Give and take: Effects of genetic admixture on mutation load in endangered Florida panthers.

Genetic admixture is a biological event inherent to genetic rescue programs aimed at the long-term conservation of endangered wildlife. Although the success of such programs can be measured by the increase in genetic diversity and fitness of subsequent admixed individuals, predictions supporting admixture costs to fitness due to the introduction of novel deleterious alleles are necessary. Here, we analyzed nonsynonymous variation from conserved genes to quantify and compare levels of mutation load (i.e. proportion of deleterious alleles and genotypes carrying these alleles) among endangered Florida panthers and non-endangered Texas pumas. Specifically, we used canonical (i.e. non-admixed) Florida panthers, Texas pumas, and F1 (canonical Florida × Texas) panthers dating from a genetic rescue program and Everglades National Park panthers with Central American ancestry resulting from an earlier admixture event. We found neither genetic drift nor selection significantly reduced overall proportions of deleterious alleles in the severely bottlenecked canonical Florida panthers. Nevertheless, the deleterious alleles identified were distributed into a disproportionately high number of homozygous genotypes due to close inbreeding in this group. Conversely, admixed Florida panthers (either with Texas or Central American ancestry) presented reduced levels of homozygous genotypes carrying deleterious alleles but increased levels of heterozygous genotypes carrying these variants relative to canonical Florida panthers. Although admixture is likely to alleviate the load of standing deleterious variation present in homozygous genotypes, our results suggest that introduced novel deleterious alleles (temporarily present in heterozygous state) in genetically rescued populations could potentially be expressed in subsequent generations if their effective sizes remain small.

Inferring the Demographic History of Inbred Species from Genome-Wide SNP Frequency Data.

Demographic inference using the site frequency spectrum (SFS) is a common way to understand historical events affecting genetic variation. However, most methods for estimating demography from the SFS assume random mating within populations, precluding these types of analyses in inbred populations. To address this issue, we developed a model for the expected SFS that includes inbreeding by parameterizing individual genotypes using beta-binomial distributions. We then take the convolution of these genotype probabilities to calculate the expected frequency of biallelic variants in the population. Using simulations, we evaluated the model's ability to coestimate demography and inbreeding using one- and two-population models across a range of inbreeding levels. We also applied our method to two empirical examples, American pumas (Puma concolor) and domesticated cabbage (Brassica oleracea var. capitata), inferring models both with and without inbreeding to compare parameter estimates and model fit. Our simulations showed that we are able to accurately coestimate demographic parameters and inbreeding even for highly inbred populations (F = 0.9). In contrast, failing to include inbreeding generally resulted in inaccurate parameter estimates in simulated data and led to poor model fit in our empirical analyses. These results show that inbreeding can have a strong effect on demographic inference, a pattern that was especially noticeable for parameters involving changes in population size. Given the importance of these estimates for informing practices in conservation, agriculture, and elsewhere, our method provides an important advancement for accurately estimating the demographic histories of these species.

Draft de novo Genome Assembly of the Elusive Jaguarundi, Puma yagouaroundi.

The Puma lineage within the family Felidae consists of 3 species that last shared a common ancestor around 4.9 million years ago. Whole-genome sequences of 2 species from the lineage were previously reported: the cheetah (Acinonyx jubatus) and the mountain lion (Puma concolor). The present report describes a whole-genome assembly of the remaining species, the jaguarundi (Puma yagouaroundi). We sequenced the genome of a male jaguarundi with 10X Genomics linked reads and assembled the whole-genome sequence. The assembled genome contains a series of scaffolds that reach the length of chromosome arms and is similar in scaffold contiguity to the genome assemblies of cheetah and puma, with a contig N50 = 100.2 kbp and a scaffold N50 = 49.27 Mbp. We assessed the assembled sequence of the jaguarundi genome using BUSCO, aligned reads of the sequenced individual and another published female jaguarundi to the assembled genome, annotated protein-coding genes, repeats, genomic variants and their effects with respect to the protein-coding genes, and analyzed differences of the 2 jaguarundis from the reference mitochondrial genome. The jaguarundi genome assembly and its annotation were compared in quality, variants, and features to the previously reported genome assemblies of puma and cheetah. Computational analyzes used in the study were implemented in transparent and reproducible way to allow their further reuse and modification.

Urbanization impacts apex predator gene flow but not genetic diversity across an urban-rural divide.

Apex predators are important indicators of intact natural ecosystems. They are also sensitive to urbanization because they require broad home ranges and extensive contiguous habitat to support their prey base. Pumas (Puma concolor) can persist near human developed areas, but urbanization may be detrimental to their movement ecology, population structure, and genetic diversity. To investigate potential effects of urbanization in population connectivity of pumas, we performed a landscape genomics study of 130 pumas on the rural Western Slope and more urbanized Front Range of Colorado, USA. Over 12,000 single nucleotide polymorphisms (SNPs) were genotyped using double-digest, restriction site-associated DNA sequencing (ddRADseq). We investigated patterns of gene flow and genetic diversity, and tested for correlations between key landscape variables and genetic distance to assess the effects of urbanization and other landscape factors on gene flow. Levels of genetic diversity were similar for the Western Slope and Front Range, but effective population sizes were smaller, genetic distances were higher, and there was more admixture in the more urbanized Front Range. Forest cover was strongly positively associated with puma gene flow on the Western Slope, while impervious surfaces restricted gene flow and more open, natural habitats enhanced gene flow on the Front Range. Landscape genomic analyses revealed differences in puma movement and gene flow patterns in rural versus urban settings. Our results highlight the utility of dense, genome-scale markers to document subtle impacts of urbanization on a wide-ranging carnivore living near a large urban center.

Evolutionary and Functional Mitogenomics Associated With the Genetic Restoration of the Florida Panther.

Florida panthers are endangered pumas that currently persist in reduced patches of habitat in South Florida, USA. We performed mitogenome reference-based assemblies for most parental lines of the admixed Florida panthers that resulted from the introduction of female Texas pumas into South Florida in 1995. With the addition of 2 puma mitogenomes, we characterized 174 single nucleotide polymorphisms (SNPs) across 12 individuals. We defined 5 haplotypes (Pco1-Pco5), one of which (Pco1) had a geographic origin exclusive to Costa Rica and Panama and was possibly introduced into the Everglades National Park, Florida, prior to 1995. Haplotype Pco2 was native to Florida. Haplotypes Pco3 and Pco4 were exclusive to Texas, whereas haplotype Pco5 had an undetermined geographic origin. Phylogenetic inference suggests that haplotypes Pco1-Pco4 diverged ~202000 (95% HPDI = 83000-345000) years ago and that haplotypes Pco2-Pco4 diverged ~61000 (95% HPDI = 9000-127000) years ago. These results are congruent with a south-to-north continental expansion and with a recent North American colonization by pumas. Furthermore, pumas may have migrated from Texas to Florida no earlier than ~44000 (95% HPDI = 2000-98000) years ago. Synonymous mutations presented a greater mean substitution rate than other mitochondrial functional regions: nonsynonymous mutations, tRNAs, rRNAs, and control region. Similarly, all protein-coding genes were under predominant negative selection constraints. We directly and indirectly assessed the presence of potential deleterious SNPs in the ND2 and ND5 genes in Florida panthers prior to and as a consequence of the introduction of Texas pumas. Screenings for such variants are recommended in extant Florida panthers.

Interactions between demography, genetics, and landscape connectivity increase extinction probability for a small population of large carnivores in a major metropolitan area.

The extinction vortex is a theoretical model describing the process by which extinction risk is elevated in small, isolated populations owing to interactions between environmental, demographic, and genetic factors. However, empirical demonstrations of these interactions have been elusive. We modelled the dynamics of a small mountain lion population isolated by anthropogenic barriers in greater Los Angeles, California, to evaluate the influence of demographic, genetic, and landscape factors on extinction probability. The population exhibited strong survival and reproduction, and the model predicted stable median population growth and a 15% probability of extinction over 50 years in the absence of inbreeding depression. However, our model also predicted the population will lose 40-57% of its heterozygosity in 50 years. When we reduced demographic parameters proportional to reductions documented in another wild population of mountain lions that experienced inbreeding depression, extinction probability rose to 99.7%. Simulating greater landscape connectivity by increasing immigration to greater than or equal to one migrant per generation appears sufficient to largely maintain genetic diversity and reduce extinction probability. We provide empirical support for the central tenet of the extinction vortex as interactions between genetics and demography greatly increased extinction probability relative to the risk from demographic and environmental stochasticity alone. Our modelling approach realistically integrates demographic and genetic data to provide a comprehensive assessment of factors threatening small populations.

Multi-generational benefits of genetic rescue.

Genetic rescue-an increase in population fitness following the introduction of new alleles-has been proven to ameliorate inbreeding depression in small, isolated populations, yet is rarely applied as a conservation tool. A lingering question regarding genetic rescue in wildlife conservation is how long beneficial effects persist in admixed populations. Using data collected over 40 years from 1192 endangered Florida panthers (Puma concolor coryi) across nine generations, we show that the experimental genetic rescue implemented in 1995-via the release of eight female pumas from Texas-alleviated morphological, genetic, and demographic correlates of inbreeding depression, subsequently preventing extirpation of the population. We present unequivocal evidence, for the first time in any terrestrial vertebrate, that genetic and phenotypic benefits of genetic rescue remain in this population after five generations of admixture, which helped increase panther abundance (> fivefold) and genetic effective population size (> 20-fold). Additionally, even with extensive admixture, microsatellite allele frequencies in the population continue to support the distinctness of Florida panthers from other North American puma populations, including Texas. Although threats including habitat loss, human-wildlife conflict, and infectious diseases are challenges to many imperiled populations, our results suggest genetic rescue can serve as an effective, multi-generational tool for conservation of small, isolated populations facing extinction from inbreeding.

A cat's tale: the impact of genetic restoration on Florida panther population dynamics and persistence.

1. Genetic restoration has been suggested as a management tool for mitigating detrimental effects of inbreeding depression in small, inbred populations, but the demographic mechanisms underlying population-level responses to genetic restoration remain poorly understood. 2. We studied the dynamics and persistence of the endangered Florida panther Puma concolor coryi population and evaluated the potential influence of genetic restoration on population growth and persistence parameters. As part of the genetic restoration programme, eight female Texas pumas P. c. stanleyana were released into Florida panther habitat in southern Florida in 1995. 3. The overall asymptotic population growth rate (λ) was 1.04 (5th and 95th percentiles: 0.95-1.14), suggesting an increase in the panther population of approximately 4% per year. Considering the effects of environmental and demographic stochasticities and density-dependence, the probability that the population will fall below 10 panthers within 100 years was 0.072 (0-0.606). 4. Our results suggest that the population would have declined at 5% per year (λ = 0.95; 0.83-1.08) in the absence of genetic restoration. Retrospective life table response experiment analysis revealed that the positive effect of genetic restoration on survival of kittens was primarily responsible for the substantial growth of the panther population that would otherwise have been declining. 5. For comparative purposes, we also estimated probability of quasi-extinction under two scenarios - implementation of genetic restoration and no genetic restoration initiative - using the estimated abundance of panthers in 1995, the year genetic restoration was initiated. Assuming no density-dependence, the probability that the panther population would fall below 10 panthers by 2010 was 0.098 (0.002-0.332) for the restoration scenario and 0.445 (0.032-0.944) for the no restoration scenario, providing further evidence that the panther population would have faced a substantially higher risk of extinction if the genetic restoration initiative had not been implemented. 6. Our results, along with those reporting increases in population size and improvements in biomedical correlates of inbreeding depression, provide strong evidence that genetic restoration substantially contributed to the observed increases in the Florida panther population.

Where do you come from, where do you go? Directional migration rates in landscape genetics.

Identifying landscape elements that influence gene flow and migration in wild species is the current main topic of landscape genetics. Most landscape genetic studies infer gene flow and migration from genetic distances among populations or individuals and statistically relate these measurements to landscape composition and configuration. This approach assumes symmetrical gene flow between pairs of populations. Such an assumption, however, will often be violated, especially in source­sink systems. Source populations provide more emigrants than they receive immigrants, and sink populations get many immigrants, but release few emigrants. Source­sink dynamics cannot be explored using common landscape genetic approaches relying on genetic distances. In this issue of Molecular Ecology, Andreasen et al. (2012) apply an alternative approach allowing them to infer asymmetrical migration. They use a Bayesian assignment test among objectively defined populations of mountain lions (Puma concolor) in western USA to estimate recent and directional migration rates. The study shows that an area with a high amount of wildlife refuges and low hunting pressure harbours a source population for mountain lion dispersal, while areas with high hunting pressures form sink populations; a result helpful in making informed decisions in conservation management.

Identification of source-sink dynamics in mountain lions of the Great Basin.

Natural and anthropogenic boundaries have been shown to affect population dynamics and population structure for many species with movement patterns at the landscape level. Understanding population boundaries and movement rates in the field for species that are cryptic and occur at low densities is often extremely difficult and logistically prohibitive; however genetic techniques may offer insights that have previously been unattainable. We analysed thirteen microsatellite loci for 739 mountain lions (Puma concolor) using muscle tissue samples from individuals in the Great Basin throughout Nevada and the Sierra Nevada mountain range to test the hypothesis that heterogeneous hunting pressure results in source-sink dynamics at the landscape scale. We used a combination of non-spatial and spatial model-based Bayesian clustering methods to identify genetic populations. We then used a recently developed Bayesian multilocus genotyping method to estimate asymmetrical rates of contemporary movement between those subpopulations and to identify source and sink populations. We identified two populations at the highest level of genetic structuring with a total of five subpopulations in the Great Basin of Nevada and the Sierra Nevada range. Our results suggest that source-sink dynamics occur at landscape scales for wide-ranging species, such as mountain lions, and that source populations may be those that are under relatively less hunting pressure and that occupy refugia.

Does genetic introgression improve female reproductive performance? A test on the endangered Florida panther.

Genetic introgression has been suggested as a management tool for mitigating detrimental effects of inbreeding depression, but the role of introgression in species conservation has been controversial, partly because population-level impacts of genetic introgressions are not well understood. Concerns about potential inbreeding depression in the endangered Florida panther (Puma concolor coryi) led to the release of eight female Texas pumas (P. c. stanleyana) into the Florida panther population in 1995. We used long-term reproductive data (1995-2008) collected from 61 female Florida panthers to estimate and model reproduction probability (probability of producing a litter) and litter size, and to investigate the influence of intentional genetic introgression on these parameters. Overall, 6-month probability of reproduction (±1SE) was 0.232 ± 0.021 and average litter size was 2.60 ± 0.09. Although F(1) admixed females had a lower reproduction probability than females with other ancestries, this was most likely because kittens born to F(1) females survive better; consequently, these females are unavailable for breeding until kittens are independent. There was no evidence for the effect of ancestry on litter size or of heterozygosity on probability of reproduction or litter size. In contrast, earlier studies have shown that genetic introgression positively affected Florida panther survival. Our results, along with those of earlier studies, clearly suggest that genetic introgression can have differential effects on components of fitness and highlight the importance of examining multiple demographic parameters when evaluating the effects of management actions.

Morphological, Ultrastructural, and Immunocytochemical Characterization and Assessment of Puma (Puma concolor Linnaeus, 1771) Cell Lines After Extended Culture and Cryopreservation.

Cell lines are valuable tools to safeguard genetic material from species threatened with extinction that is mainly due to human action. In this scenario, the puma constitutes a species whose population is being rapidly reduced in the ecosystems it inhabits. For the first time, we characterized puma skin-derived cell lines and assessed these cells after extended culture (experiment 1) and cryopreservation (experiment 2). Initially, we identified and characterized four dermal fibroblast lines using morphology, ultrastructure, and immunofluorescence assays. Moreover, we evaluated the effects of culture time (1st, 3rd, and 10th passages) and cryopreservation on their morphology, ultrastructure, viability, metabolism, proliferative activity, reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨm), and apoptosis. The cells showed a typical spindle-shaped morphology with centrally located oval nuclei. The cells were identified as fibroblasts by staining for vimentin. In vitro culture after the 1st, 3rd, and 10th passages did not alter most of the evaluated parameters. Cells in the 3rd and 10th passages showed a reduction in ROS levels (p < 0.05). The ultrastructure revealed morphological damage in the prolongments, and nuclei of cells derived from the 3rd and 10th passages. Moreover, cryopreservation resulted in a reduction in ΔΨm compared with that of noncryopreserved cells, suggesting that the optimization of cryopreservation methods for puma fibroblasts is essential. In conclusion, we found that viable fibroblasts could be obtained from puma skin, with slight changes after the 10th passage in in vitro culture and cryopreservation. This is the first report on the development of cell lines derived from pumas.

Mountain lions avoid burned areas and increase risky behavior after wildfire in a fragmented urban landscape.

Urban environments are high risk areas for large carnivores, where anthropogenic disturbances can reduce fitness and increase mortality risk.1 When catastrophic events like large wildfires occur, trade-offs between acquiring resources and avoiding risks of the urban environment are intensified. This landscape context could lead to an increase in risk-taking behavior by carnivores if burned areas do not allow them to meet their energetic needs, potentially leading to human-wildlife conflict.2,3 We studied mountain lion behavior using GPS location and accelerometer data from 17 individuals tracked before and after a large wildfire (the 2018 Woolsey Fire) within a highly urbanized area (Los Angeles, California, USA). After the wildfire, mountain lions avoided burned areas and increased behaviors associated with anthropogenic risk, including more frequent road and freeway crossings (mean crossings increased from 3 to 5 per month) and greater activity during the daytime (means from increased 10% to 16% of daytime active), a time when they are most likely to encounter humans. Mountain lions also increased their amount of space used, distance traveled (mean distances increased from 250 to 390 km per month), and intrasexual overlap, potentially putting them at risk of intraspecific conflict. Joint pressures from urbanization and severe wildfire, alongside resulting risk-taking, could thus increase mortality and extinction risk for populations already suffering from low genetic diversity, necessitating increased connectivity in fire-prone areas.

Accurate and fast clade assignment via deep learning and frequency chaos game representation.

BACKGROUND: Since the beginning of the coronavirus disease 2019 pandemic, there has been an explosion of sequencing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, making it the most widely sequenced virus in the history. Several databases and tools have been created to keep track of genome sequences and variants of the virus; most notably, the GISAID platform hosts millions of complete genome sequences, and it is continuously expanding every day. A challenging task is the development of fast and accurate tools that are able to distinguish between the different SARS-CoV-2 variants and assign them to a clade. RESULTS: In this article, we leverage the frequency chaos game representation (FCGR) and convolutional neural networks (CNNs) to develop an original method that learns how to classify genome sequences that we implement into CouGaR-g, a tool for the clade assignment problem on SARS-CoV-2 sequences. On a testing subset of the GISAID, CouGaR-g achieved an $96.29\%$ overall accuracy, while a similar tool, Covidex, obtained a $77,12\%$ overall accuracy. As far as we know, our method is the first using deep learning and FCGR for intraspecies classification. Furthermore, by using some feature importance methods, CouGaR-g allows to identify k-mers that match SARS-CoV-2 marker variants. CONCLUSIONS: By combining FCGR and CNNs, we develop a method that achieves a better accuracy than Covidex (which is based on random forest) for clade assignment of SARS-CoV-2 genome sequences, also thanks to our training on a much larger dataset, with comparable running times. Our method implemented in CouGaR-g is able to detect k-mers that capture relevant biological information that distinguishes the clades, known as marker variants. AVAILABILITY: The trained models can be tested online providing a FASTA file (with 1 or multiple sequences) at https://huggingface.co/spaces/BIASLab/sars-cov-2-classification-fcgr. CouGaR-g is also available at https://github.com/AlgoLab/CouGaR-g under the GPL.

Intentional genetic introgression influences survival of adults and subadults in a small, inbred felid population.

1. Inbreeding and low genetic diversity can cause reductions in individual fitness and increase extinction risk in animal populations. Intentional introgression, achieved by releasing genetically diverse individuals into inbred populations, has been used as a conservation tool to improve demographic performance in endangered populations. 2. By the 1980s, Florida panthers (Puma concolor coryi) had been reduced to a small, inbred population that appeared to be on the brink of extinction. In 1995, female pumas from Texas (P. c. stanleyana) were released in occupied panther range as part of an intentional introgression programme to restore genetic variability and improve demographic performance of panthers. 3. We used 25 years (1981-2006) of continuous radiotelemetry and genetic data to estimate and model subadult and adult panther survival and cause-specific mortality to provide rigorous sex and age class-specific survival estimates and evaluate the effect of the introgression programme on these parameters. 4. Genetic ancestry influenced annual survival of subadults and adults after introgression, as F(1) generation admixed panthers ( = 0·98) survived better than pre-introgression type panthers ( = 0·77) and other admixed individuals ( = 0·82). Furthermore, heterozygosity was higher for admixed panthers relative to pre-introgression type panthers and positively influenced survival. 5. Our results are consistent with hybrid vigour; however, extrinsic factors such as low density of males in some areas of panther range may also have contributed to higher survival of F(1) panthers. Regardless, improved survival of F(1) subadults and adults likely contributed to the numerical increase in panthers following introgression, and our results indicate that intentional admixture, achieved here by releasing individuals from another population, appears to have been successful in improving demographic performance in this highly endangered population.

Comparison of Bayesian clustering and edge detection methods for inferring boundaries in landscape genetics.

Recently, techniques available for identifying clusters of individuals or boundaries between clusters using genetic data from natural populations have expanded rapidly. Consequently, there is a need to evaluate these different techniques. We used spatially-explicit simulation models to compare three spatial Bayesian clustering programs and two edge detection methods. Spatially-structured populations were simulated where a continuous population was subdivided by barriers. We evaluated the ability of each method to correctly identify boundary locations while varying: (i) time after divergence, (ii) strength of isolation by distance, (iii) level of genetic diversity, and (iv) amount of gene flow across barriers. To further evaluate the methods' effectiveness to detect genetic clusters in natural populations, we used previously published data on North American pumas and a European shrub. Our results show that with simulated and empirical data, the Bayesian spatial clustering algorithms outperformed direct edge detection methods. All methods incorrectly detected boundaries in the presence of strong patterns of isolation by distance. Based on this finding, we support the application of Bayesian spatial clustering algorithms for boundary detection in empirical datasets, with necessary tests for the influence of isolation by distance.

The differential genetic signatures related to climatic landscapes for jaguars and pumas on a continental scale.

Modern and paleoclimate changes may have altered species dynamics by shifting species' niche suitability over space and time. We analyze whether the current genetic structure and isolation of the two large American felids, jaguar (Panthera onca) and puma (Puma concolor), are mediated by changes in climatic suitability and connection routes over modern and paleoclimatic landscapes. We estimate species distribution under 5 climatic landscapes (modern, Holocene, last maximum glaciations [LMG], average suitability, and climatic instability) and correlate them with individuals' genetic isolation through causal modeling on a resemblance matrix. Both species exhibit genetic isolation patterns correlated with LMG climatic suitability, suggesting that these areas may have worked as "allele refuges." However, the jaguar showed higher vulnerability to climate changes, responding to modern climatic suitability and connection routes, whereas the puma showed a continuous and gradual transition of genetic variation. Despite differential responsiveness to climate change, both species are subjected to the climatic effects on genetic configuration, which may make them susceptible to future climatic changes, since these are progressing faster and with higher intensity than changes in the paleoclimate. Thus, the effects of climatic changes should be considered in the design of conservation strategies to ensure evolutionary and demographic processes mediated by gene flow for both species.

Host relatedness and landscape connectivity shape pathogen spread in the puma, a large secretive carnivore.

Urban expansion can fundamentally alter wildlife movement and gene flow, but how urbanization alters pathogen spread is poorly understood. Here, we combine high resolution host and viral genomic data with landscape variables to examine the context of viral spread in puma (Puma concolor) from two contrasting regions: one bounded by the wildland urban interface (WUI) and one unbounded with minimal anthropogenic development (UB). We found landscape variables and host gene flow explained significant amounts of variation of feline immunodeficiency virus (FIV) spread in the WUI, but not in the unbounded region. The most important predictors of viral spread also differed; host spatial proximity, host relatedness, and mountain ranges played a role in FIV spread in the WUI, whereas roads might have facilitated viral spread in the unbounded region. Our research demonstrates how anthropogenic landscapes can alter pathogen spread, providing a more nuanced understanding of host-pathogen relationships to inform disease ecology in free-ranging species.

Heterologous microsatellite primers are informative for paca (Cuniculus paca), a large rodent with economic and ecological importance.

OBJECTIVE: This study was designed to facilitate genetic studies that would allow information on population structure and genetic diversity of natural or captive stocks of paca (Cuniculus paca), a species of ecological and socioeconomic importance, by testing cross-amplification of 20 heterologous microsatellite primer pairs developed for guinea pigs (Cavia porcellus) and capybara (Hydrochoerus hydrochaeris). RESULTS: Those primers that showed the best amplification profile in blood samples were subsequently applied to scats and saliva samples, to evaluate their efficiency. Of the 13 microsatellite pairs that amplified in blood, one-third (32%) were successfully amplified in saliva and scat samples. This initial work demonstrates successful cross-amplification in paca providing a solid and promising foundation for future genetic studies with this species. The ability to quantify genetic diversity using noninvasive samples from free-ranging paca is essential to developing applied management strategies for this large neotropical rodent that is not only a prey favored by wide-ranging carnivores [e.g., jaguar (Panthera onca), puma (Puma concolor)], but is also a species targeted by illegal hunting and wildlife trade.

Development and testing of an optimized method for DNA-based identification of jaguar (Panthera onca) and puma (Puma concolor) faecal samples for use in ecological and genetic studies.

The elusive nature and endangered status of most carnivore species imply that efficient approaches for their non-invasive sampling are required to allow for genetic and ecological studies. Faecal samples are a major potential source of information, and reliable approaches are needed to foster their application in this field, particularly in areas where few studies have been conducted. A major obstacle to the reliable use of faecal samples is their uncertain species-level identification in the field, an issue that can be addressed with DNA-based assays. In this study we describe a sequence-based approach that efficiently distinguishes jaguar versus puma scats, and that presents several desirable properties: (1) considerably high amplification and sequencing rates; (2) multiple diagnostic sites reliably differentiating the two focal species; (3) high information content that allows for future application in other carnivores; (4) no evidence of amplification of prey DNA; and (5) no evidence of amplification of a nuclear mitochondrial DNA insertion known to occur in the jaguar. We demonstrate the reliability and usefulness of this approach by evaluating 55 field-collected samples from four locations in the highly fragmented Atlantic Forest biome of Brazil and Argentina, and document the presence of one or both of these endangered felids in each of these areas.