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.     

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

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

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.     

Estimating population size using single‐nucleotide polymorphism‐based pedigree data

Reliable population estimates are an important aspect of sustainable wildlife management and conservation but can be difficult to obtain for rare and elusive species. Here, we test a new census method based on pedigree reconstruction recently developed by Creel and Rosenblatt (2013). Using a panel of 96 single‐nucleotide polymorphisms (SNPs), we genotyped fecal samples from two Swedish brown bear populations for pedigree reconstruction. Based on 433 genotypes from central Sweden (CS) and 265 from northern Sweden (NS), the population estimates (N = 630 for CS, N = 408 for NS) fell within the 95% CI of the official estimates. The precision and accuracy improved with increasing sampling intensity. Like genetic capture–mark–recapture methods, this method can be applied to data from a single sampling session. Pedigree reconstruction combined with noninvasive genetic sampling may thus augment population estimates, particularly for rare and elusive species for which sampling may be challenging.     

Estimating population size by spatially explicit capture–recapture

The number of animals in a population is conventionally estimated by capture–recapture without modelling the spatial relationships between animals and detectors. Problems arise with non‐spatial estimators when individuals differ in their exposure to traps or the target population is poorly defined. Spatially explicit capture–recapture (SECR) methods devised recently to estimate population density largely avoid these problems. Some applications require estimates of population size rather than density, and population size in a defined area may be obtained as a derived parameter from SECR models. While this use of SECR has potential benefits over conventional capture–recapture, including reduced bias, it is unfamiliar to field biologists and no study has examined the precision and robustness of the estimates. We used simulation to compare the performance of SECR and conventional estimators of population size with respect to bias and confidence interval coverage for several spatial scenarios. Three possible estimators for the sampling variance of realised population size all performed well. The precision of SECR estimates was nearly the same as that of the null‐model conventional population estimator. SECR estimates of population size were nearly unbiased (relative bias 0–10%) in all scenarios, including surveys in randomly generated patchy landscapes. Confidence interval coverage was near the nominal level. We used SECR to estimate the population of a species of skink Oligosoma infrapunctatum from pitfall trapping. The estimated number in the area bounded by the outermost traps differed little between a homogeneous density model and models with a quadratic trend in density or a habitat effect on density, despite evidence that the latter models fitted better. Extrapolation of trend models to a larger plot may be misleading. To avoid extrapolation, a large region of interest should be sampled throughout, either with one continuous trapping grid or with clusters of traps dispersed widely according to a probability‐based and spatially representative sampling design.     

Noninvasive Hair Sampling and Genetic Tagging of Co-Distributed Fishers and American Martens

ABSTRACT 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.     

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

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

Pest fencing or pest trapping: A bio‐economic analysis of cost‐effectiveness

Scofield et al. discredited the utility of pest‐exclusion fences for restoring biodiversity partly on the grounds of unquantified costs and benefits. We estimated the discounted costs of mammal exclusion fences, semi‐permeable (‘leaky’) fences and trapping, over 50 years and adjusted costs by their observed effectiveness at reducing mammalian predator abundance. We modelled data from two large predator management programmes operated by the New Zealand Department of Conservation. Using typical baseline costs and predator control efficacies (scale 0 to 1), the model predicted that an exclusion fence (efficacy 1.0) is the cheapest and most cost‐effective option for areas below about 1 ha, a leaky fence (efficacy 0.9) is most cost‐effective for 1–219 ha, and trapping (efficacy 0.6, based on 0.2 traps per hectare and a 1500‐m buffer to reduce predator reinvasion) for areas above 219 ha. This ranking was insensitive to adjustments in efficacy, but reducing efficacy of leaky fences to 0.8 or increasing trapping efficacy to 0.7 reduced the cost‐effective range of leaky fences by about 90 ha. Reducing trap maintenance costs from $300 to $100 per trap per year (e.g. using long‐life lures), or reducing trap buffer widths to 500 m, significantly elevated trapping as the most cost‐effective method for areas greater than 11–15 ha. These results were largely consistent with an ecological measure of effectiveness based on observed rates of recovery of two indigenous skink species inside exclusion fences or with trapping. The results support criticisms that exclusion fences are generally not cost‐effective, but highlight the value of considering cheaper leaky designs for small‐ to medium‐sized areas. Because this study is based largely on reductions in predator abundance, it has general application to broader biodiversity protection interests, but not to indigenous species that are highly sensitive to predation and only ever adequately protected on the mainland by exclusion fences.     

Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization

DNA extraction from environmental samples (environmental DNA; eDNA) for metabarcoding‐based biodiversity studies is gaining popularity as a noninvasive, time‐efficient, and cost‐effective monitoring tool. The potential benefits are promising for marine conservation, as the marine biome is frequently under‐surveyed due to its inaccessibility and the consequent high costs involved. With increasing numbers of eDNA‐related publications have come a wide array of capture and extraction methods. Without visual species confirmation, inconsistent use of laboratory protocols hinders comparability between studies because the efficiency of target DNA isolation may vary. We determined an optimal protocol (capture and extraction) for marine eDNA research based on total DNA yield measurements by comparing commonly employed methods of seawater filtering and DNA isolation. We compared metabarcoding results of both targeted (small taxonomic group with species‐level assignment) and universal (broad taxonomic group with genus/family‐level assignment) approaches obtained from replicates treated with the optimal and a low‐performance capture and extraction protocol to determine the impact of protocol choice and DNA yield on biodiversity detection. Filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit outperformed other combinations of capture and extraction methods, showing a ninefold improvement in DNA yield over the poorest performing methods. Use of optimized protocols resulted in a significant increase in OTU and species richness for targeted metabarcoding assays. However, changing protocols made little difference to the OTU and taxon richness obtained using universal metabarcoding assays. Our results demonstrate an increased risk of false‐negative species detection for targeted eDNA approaches when protocols with poor DNA isolation efficacy are employed. Appropriate optimization is therefore essential for eDNA monitoring to remain a powerful, efficient, and relatively cheap method for biodiversity assessments. For seawater, we advocate filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit or phenol‐chloroform‐isoamyl for successful implementation of eDNA multi‐marker metabarcoding surveys.     

Common species affects the utility of non‐invasive genetic monitoring of a cryptic endangered mammal: The bridled nailtail wallaby

Non‐invasive methods of monitoring wild populations (such as genotyping faeces or hair) are now widely used and advocated. The potential advantages of such methods over traditional direct monitoring (such as live capture) are that accuracy improves because sampling of non‐trappable individuals may be possible, species in difficult and remote terrain can be surveyed more efficiently, and disturbance to animals is minimal. Few studies have assessed the effects of interactions between species on remote sampling success. We test the use of non‐invasive monitoring for the cryptic, forest‐dwelling, solitary and endangered bridled nailtail wallaby (Onychogalea fraenata) that is sympatric with the ecologically similar and more common black‐striped wallaby (Macropus dorsalis). Six types of hair traps were tested for 3668 trap days, and hairs were caught with about a 10% success rate. Camera traps showed that baited hair traps targeted both wallaby species. We microscopically identified hair as bridled nailtail wallaby or black‐striped wallaby. We compared these hairs and their genotypes (using seven microsatellite loci) with known bridled nailtail wallaby hairs and genotypes derived from animal trapping. Trapped bridled nailtail wallaby hairs had characteristics that could be mistaken for black‐stripe wallaby hairs; characteristics were not diagnostic. Genetic assignment tests consistently differentiated the known bridled nailtail wallaby samples from identified black‐striped wallaby samples, however genetic overlap between most of the microsatellite markers means that they are not suitable for species identification of single samples, with the possible exception of the microsatellite locus B151. With similar trapping effort and within the same area, live‐capture mark‐recapture techniques estimated 40–60 individuals and non‐invasive methods only detected 14 genotypes. A species‐specific genetic marker would allow more efficient targeting of bridled nailtail wallaby samples and increase capture rates.     

Evaluation of three field monitoring-density estimation protocols and their relevance to Komodo dragon conservation

Finding practical ways to robustly estimate abundance or density trends in threatened species is a key facet for effective conservation management. Further identifying less expensive monitoring methods that provide adequate data for robust population density estimates can facilitate increased investment into other conservation initiatives needed for species recovery. Here we evaluated and compared inference-and cost-effectiveness criteria for three field monitoring-density estimation protocols to improve conservation activities for the threatened Komodo dragon (Varanus komodoensis). We undertook line-transect counts, cage trapping and camera monitoring surveys for Komodo dragons at 11 sites within protected areas in Eastern Indonesia to collect data to estimate density using distance sampling methods or the Royle–Nichols abundance induced heterogeneity model. Distance sampling estimates were considered poor due to large confidence intervals, a high coefficient of variation and that false absences were obtained in 45 % of sites where other monitoring methods detected lizards present. The Royle–Nichols model using presence/absence data obtained from cage trapping and camera monitoring produced highly correlated density estimates, obtained similar measures of precision and recorded no false absences in data collation. However because costs associated with camera monitoring were considerably less than cage trapping methods, albeit marginally more expensive than distance sampling, better inference from this method is advocated for ongoing population monitoring of Komodo dragons. Further the cost-savings achieved by adopting this field monitoring method could facilitate increased expenditure on alternative management strategies that could help address current declines in two Komodo dragon populations.     

Evaluating next‐generation sequencing (NGS) methods for routine monitoring of wild bees: Metabarcoding,mitogenomics or NGS barcoding

Implementing cost‐effective monitoring programs for wild bees remains challenging due to the high costs of sampling and specimen identification. To reduce costs, next‐generation sequencing (NGS)‐based methods have lately been suggested as alternatives to morphology‐based identifications. To provide a comprehensive presentation of the advantages and weaknesses of different NGS‐based identification methods, we assessed three of the most promising ones, namely metabarcoding, mitogenomics and NGS barcoding. Using a regular monitoring data set (723 specimens identified using morphology), we found that NGS barcoding performed best for both species presence/absence and abundance data, producing only few false positives (3.4%) and no false negatives. In contrast, the proportion of false positives and false negatives was higher using metabarcoding and mitogenomics. Although strong correlations were found between biomass and read numbers, abundance estimates significantly skewed the communities' composition in these two techniques. NGS barcoding recovered the same ecological patterns as morphology. Ecological conclusions based on metabarcoding and mitogenomics were similar to those based on morphology when using presence/absence data, but different when using abundance data. In terms of workload and cost, we show that metabarcoding and NGS barcoding can compete with morphology, but not mitogenomics which was consistently more expensive. Based on these results, we advocate that NGS barcoding is currently the seemliest NGS method for monitoring of wild bees. Furthermore, this method has the advantage of potentially linking DNA sequences with preserved voucher specimens, which enable morphological re‐examination and will thus produce verifiable records which can be fed into faunistic databases.     

Trapping initiates stress response in breeding and non-breeding house sparrows Passer domesticus: implications for using unmonitored traps in field studies

In response to a variety of unpredictable conditions, birds secrete the steroid hormone corticosterone, which has numerous effects on physiology and behavior. A standardized protocol of handling and restraint has been demonstrated to elicit a robust corticosterone response in many species of birds. In contrast, comparatively little is known about the effects of capture technique on corticosterone secretion in wild birds. Setting up multiple live traps checked at regular intervals allows field researchers to capture many birds in a short period of time. However, one potential drawback of this technique is that birds may spend unknown lengths of time in traps prior to sampling. Many birds appear to remain calm and/or feed during this period, potentially leading researchers to assume that corticosterone secretion is unaffected by trapping. We assessed the impact of being left in traps for up to 30 minutes on baseline corticosterone and subsequent corticosterone responses to restraint in non‐breeding and breeding house sparrows Passer domesticus. Traps were baited with seed, and birds were either removed immediately after entry (controls), or left in the trap undisturbed for 15 or 30 min. Upon removal, birds were subjected to a standardized handling/restraint protocol in which blood samples were collected within 3 min, and again at regular intervals for 60 min. Analysis of blood samples revealed that both non‐breeding and breeding sparrows that were held in the traps had significantly higher baseline corticosterone than controls, and showed no further increase in corticosterone secretion in response to handling. However, corticosterone responses to trapping differed seasonally. Our study indicates that although birds did not exhibit prolonged escape behavior while trapped, entry into a walk‐in trap initiated a robust stress response. Taken together with data from a previous study, our data suggest that ornithologists should consider species‐specific and stage‐specific effects of trapping on physiology when designing field studies.     

Is genomic diversity a useful proxy for census population size? Evidence from a species‐rich community of desert lizards

Species abundance data are critical for testing ecological theory, but obtaining accurate empirical estimates for many taxa is challenging. Proxies for species abundance can help researchers circumvent time and cost constraints that are prohibitive for long‐term sampling. Under simple demographic models, genetic diversity is expected to correlate with census size, such that genome‐wide heterozygosity may provide a surrogate measure of species abundance. We tested whether nucleotide diversity is correlated with long‐term estimates of abundance, occupancy and degree of ecological specialization in a diverse lizard community from arid Australia. Using targeted sequence capture, we obtained estimates of genomic diversity from 30 species of lizards, recovering an average of 5,066 loci covering 3.6 Mb of DNA sequence per individual. We compared measures of individual heterozygosity to a metric of habitat specialization to investigate whether ecological preference exerts a measurable effect on genetic diversity. We find that heterozygosity is significantly correlated with species abundance and occupancy, but not habitat specialization. Demonstrating the power of genomic sampling, the correlation between heterozygosity and abundance/occupancy emerged from considering just one or two individuals per species. However, genetic diversity does no better at predicting abundance than a single day of traditional sampling in this community. We conclude that genetic diversity is a useful proxy for regional‐scale species abundance and occupancy, but a large amount of unexplained variation in heterozygosity suggests additional constraints or a failure of ecological sampling to adequately capture variation in true population size.     

Toward an optimal sampling protocol for Hemiptera on understorey plants

There are no standardised sampling protocols for inventorying Hemiptera from understorey or canopy plants. This paper proposes an optimal protocol for the understorey, after evaluating the efficiency of seven methods to maximise the richness of Hemiptera collected from plants with minimal field and laboratory time. The methods evaluated were beating, chemical knockdown, sweeping, branch clipping, hand collecting, vacuum sampling and sticky trapping. These techniques were tested at two spatial scales: 1 ha sites and individual plants. In addition, because efficiency may differ with vegetation structure, sampling of sites was conducted in three disparate understorey habitats, and sampling of individual plants was conducted across 33 plant species. No single method sampled the majority of hemipteran species in the understorey. Chemical knockdown, vacuum sampling and beating yielded speciose samples (61, 61 and 30 species, respectively, representing 53, 53 and 26% of total species collected). The four remaining methods provided species-poor samples (<18 species or <16% of total species collected). These methods also had biases towards particular taxa (e.g., branch clipping and hand collecting targeted sessile Hemiptera, and sticky trapping were dominated by five species of Psyllidae). The most time-efficient methods were beating, sweeping and hand collecting (200 minutes of field and laboratory time yielded >7 species for each technique). By comparison, vacuum sampling, sticky trapping, branch clipping and chemical knockdown yielded <5 species for the same period. Chemical knockdown had further disadvantages; high financial cost and potential spray drift. The most effective methods for a standardised sampling protocol to inventory Hemiptera from the understorey are beating and vacuum sampling. If used in combination, these methods optimise the catch of understorey hemipteran species, as their samples have high complementarity.     

An evaluation of field and noninvasive genetic methods for estimating Eurasian otter population size

Successful conservation and management of rare and elusive species requires reliable estimates of population size, but acquisition of such data is often challenging. We compare the two most frequently used methods of assessing abundance of Eurasian otter (Lutra lutra) populations, noninvasive genetic sampling (NGS) based on genotyping of faeces and field surveys using snow tracking. In a 100-km² oligotrophic otter habitat with linear water bodies, both methods yielded very similar estimates (10–12 individuals). However, in a 100-km² fishpond area, consisting of a complex network of rivers, fishponds, channels and marshes, genotyping of faeces revealed the presence of a higher number of individuals (46–50 genotypes) than the snow survey (38 individuals). NGS data analysed by capture-mark-recapture (CMR)-based software CAPWIRE provided even higher estimates, being twice the number assessed through snow tracking (76–81 individuals, CI_95% = 49–96 and 55–89). Our results suggest that the performance of both NGS and snow tracking is comparable in simple linear habitats, but in complex habitats with very high otter density a combination of genetic and field methods, or CMR analysis using genetic data, is recommended. We emphasise that to obtain reliable estimates using NGS it is necessary to follow strict protocols for detection and elimination of genotyping errors. Based on a literature review and our experience, we suggest improvements that may increase the success rate and efficiency of NGS for otters.     

Sequence capture and next‐generation sequencing of ultraconserved elements in a large‐genome salamander

Amidst the rapid advancement in next‐generation sequencing (NGS) technology over the last few years, salamanders have been left behind. Salamanders have enormous genomes—up to 40 times the size of the human genome—and this poses challenges to generating NGS data sets of quality and quantity similar to those of other vertebrates. However, optimization of laboratory protocols is time‐consuming and often cost prohibitive, and continued omission of salamanders from novel phylogeographic research is detrimental to species facing decline. Here, we use a salamander endemic to the southeastern United States, Plethodon serratus, to test the utility of an established protocol for sequence capture of ultraconserved elements (UCEs) in resolving intraspecific phylogeographic relationships and delimiting cryptic species. Without modifying the standard laboratory protocol, we generated a data set consisting of over 600 million reads for 85 P. serratus samples. Species delimitation analyses support recognition of seven species within P. serratus sensu lato, and all phylogenetic relationships among the seven species are fully resolved under a coalescent model. Results also corroborate previous data suggesting nonmonophyly of the Ouachita and Louisiana regions. Our results demonstrate that established UCE protocols can successfully be used in phylogeographic studies of salamander species, providing a powerful tool for future research on evolutionary history of amphibians and other organisms with large genomes.     

A method for estimating population sex ratio for sage‐grouse using noninvasive genetic samples

Population sex ratio is an important metric for wildlife management and conservation, but estimates can be difficult to obtain, particularly for sexually monomorphic species or for species that differ in detection probability between the sexes. Noninvasive genetic sampling (NGS) using polymerase chain reaction (PCR) has become a common method for identifying sex from sources such as hair, feathers or faeces, and is a potential source for estimating sex ratio. If, however, PCR success is sex‐biased, naively using NGS could lead to a biased sex ratio estimator. We measured PCR success rates and error rates for amplifying the W and Z chromosomes from greater sage‐grouse (Centrocercus urophasianus) faecal samples, examined how success and error rates for sex identification changed in response to faecal sample exposure time, and used simulation models to evaluate precision and bias of three sex assignment criteria for estimating population sex ratio with variable sample sizes and levels of PCR replication. We found PCR success rates were higher for females than males and that choice of sex assignment criteria influenced the bias and precision of corresponding sex ratio estimates. Our simulations demonstrate the importance of considering the interplay between the sex bias of PCR success, number of genotyping replicates, sample size, true population sex ratio and accuracy of assignment rules for designing future studies. Our results suggest that using faecal DNA for estimating the sex ratio of sage‐grouse populations has great potential and, with minor adaptations and similar marker evaluations, should be applicable to numerous species.     

Cost‐efficiency of Subsampling Protocols to Evaluate Oribatid‐Mite Communities in an Amazonian Savanna

Sampling oribatid mites in large areas using conventional methods is expensive, time‐consuming, and this constrains their use in environmental monitoring programs. We used samples collected in 38 plots of 3.75 ha spread over 30,000 ha in an Amazonian savanna to evaluate the reduction in costs and person‐hours in sampling and sorting and to elaborate cost‐effective protocols. Ten samples per plot were collected and extracted using a Berlese‐Tullgren apparatus. In the laboratory, samples were reduced to 50, 25, 12.5, and 6.25 percent of the initial content. Field‐effort reduction was estimated by reducing the number of subsamples per plot. Dissimilarity matrices were generated using Bray–Curtis, Sørensen, and Chao–Sørensen indices. Correlations between each reduced‐effort dissimilarity matrix and 100 or 50 percent sorting were used as an index of how much information was retained in reduced‐effort sampling, and could still be used in multivariate analyses. The effects of most predictor variables on mite composition were detected in data based on every level of sample reduction. The intensive sampling was insufficient to reveal the full oribatid‐mite fauna in the savanna; as more plots were sampled, more species were recorded. Our data indicate subsampling protocols for biodiversity assessment of oribatid mites in savanna that increase field and laboratory efficiency, and optimize both taxonomic and ecological aspects of the investigation.     

Spatial density estimates of Eurasian lynx (Lynx lynx) in the French Jura and Vosges Mountains

Obtaining estimates of animal population density is a key step in providing sound conservation and management strategies for wildlife. For many large carnivores however, estimating density is difficult because these species are elusive and wide‐ranging. Here, we focus on providing the first density estimates of the Eurasian lynx (Lynx lynx) in the French Jura and Vosges mountains. We sampled a total of 413 camera trapping sites (with two cameras per site) between January 2011 and April 2016 in seven study areas across seven counties of the French Jura and Vosges mountains. We obtained 592 lynx detections over 19,035 trap days in the Jura mountains and 0 detection over 6,804 trap days in the Vosges mountains. Based on coat patterns, we identified a total number of 92 unique individuals from photographs, including 16 females, 13 males, and 63 individuals of unknown sex. Using spatial capture–recapture (SCR) models, we estimated abundance in the study areas between 5 (SE = 0.1) and 29 (0.2) lynx and density between 0.24 (SE = 0.02) and 0.91 (SE = 0.03) lynx per 100 km². We also provide a comparison with nonspatial density estimates and discuss the observed discrepancies. Our study is yet another example of the advantage of combining SCR methods and noninvasive sampling techniques to estimate density for elusive and wide‐ranging species, like large carnivores. While the estimated densities in the French Jura mountains are comparable to other lynx populations in Europe, the fact that we detected no lynx in the Vosges mountains is alarming. Connectivity should be encouraged between the French Jura mountains, the Vosges mountains, and the Palatinate Forest in Germany where a reintroduction program is currently ongoing. Our density estimates will help in setting a baseline conservation status for the lynx population in France.