River otter population size estimation using noninvasive latrine surveys

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

Lessons from the use of non-invasive genetic sampling as a way to estimate Eurasian otter population size and sex ratio

Given the difficulties in establishing population parameters of elusive animals in the wild by traditional methods, such as trapping, much attention has been given in recent years to non-invasive genetic sampling. Our work compared estimates of population size and sex ratio derived from genetic sampling with the known number and sex of animals released during an otter reintroduction and reports on the pitfalls and opportunities that may be encountered in studies of this kind. This study makes use of 121 samples of otter spraints (faeces) collected over 7 months during a reintroduction in the Upper Thames (UK) where a total of 17 otters was released in two consecutive phases. Spraints were processed with a multiple tubes approach and seven microsatellites were used. Of all collected samples, 19 % were complete for at least five loci, the minimum required for discrimination between individuals. Six out of nine of the otters that were released in the first phase were detected, four males and two females, while none of the otters released in the second phase was detected probably due to a combination of sampling pitfalls and otter behaviour. In particular, the specific sex (mostly females) and dominance composition (lower) of the individuals in the second release group may explain our failure to detect individuals in this group. Taken together, our results add further evidence that genetic sampling approaches represent a potentially accurate and non-invasive route to census populations of otters but that the sampling design should take into account factors like the sex ratio and dominance composition of the population in order to maximise detection and minimise error.     

A comparison of methods for estimating plant population size

Assessing local population size is one of the most common tasks in biodiversity monitoring. Population size estimates are not only important for conservation management and population threat assessment; they also enter many other analyses in landscape ecology and conservation. It is therefore concerning that methods for estimating plant population sizes are not standardized. We surveyed the literature and found that the most commonly used methods are counting either all or only flowering individuals on a site, as well as counting individuals in random plots or plots on transects. Sometimes, these methods are combined in the same study, without assurance that they produce comparable results. We therefore conducted a field study, in which we obtained population size estimates from all four methods for six different calcareous grassland species at 18 study sites. Our results demonstrate not only substantial differences between overall count rates generated by the different methods, but methods that surveyed the whole population also systematically yielded lower counts when species were less visible and when the area was larger, suggesting that these methods suffer from biases that could distort species and site comparisons. We conclude that estimates from different methods should not be mixed, and that plot or transect based surveys have likely smaller biases for large areas or poorly visible individuals, and are therefore preferable.     

Evaluating methods for estimating local effective population size with and without migration

Effective population size is a fundamental parameter in population genetics, evolutionary biology, and conservation biology, yet its estimation can be fraught with difficulties. Several methods to estimate N_e from genetic data have been developed that take advantage of various approaches for inferring N_e. The ability of these methods to accurately estimate N_e, however, has not been comprehensively examined. In this study, we employ seven of the most cited methods for estimating N_e from genetic data (Colony2, CoNe, Estim, MLNe, ONeSAMP, TMVP, and NeEstimator including LDNe) across simulated datasets with populations experiencing migration or no migration. The simulated population demographies are an isolated population with no immigration, an island model metapopulation with a sink population receiving immigrants, and an isolation by distance stepping stone model of populations. We find considerable variance in performance of these methods, both within and across demographic scenarios, with some methods performing very poorly. The most accurate estimates of N_e can be obtained by using LDNe, MLNe, or TMVP; however each of these approaches is outperformed by another in a differing demographic scenario. Knowledge of the approximate demography of population as well as the availability of temporal data largely improves N_e estimates.     

Similar estimates of population genetic composition and sex ratio derived from carcasses and faeces of Eurasian otter Lutra lutra

Collecting faeces is viewed as a potentially efficient way to sample elusive animals. Nonetheless, any biases in estimates of population composition associated with such sampling remain uncharacterized. The goal of this study was to compare estimates of genetic composition and sex ratio derived from Eurasian otter Lutra lutra spraints (faeces) with estimates derived from carcasses. Twenty per cent of 426 wild-collected spraints from SW England yielded composite genotypes for 7-9 microsatellites and the SRY gene. The expected number of incorrect spraint genotypes was negligible, given the proportions of allele dropout and false allele detection estimated using paired blood and spraint samples of three captive otters. Fifty-two different spraint genotypes were detected and compared with genotypes of 70 otter carcasses from the same area. Carcass and spraint genotypes did not differ significantly in mean number of alleles, mean unbiased heterozygosity or sex ratio, although statistical power to detect all but large differences in sex ratio was low. The genetic compositions of carcass and spraint genotypes were very similar according to confidence intervals of theta and two methods for assigning composite genotypes to groups. A distinct group of approximately 11 carcass and spraint genotypes was detected using the latter methods. The results suggest that spraints can yield unbiased estimates of population genetic composition and sex ratio.     

A genetic analogue of 'mark-recapture' methods for estimating population size: an approach based on molecular parentage assessments

Molecular polymorphisms have been used in a variety of ways to estimate both effective and local census population sizes in nature. A related approach for estimating the current size of a breeding population, explored here for the first time, is the use of genetic 'marks' reconstructed for otherwise unknown parents in paternity or maternity analyses of progeny arrays. This method provides interesting similarities and contrasts to traditional mark-recapture methods based on physical tags. To illustrate, this genetic method is applied to a population of painted turtles on the Mississippi River to estimate the number of successfully breeding males. Non-genetic mark-recapture approaches were also applied to animals trapped at this location. Results demonstrate that such genetic data on parentage can be helpful not only in estimating contemporary population sizes, but also in providing additional information, not present in customary mark-recapture data, about possible extended movements of breeding individuals and the size of the pool of mates which they encounter.     

Empirical Bayes procedure for estimating genetic distance between populations and effective population size

We developed an empirical Bayes procedure to estimate genetic distances between populations using allele frequencies. This procedure makes it possible to describe the skewness of the genetic distance while taking full account of the uncertainty of the sample allele frequencies. Dirichlet priors of the allele frequencies are specified, and the posterior distributions of the various composite parameters are obtained by Monte Carlo simulation. To avoid overdependence on subjective priors, we adopt a hierarchical model and estimate hyperparameters by maximizing the joint marginal-likelihood function. Taking advantage of the empirical Bayesian procedure, we extend the method to estimate the effective population size using temporal changes in allele frequencies. The method is applied to data sets on red sea bream, herring, northern pike, and ayu broodstock. It is shown that overdispersion overestimates the genetic distance and underestimates the effective population size, if it is not taken into account during the analysis. The joint marginal-likelihood function also estimates the rate of gene flow into island populations.     

Genetic variability and size estimates of the Eurasian otter (Lutra lutra) population in the Bohemian Forest Ecosystem

Even though recent years have shown a slow recovery of the Eurasian otter (Lutra lutra) populations from their previous lows, the species is still highly endangered in most parts of its European distribution range. Surprisingly, only a few studies have so far assessed the species’ genetic variability and population density, and they have mostly been carried out only in small territories. In Germany, most otter populations live in protected areas whose management urgently needs data on population sizes and densities as well as on genetic variability of the species under their custody. Thus, we analyzed genetic variability and assessed size and density of the otter population in the Bohemian Forest Ecosystem, an area that had not been included in the few previous molecular studies. The study area comprised of 1500 km², divided into fifteen squares of 10 × 10 km², each of which was sampled in two collection periods. Overall we collected 261 fecal samples (spraints), of which 60 (23%) could be genotyped at least at eight microsatellite loci, yielding 38 distinct otter genotypes. The low genotyping success rate was the result of high ambient temperature at the time of sampling rather than that of high humidity. The population did not show signs of a past bottleneck, indicating a small yet stable population size. Population size was estimated to be 118 (CI_95% 64–163) individuals, with a mean density of 1 animal per 8.5 km² or 3.1 km river length. Our results imply that hunting, requested by local fishpond owners, should remain banned to avoid a decline in (effective) population size.     

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.     

The reintroduction of the Eurasian otter (Lutra lutra) into the Netherlands: hidden life revealed by noninvasive genetic monitoring

The last recorded presence of the Eurasian otter (Lutra lutra) in the Netherlands dates from 1989 and concerned a dead individual. In 2002 a reintroduction programme was started, and between June 2002 and April 2008 a total of 30 individuals (10 males and 20 females) were released into a lowland peat marsh in the north of the Netherlands. Noninvasive genetic monitoring based on the genetic profiles obtained from DNA extracted from otter faeces (spraints) was chosen for the post-release monitoring of the population. To this end, the founding individuals were genotyped before release and spraints were collected in the release area each winter from 2002 to 2008. From June 2002 to April 2008 we analysed the genetic profile of 1,265 spraints on the basis of 7–15 microsatellite loci, 582 of which (46%) were successfully assigned to either released or newly identified genotypes. We identified 54 offspring (23 females and 31 males): the females started to reproduce after 2 years and the males after 4 years. The mating and reproductive success among males was strongly skewed, with a few dominant males fathering two-thirds of the offspring, but the females had a more even distribution. The effective population size (Ne) was only about 30% of the observed density (N), mainly because of the large variance in reproductive success among males. Most juvenile males dispersed to surrounding areas on maturity, whereas juvenile females stayed inside the area next to the mother’s territory. The main cause of mortality was traffic accidents. Males had a higher mortality rate (22 out of 41 males (54%) vs. 9 out of 43 females (21%)). During winter 2007/08 we identified 47 individuals, 41 of which originated from mating within the release area. This study demonstrates that noninvasive molecular methods can be used efficiently in post-release monitoring studies of elusive species to reveal a comprehensive picture of the state of the population.     

Noninvasive genetic analyses for estimating population size and genetic diversity of the remaining Far Eastern leopard (Panthera pardus orientalis) population

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

An evaluation of the sampling variation when estimating the population of Verticillium dahliae in field soil

This paper describes a study designed to find a reliable sampling procedure for estimating the population of Verticillium duhliue in field plots that would not be too demanding in terms of the time required to assay soil samples. In the soil under study, the amount of time needed to obtain reliable estimates of the inoculum density was reduced by taking the larger plot sizes in the range considered, generally taking more plots with fewer samples per plot, and mixing the samples within a plot before redividing for assaying.     

完达山东部欧亚水獭种群数量及出现频次影响因素

水獭作为淡水生态系统健康的指示种和旗舰物种,在维持水生生态系统平衡与稳定中发挥着重要作用。然而目前对于完达山东部地区水獭种群数量、分布及其生境选择的影响因素的研究较为匮乏,严重影响了对该物种的野外保护与管理工作。于2021年12月至2022年4月冬季河流封冻期,采用沿河随机样线调查和红外相机监测相结合的方法对完达山东部地区8条主河和23条支流内的欧亚水獭(Lutra lutra)种群数量、分布现状进行了调查,并利用广义可加模型探究水獭出现频次与环境因子的关系。研究结果表明:(1)欧亚水獭在完达山东部水獭种群数量为571-661只,水獭种群密度为沿河(0.5559±0.2898)只/km,呈现中间高,四周低的趋势;(2)环境因子对水獭出现频次影响分析表明,河流深度、距农田距离、距居民区距离和距道路距离是影响水獭出现频次的关键因素。水獭出现频次与河流深度(0-40 cm)呈线性正相关,当河流深度达到40-50 cm时,水獭出现频次最高,之后随着河流深度的增加,出现频次降低;水獭出现频次与距农田距离(0-1.5 km)呈非线性正相关,在距农田距离为1.5-2 km范围内,水獭出现频次最高,之后降低;水獭出现频次与距居民区距离呈线性正相关;水獭出现频次与距道路距离呈非线性关系,当距离>7 km时,二者之间呈现为正相关,反之呈现为负相关。因此,水獭选择栖息地偏向于河流深(40-50 cm),远离农田(1.5-2 km)、居民区和道路(>7 km)的水域。研究为完达山东部欧亚水獭物种保护提供了基础数据和理论依据,建议通过河岸土地覆盖类型的管理、在河岸周围建立森林缓冲区及加强水獭保护宣传力度等措施实现对水獭物种及其栖息地的维持和保护。     

An evaluation of the methods to estimate effective population size from measures of linkage disequilibrium

In 1971, John Sved derived an approximate relationship between linkage disequilibrium (LD) and effective population size for an ideal finite population. This seminal work was extended by Sved and Feldman (Theor Pop Biol 4, 129, 1973) and Weir and Hill (Genetics 95, 477, 1980) who derived additional equations with the same purpose. These equations yield useful estimates of effective population size, as they require a single sample in time. As these estimates of effective population size are now commonly used on a variety of genomic data, from arrays of single nucleotide polymorphisms to whole genome data, some authors have investigated their bias through simulation studies and proposed corrections for different mating systems. However, the cause of the bias remains elusive. Here, we show the problems of using LD as a statistical measure and, analogously, the problems in estimating effective population size from such measure. For that purpose, we compare three commonly used approaches with a transition probability‐based method that we develop here. It provides an exact computation of LD. We show here that the bias in the estimates of LD and effective population size are partly due to low‐frequency markers, tightly linked markers or to a small total number of crossovers per generation. These biases, however, do not decrease when increasing sample size or using unlinked markers. Our results show the issues of such measures of effective population based on LD and suggest which of the method here studied should be used in empirical studies as well as the optimal distance between markers for such estimates.     

Sampling strategies for estimating brook trout effective population size

The influence of sampling strategy on estimates of effective population size (N _e ) from single-sample genetic methods has not been rigorously examined, though these methods are increasingly used. For headwater salmonids, spatially close kin association among age-0 individuals suggests that sampling strategy (number of individuals and location from which they are collected) will influence estimates of N _e through family representation effects. We collected age-0 brook trout by completely sampling three headwater habitat patches, and used microsatellite data and empirically parameterized simulations to test the effects of different combinations of sample size (S = 25, 50, 75, 100, 150, or 200) and number of equally-spaced sample starting locations (SL = 1, 2, 3, 4, or random) on estimates of mean family size and effective number of breeders (N _b ). Both S and SL had a strong influence on estimates of mean family size and [^(N)]_b , \hat{N}_{b} , however the strength of the effects varied among habitat patches that varied in family spatial distributions. The sampling strategy that resulted in an optimal balance between precise estimates of N _b and sampling effort regardless of family structure occurred with S = 75 and SL = 3. This strategy limited bias by ensuring samples contained individuals from a high proportion of available families while providing a large enough sample size for precise estimates. Because this sampling effort performed well for populations that vary in family structure, it should provide a generally applicable approach for genetic monitoring of iteroparous headwater stream fishes that have overlapping generations.     

Hematological and biochemical reference intervals for wild caught Eurasian otter from Spain

Hematologic and serum chemistry reference intervals were determined from 33 wild caught Eurasian otters (Lutra lutra lutra) between November 1995 and May 1998 during a reintroduction project. Blood was obtained by jugular venipuncture after administration of ketamine and medetomidine. The mean, standard deviation, and range for 19 hematology parameters and 28 serum chemistry values are presented. There were no significant differences between sexes in most analytes. The results are in agreement with those reported previously for Eurasian otters with the exception of higher leukocyte and neutrophil counts, lower eosinophil and lymphocyte counts and higher activities for aspartate aminotransferase and creatine kinase. The Eurasian otters have lower erythrocyte counts but higher mean corpuscular volume and mean corpuscular hemoglobin values than the river otter (Lutra canadensis) in North America.