Fría, in Aguascalientes, Mexico. Scat collection was undertaken via transects using the direct search method, while the seasonal phenology of A. pungens and J. deppeana was evaluated by recording flower and fruit abundance on both the plant and the surrounding forest floor ground. Seed viability was assessed by optical densitometry via X‐ray and a germination test. It was found that the gray fox, coyote, ringtail (Bassariscus astutus), and bobcat (Lynx rufus) disseminated seeds of A. pungens (212 ± 48.9 seeds/scat) and J. deppeana (23.6 ± 4.9 seeds/scat), since a large proportion of the collected scat of these species contained seeds (28/30 = 93.33%, 12/43 = 27.9%, 6/12 = 50% and 7/25 = 28% respectively). The gray fox, coyote, ringtail, and bobcat presented an average of seed dispersion of both plant species of 185.4 ± 228.7, 4.0 ± 20.0, 12.1 ± 30.4, and 0.8 ± 1.5 per scat; the seed proportions in the gray fox, coyote, ringtail, and bobcat were 89.6/10.4%, 82.3/17.7%, 90.4/9.6%, and 38.1/61.9% for A. pungens and J. deppeana, respectively. The phenology indicated a finding related to the greater abundance of ripe fruit in autumn and winter (p < .01). This coincided with the greater abundance of seeds found in scats during these seasons. Endozoochory and diploendozoochory enhanced the viability and germination of the seeds (p > .05), except in those of A. pungens dispersed by coyote. These results suggest that carnivores, particularly the gray fox, the coyote, and the bobcat, play an important role in forest seed dissemination, and thus forest regeneration, by making both a quantitative and qualitative contribution to the dispersal of the two pioneer species under study.     

Investigating a simplified method for noninvasive genetic sampling in East African mammals using silica dried scat swabs

Swabbing scat has proved to be an effective noninvasive method to collect DNA from mammals in the field. Previously, this method has relied on preservative liquids or freezing to preserve the DNA collected on swabs. In this study, we determine the effectiveness of using silica to simply dry the swab in field as an alternative way to prevent DNA degredation. Four species were included in the study; reticulated giraffe, impala, fringe‐eared oryx, and lion. Swabs were taken at multiple time points for giraffe and impala scat samples, with the lion and oryx sampled opportunistically. Mitochondrial DNA was successfully amplified and sequenced from scat swabs from all species; however, effectiveness varied between species, with 81.8% amplification success rate from swabs taken from impala scat compared to 25% amplification success rate in giraffe. This variation in success rate was overcome by taking multiple swabs, thus increasing the probability of a successful amplification. The true merit of this method is in its simplicity and cheapness; no preservative liquids were required to be brought into the field, at no stage in the 2 weeks of field sampling were samples frozen, and no commercial kits were used for DNA extraction.     

Impacts of sampling location within a faeces on DNA quality in two carnivore species

We investigated the influence of sampling location within a faeces on DNA quality by sampling from both the outside and inside of 25 brown bear (Ursus arctos) scats and the side and the tip of 30 grey wolf (Canis lupus) scats. The outside of the bear scat and side of the wolf scat had significantly lower nuclear DNA microsatellite allelic dropout error rates (U. arctos: P = 0.017; C. lupus: P = 0.025) and significantly higher finalized genotyping success rates (U. arctos: P = 0.017; C. lupus: P = 0.012) than the tip and inside of the scat. A review of the faecal DNA literature indicated that <45% of studies report the sampling location within a faeces indicating that this methodological consideration is currently underappreciated. Based on our results, we recommend sampling from the side of canid scats and the outside portion of ursid scats to obtain higher quality DNA samples. The sampling location within a faeces should be carefully considered and reported as it can directly influence laboratory costs and efficiency, as well as the ability to obtain reliable genotypes.     

Long‐term persistence of horse fecal DNA in the environment makes equids particularly good candidates for noninvasive sampling

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.     

Evaluating noninvasive genetic sampling techniques to estimate large carnivore abundance

Monitoring large carnivores is difficult because of intrinsically low densities and can be dangerous if physical capture is required. Noninvasive genetic sampling (NGS) is a safe and cost‐effective alternative to physical capture. We evaluated the utility of two NGS methods (scat detection dogs and hair sampling) to obtain genetic samples for abundance estimation of coyotes, black bears and Canada lynx in three areas of Newfoundland, Canada. We calculated abundance estimates using program capwire , compared sampling costs, and the cost/sample for each method relative to species and study site, and performed simulations to determine the sampling intensity necessary to achieve abundance estimates with coefficients of variation (CV) of <10%. Scat sampling was effective for both coyotes and bears and hair snags effectively sampled bears in two of three study sites. Rub pads were ineffective in sampling coyotes and lynx. The precision of abundance estimates was dependent upon the number of captures/individual. Our simulations suggested that ~3.4 captures/individual will result in a < 10% CV for abundance estimates when populations are small (23–39), but fewer captures/individual may be sufficient for larger populations. We found scat sampling was more cost‐effective for sampling multiple species, but suggest that hair sampling may be less expensive at study sites with limited road access for bears. Given the dependence of sampling scheme on species and study site, the optimal sampling scheme is likely to be study‐specific warranting pilot studies in most circumstances.     

Scat on the doorstep: Refuge choice in a group‐living lizard is influenced by the presence of scat piles

Group living often requires strong levels of communication between individuals. This communication is usually studied in the context of visual or auditory communication. However, chemical communication is the most widely used form of communication. We examined the role of chemical communication in mediating social decisions in a group‐living lizard, Egernia stokesii. Specifically, we examined the extent to which scat‐piling, a behaviour by which individuals deposit scat in a communal area, affected the refuge choice of individual E. stokesii. To achieve this, we examined individual refuge choice in response to scat piles or single scats and against two types of scat stimuli, one being their own scat and the other being scat belonging to an unrelated and unfamiliar conspecific. We show that lizards behave differently when presented with a scat pile compared with a single scat, and whether the scat stimulus was their own or sourced from an unfamiliar conspecific. When scats were in piles, individuals spent more time inspecting, more time in, and more often chose the treatment refuge as their final refuge choice, at a trial’s end, when the treatment was their own scat compared with when the treatment was the refuge with the unfamiliar scat. In contrast, for individual scat treatments, individuals spent more time inspecting and more often ended up in the treatment refuge with an unfamiliar scat compared with when the treatment was their own scat. These results suggest that individuals are responding to information contained within multiple components of the scats – both their volume and their source. These results have implications for understanding how social aggregations are maintained within squamates, where sociality has evolved independently from other vertebrate lineages.     

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.     

Fresh is best: Accurate SNP genotyping from koala scats

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.     

Detecting rare carnivores using scats: Implications for monitoring a fox incursion into Tasmania

The ability to detect the incursion of an invasive species or destroy the last individuals during an eradication program are some of the most difficult aspects of invasive species management. The presence of foxes in Tasmania is a contentious issue with recent structured monitoring efforts, involving collection of carnivore scats and testing for fox DNA, failing to detect any evidence of foxes. Understanding the likelihood that monitoring efforts would detect fox presence, given at least one is present, is therefore critical for understanding the role of scat monitoring for informing the response to an incursion. We undertook trials to estimate the probability of fox scat detection through monitoring by scat‐detector dogs and person searches and used this information to critically evaluate the power of scat monitoring efforts for detecting foxes in the Tasmanian landscape. The probability of detecting a single scat present in a 1‐km² survey unit was highest for scat‐detector dogs searches (0.053) compared with person searches () for each 10 km of search effort. Simulation of the power of recent scat monitoring efforts undertaken in Tasmania from 2011 to 2015 suggested that single foxes would have to be present in at least 20 different locations or fox breeding groups present in at least six different locations, in order to be detected with a high level of confidence (>0.80). We have shown that highly structured detection trials can provide managers with the quantitative tools needed to make judgments about the power of large‐scale scat monitoring programs. Results suggest that a fox population, if present in Tasmania, could remain undetected by a large‐scale, structured scat monitoring program. Therefore, it is likely that other forms of surveillance, in conjunction with scat monitoring, will be necessary to demonstrate that foxes are absent from Tasmania with high confidence.     

Environmental DNA facilitates accurate,inexpensive, and multiyear population estimates of millions of anadromous fish

Although environmental DNA shed from an organism is now widely used for species detection in a wide variety of contexts, mobilizing environmental DNA for management requires estimation of population size and trends in addition to assessing presence or absence. However, the efficacy of environmental‐DNA‐based indices of abundance for long‐term population monitoring have not yet been assessed. Here we report on the relationship between six years of mark‐recapture population estimates for eulachon (Thaleichthys pacificus) and “eDNA rates” which are calculated from the product of stream flow and DNA concentration. Eulachon are a culturally and biologically important anadromous fish that have significantly declined in the southern part of their range but were historically rendered into oil and traded. Both the peak eDNA rate and the area under the curve of the daily eDNA rate were highly predictive of the mark‐recapture population estimate, explaining 84.96% and 92.53% of the deviance, respectively. Even in the absence of flow correction, the peak of the daily eDNA concentration explained an astonishing 89.53% while the area under the curve explained 90.74% of the deviance. These results support the use of eDNA to monitor eulachon population trends and represent a >80% cost savings over mark‐recapture, which could be further increased with automated water sampling, reduced replication, and focused temporal sampling. Due to its logistical ease and affordability, eDNA sampling can facilitate monitoring a larger number of rivers and in remote locations where mark‐recapture is infeasible.     

Evaluating the predictive power of field variables for species and individual molecular identification on wolf noninvasive samples

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.     

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.     

Diet estimation based on an integrated mixed prey feeding experiment using Arctocephalus seals

Food web models depend on identifying which taxa are eaten and in what proportion they are consumed. Arctocephalus seals are generalist foragers and are an ongoing focus of Southern Hemisphere marine ecosystem research. This is the first feeding experiment to use Arctocephalus spp. to assess the utility of hard part scat analysis for diet estimation, based on mixed prey diets integrated over several days. Recovery rates of otoliths were extremely low for all taxa (0-9%). Although we could not collect scats produced during a 90 min period each day, during which the seals had access to a large pool, this result could not be attributed to otolith robustness, pinniped species or class, activity level, meal size or frequency, or fat content of the diet. We conclude that the unusually low recovery rates in this study may be due to unaccounted scats produced during 90 min of each day, if they contained otolith numbers an order of magnitude greater than all otoliths retrieved from scats produced during the other 22.5 h of each day, and/or may be related to the digestive processing of a mixed prey diet. Our study demonstrates the inadequacy of using otoliths in field collected scats for diet estimation due to the high level of unexplained variability of otolith occurrence in scats. We also identify two new potential sources of this variability. These are variability in numbers of otoliths per scat depending on activity level when a scat is excreted, and variability in recovery rates of otoliths as a function of the complexity of the diet.     

Detection distance and environmental factors in conservation detection dog surveys

Surveys using conservation detection dogs have grown increasingly popular as an efficient means to gather monitoring data, particularly for elusive and low-density species such as carnivores. Working with dogs can greatly increase the area surveyed for wildlife and the detection rate of survey targets. Due to the confounding effects of scent dispersion and dog movement, however, it can be difficult to estimate the area searched in a survey. Additionally, although detection dogs have been used in studies under a wide range of air temperature, humidity, and wind conditions, little research has examined how environmental factors affect detection dogs' effectiveness for wildlife surveys. Between 2003 and 2005, we trained 2 dogs to assist us with surveys for mammalian carnivore scats in northern California. We conducted controlled search trials to assess how the dogs' scat detection rates were affected by the distance of scats from the transect search line, as well as variation in six environmental factors. Both dogs detected >75% of scats located within 10 m, and the dogs' detection rates decreased with increasing distance of scats from the transect line. Among environmental factors, precipitation was the most important variable explaining variation in scat detection rates for both dogs. Precipitation likely degrades or removes scats from the landscape over time, and detection rates increase as scat begins to accumulate following the last substantial (>5 mm) rain event of the year. If scat accumulation is not controlled for in ecosystems with a strong seasonal pattern of rainfall, it could lead to considerable bias in study results. We recommend that researchers report the conditions under which conservation detection dog surveys took place and analyze how detection rates vary as a function of distance, temperature, precipitation, humidity, wind, and other locally important environmental factors. © 2010 The Wildlife Society.     

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.