Evaluation of noninvasive genetic sampling methods for cougars in Yellowstone National Park

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

Heterozygosity-fitness correlations in a bottlenecked island species: a case study on the Seychelles warbler

We used capture-mark-recapture models to investigate the effects of both individual and parental heterozygosity, measured at microsatellite loci on the survival of Seychelles warblers (Acrocephalus sechellensis), an endemic island species which went through a severe population bottleneck in the middle of the last century. We found that an individual's survival was not correlated with multilocus heterozygosity, or with heterozygosity at any specific locus. However, maternal, but not paternal, multilocus heterozygosity was positively associated with offspring survival, but only in years with low survival probabilities. A nestling cross-fostering experiment showed that this was a direct maternal effect as there was an effect of the genetic mother's, but not of the social mother's, heterozygosity. Heterozygosity-fitness correlations at microsatellite markers were generally assumed to reflect genome-wide effects. Although this might be true in partially inbred populations, such correlations may also arise as a result of local effects with specific markers being closely linked to genes which determine fitness. However, heterozygosity at the individual microsatellite loci was not correlated and therefore does not seem to reflect genome-wide heterozygosity. This suggests that even in a small bottlenecked population, heterozygosity-fitness correlations may not be caused by genome-wide effects. Support for the local effects hypothesis was also equivocal; although three specific loci were associated with offspring survival, including all single-locus heterozygosities as independent predictors for the variation in survival was not supported by the data. Furthermore, in contrast to the local effects hypothesis, the loci which contributed most to the heterozygosity-survival relationship were not more polymorphic than the other loci. This study highlights the difficulties in distinguishing between the two hypotheses.     

A new method for estimating the size of small populations from genetic mark-recapture data

The use of non-invasive genetic sampling to estimate population size in elusive or rare species is increasing. The data generated from this sampling differ from traditional mark-recapture data in that individuals may be captured multiple times within a session or there may only be a single sampling event. To accommodate this type of data, we develop a method, named capwire, based on a simple urn model containing individuals of two capture probabilities. The method is evaluated using simulations of an urn and of a more biologically realistic system where individuals occupy space, and display heterogeneous movement and DNA deposition patterns. We also analyse a small number of real data sets. The results indicate that when the data contain capture heterogeneity the method provides estimates with small bias and good coverage, along with high accuracy and precision. Performance is not as consistent when capture rates are homogeneous and when dealing with populations substantially larger than 100. For the few real data sets where N is approximately known, capwire's estimates are very good. We compare capwire's performance to commonly used rarefaction methods and to two heterogeneity estimators in program capture: Mh-Chao and Mh-jackknife. No method works best in all situations. While less precise, the Chao estimator is very robust. We also examine how large samples should be to achieve a given level of accuracy using capwire. We conclude that capwire provides an improved way to estimate N for some DNA-based data sets.     

Are extra-pair young better than within-pair young? A comparison of survival and dominance in alpine marmot

1. In socially monogamous species, females may seek extra-pair copulation to gain genetic benefits. In order to test this 'genetic quality' hypothesis, one must compare the performance of extra-pair young (EPY) and within-pair young (WPY). Such tests, however, are scarce and results published so far are inconclusive. 2. Here, we test the 'genetic quality' hypothesis using multistate capture-recapture models to compare age-specific survival and access to dominance between EPY and WPY in the alpine marmot Marmota marmota, a socially monogamous mammal showing extra-pair paternities. 3. When compared with WPY, survival of EPY was higher by 15%, 10% and 30%, for juveniles, yearlings and 2-year-old individuals, respectively. Survival at older ages did not differ. 4. Survival corresponded to true survival for yearlings and juveniles as dispersal does not occur before 2 years of age in marmots. For older individuals, survival estimates included a mixture of survival and dispersal. The 30% increase of the 2-year-old EPY survival might reflect delayed dispersal rather than high survival of EPY as compared with WPY. 5. WPY and EPY had the same probability (0.28) to access dominance at 2 years of age, but EPY were more successful at older ages than WPY (0.46 vs. 0.10). 6. Both survival and reproductive performance were higher in EPY than in WPY. The fitness advantages of adopting such a mixed mating tactic are thus likely to be high for marmot females. We suggest that obtaining genetic benefits is the main evolutionary force driving extra-pair paternity in alpine marmots.     

Estimating the effect of inbreeding on survival

Conservation biologists need to be able to estimate reliably the effects of inbreeding on survival, and need to be able to do so with a range of different data types. Kalinowski and Hedrick described a non-linear maximum likelihood estimation procedure for modelling relationships between survivorship and inbreeding. Although their method is useful for illustrating the concepts involved in modelling such relationships, it is only applicable to simple datasets. We illustrate that the parameter estimates generated by Kalinowski and Hedrick's method are easily obtained using generalized linear modelling procedures available in standard statistical packages, and that these offer several advantages even with simple datasets. We suggest procedures that can be used for modelling relationships between survival and inbreeding with more complex data types, including datasets with multiple and ragged encounters, uncertain detection and random effects.     

Compatible genetic and ecological estimates of dispersal rates in insect (Coenagrion mercuriale: Odonata: Zygoptera) populations: analysis of 'neighbourhood size' using a more precise estimator

Genetic and demographic estimates of dispersal are often thought to be inconsistent. In this study, we use the damselfly Coenagrion mercuriale (Odonata: Zygoptera) as a model to evaluate directly the relationship between estimates of dispersal rate measured during capture-mark-recapture fieldwork with those made from the spatial pattern of genetic markers in linear and two-dimensional habitats. We estimate the 'neighbourhood size' (Nb) - the product of the mean axial dispersal rate between parent and offspring and the population density - by a previously described technique, here called the regression method. Because C. mercuriale is less philopatric than species investigated previously by the regression method we evaluate a refined estimator that may be more applicable for relatively mobile species. Results from simulations and empirical data sets reveal that the new estimator performs better under most situations, except when dispersal is very localized relative to population density. Analysis of the C. mercuriale data extends previous results which demonstrated that demographic and genetic estimates of Nb by the regression method are equivalent to within a factor of two at local scales where genetic estimates are less affected by habitat heterogeneity, stochastic processes and/or differential selective regimes. The corollary is that with a little insight into a species' ecology the pattern of spatial genetic structure provides quantitative information on dispersal rates and/or population densities that has real value for conservation management.     

Testing the reliability of noninvasive genetic sampling by comparing analyses of blood and fecal samples in Barbary macaques (Macaca sylvanus).

Genetic studies of wild animal populations are often hindered by difficulties in obtaining blood samples. Recent advances in molecular biology have allowed the use of noninvasive samples as sources of DNA (e.g., hair or feces), but such samples may provide low-quality DNA and prevent the determination of true genotypes in subsequent DNA analysis. We present a preliminary study aimed at assessing the reliability of using fecal samples for genotyping in Barbary macaques (Macaca sylvanus). The test was performed on samples of blood and feces from 11 captive animals, using three dinucleotide microsatellites. The CTAB DNA extraction method was found to be the most relevant for Barbary macaque feces, yielding successful amplification at all loci for 70% of PCRs. All the fecal samples tested gave correct genotypes at least once for each locus when referenced against blood-derived genotypes. An average of 18.3% of PCRs displayed spurious genotypes (false homozygous or false allele). The minimum theoretical probability required to obtain a 100% accurate genotype is 0.74, based on the criterion that a correct genotype is assessed only if it was observed at least twice. The observed probability of obtaining a correct genotype from three PCRs, based on our genotyping results, was greater (0.81 on average) than the minimum threshold. In conclusion, our comparison of blood and fecal samples showed that fecal sampling is a reliable tool for the further study of wild Barbary macaque populations.     

Age-specific effect of heterozygosity on survival in alpine marmots, Marmota marmota

The fitness consequences of heterozygosity and the mechanisms underpinning them are still highly controversial. Using capture–mark–recapture models, we investigated the effects of individual heterozygosity, measured at 16 microsatellite markers, on age-dependent survival and access to dominance in a socially monogamous mammalian species, the alpine marmot. We found a positive correlation between standardized multilocus heterozygosity and juvenile survival. However, there was no correlation between standardized multilocus heterozygosity and either survival of older individuals or access to dominance. The disappearance of a significant heterozygosity fitness correlation when individuals older than juveniles are considered is consistent with the prediction that differences in survival among individuals are maximal early in life. The lack of a correlation between heterozygosity and access to dominance may be a consequence of few homozygous individuals attaining the age at which they might reach dominance. Two hypotheses have been proposed to explain heterozygosity-fitness correlations: genome-wide effects reflected by all markers or local effects of specific markers linked to genes that determine fitness. In accordance with genome-wide effects of heterozygosity, we found significant correlations between heterozygosities calculated across single locus or across two sets of eight loci. Thus, the genome-wide heterozygosity effect seems to explain the observed heterozygosity-fitness correlation in the alpine marmot.     

Recruitment to a seabird population depends on environmental factors and on population size

1. A novel capture-mark-recapture (CMR) method was used to build a multistate model of recruitment by young birds to a breeding population of common guillemots Uria aalge on the Isle of May, Scotland. Recruitment of a total of 2757 individually marked guillemots over 17 years was modelled as a process where individuals had to move from an unobservable state at sea, through a nonbreeding state present in the colony, to the breeding state. The probabilities of individuals returning to the colony in a given year, at age 2 and 3-4 years, were positively correlated with an environmental covariate, the winter North Atlantic Oscillation index (WNAO) in the previous years. 2. For 2 year olds, there was a negative relationship with breeding population size, suggesting that density dependence operated in this colony through limitation of food or some other resource. 3. Survival over the first 2 years of life varied with cohort, but was unrelated to the WNAO. Mean survival over this 2-year period was high at 0.576 (95% CI: 0.444; 0.708). 4. This high survival, combined with a low 'local' survival after age 5 years of 0.695 (0-654; 0.733) and observations of Isle of May chicks at other colonies, suggests that most surviving chicks return to the natal colony before deciding whether to recruit there or move elsewhere.     

Effective population sizes and migration rates in fragmented populations of an endangered insect (Coenagrion mercuriale: Odonata)

1. Effective population sizes (N(e)) and migration rates (m) are critical evolutionary parameters that impact on population survival and determine the relative influence of selection and genetic drift. While the parameter m is well-studied in animal populations, N(e) remains challenging to measure and consequently is only rarely estimated, particularly in insect taxa. 2. We used demographic and genetic methods to estimate N(e) and m in a fragmented population of the endangered damselfly Coenagrion mercuriale to better understand the contrast between genetic and field estimates of these parameters and also to identify the spatial scale over which populations may become locally adapted. 3. We found a contrast between demographic- and genetic-based estimates of these parameters, with the former apparently providing overestimates of N(e), owing to substantial underestimation of the variance in reproductive success, and the latter overestimating m, because spatial genetic structure is weak. 4. The overall N(e) of sites within the population network at Beaulieu Heath, the largest C. mercuriale site in the UK, was estimated to vary between approximately 60 and 2700. 5. While N(e) was not correlated with either the total numbers of adults (N) or the area of habitat, this parameter was always less than N, because of substantial variance in reproductive success. The ratio N(e)/N varied between 0.006 and 0.42 and was generally larger in smaller populations, possibly representing some 'genetic compensation'. 6. From a simple genetic model and these data on N(e) and m, it seems that populations of C. mercuriale have the potential to respond to localized spatial variation in selection and this would need to be considered for future genetic management of this endangered species.     

Delayed maturation in female bank voles: optimal decision or social constraint?

1. Density, maturation and survival of female bank vole ( Clethrionomys glareolus ) in the northern taiga of Finnish Lapland were studied using long-term capture–mark–recapture data from two large grids, one food-addition grid and one control grid, in 1982–94.
2. The density on the food grid was consistently higher than the density on the control grid.
3. Females born early in the breeding season usually matured, except at very high densities. Those born later in the summer season commonly delayed maturation to the following spring.
4. Winter survival of sub-adult (having delayed maturation) females was significantly higher than survival of adult (breeding) females. However, empirical values of sub-adult and adult survival, as well as difference between them, were not consistent with survival values assumed in theoretical models on optimal deferred breeding.
5. There was a density-dependent relationship between the maturation rate of young voles and the density of already established breeding females (both bank voles and all Clethrionomys together; C. rutilus and C. rufocanus occasionally occurred on the study grids). This density dependence was different for the two grids (weaker on the food-addition grid).
6. These findings are discussed within an evolutionary context: we have, on the basis of these findings, no evidence suggesting that the observed delayed maturation represents an evolutionary optimal strategy. Rather, there is evidence suggesting that the delay is due to social constraints.     

Comparison of social networks derived from ecological data: implications for inferring infectious disease dynamics

1.  Social network analyses tend to focus on human interactions. However, there is a burgeoning interest in applying graph theory to ecological data from animal populations. Here we show how radio-tracking and capture–mark–recapture data collated from wild rodent populations can be used to generate contact networks.
2.  Both radio-tracking and capture–mark–recapture were undertaken simultaneously. Contact networks were derived and the following statistics estimated: mean-contact rate, edge distribution, connectance and centrality.
3.  Capture–mark–recapture networks produced more informative and complete networks when the rodent density was high and radio-tracking produced more informative networks when the density was low. Different data collection methods provide more data when certain ecological characteristics of the population prevail.
4.  Both sets of data produced networks with comparable edge (contact) distributions that were best described by a negative binomial distribution. Connectance and closeness were statistically different between the two data sets. Only betweenness was comparable. The differences between the networks have important consequences for the transmission of infectious diseases. Care should be taken when extrapolating social networks to transmission networks for inferring disease dynamics.     

Balancing bias and precision in capture-recapture estimates of abundance

Capture‐recapture estimates of abundance using photographic identification data are sensitive to the quality of photographs used and distinctiveness of individuals in the population. Here analyses are presented for examining the effects of photographic quality and individual animal distinctiveness scores and for objectively selecting a subset of data to use for capture‐recapture analyses using humpback whale (Megaptera novaeangliae) data from a 2‐year study in the North Atlantic. Photographs were evaluated for their level of quality and whales for their level of individual distinctiveness. Photographic quality scores had a 0.21 probability of changing by a single‐quality level, and there were no changes by two or more levels. Individual distinctiveness scores were not independent of photographic quality scores. Estimates of abundance decreased as poor‐quality photographs were removed. An appropriate balance between precision and bias in abundance estimates was achieved by removing the lowest‐quality photographs and those of incompletely photographed flukes given our assumptions about the true population abundance. A simulation of the selection process implied that, if the estimates are negatively biased by heterogeneity, the increase in bias produced by decreasing the sample size is not more than 2%. Capture frequencies were independent of individual distinctiveness scores.