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Advancing ecological understandings through technological transformations in noninvasive genetics
Abstract
Noninvasive genetic approaches continue to improve studies in molecular ecology, conservation genetics and related disciplines such as forensics and epidemiology. Noninvasive sampling allows genetic studies without disturbing or even seeing the target individuals. Although noninvasive genetic sampling has been used for wildlife studies since the 1990s, technological advances continue to make noninvasive approaches among the most used and rapidly advancing areas in genetics. Here, we review recent advances in noninvasive genetics and how they allow us to address important research and management questions thanks to improved techniques for DNA extraction, preservation, amplification and data analysis. We show that many advances come from the fields of forensics, human health and domestic animal health science, and suggest that molecular ecologists explore literature from these fields. Finally, we discuss how the combination of advances in each step of a noninvasive genetics study, along with fruitful areas for future research, will continually increase the power and role of noninvasive genetics in molecular ecology and conservation genetics.
... As the Balkan chamois is a threatened species in Greek territory, the sampling strategy was focused in non-invasive techniques, sampling the animals without capture or handling them (Pauli et al., 2010). A great variety of biological material can now be used as a source for DNA extraction such as urine, buccal cells form chewed food remnants, sloughed skin from cetaceans, reptiles' epidermis, feathers and eggshells of birds, owl pellets and most often bones, hair and feces (Piggott & Taylor, 2003;Broquet et al., 2007;Beja-Pereira et al., 2009). Especially for elusive and rare or endangered species, non-invasive sampling might be the best option to get hold of plenty samples without disturbing the animals in their natural habitat or causing any harm to them. ...
... It is necessary to remove water in order to deactivate nuclease activity. For feces preservation, ethanol (ETOH) and silica protocols are most frequent used (Beja-Pereira et al., 2009). In our study, silica was preferred as it is widely tested, provides easy handling in the field and of course it can be delivered much more safely compared to ETOH. ...
... Especially, in nucleic acid extracted from fecal material complications tend to increase. We applied strict screening process trying to mitigate at an early stage the difficulties from excremental DNA like allelic dropout, false alleles etc. (Beja-Pereira et al., 2009;Broquet et al., 2007;Waits & Paetkau, 2005;Taberlet, 1999). Firstly, samples with very low quantity and quality were left out of downstream handling, as various studies have shown high levels of incorrect amplification and genotyping (Zemanová, 2014). ...
Balkan chamois is an ungulate mammal of Rupicapra rupicapra species and in particular of the subspecies R. r. balcanica. The overall chamois’ population in Greece is estimated between 1330 and 1765 individuals with four populations numbering more than 150 individuals. Chamois has an island-like distribution and in Greece occurs in Pindos Mountain range, Central Greece, Olympus, Rhodopes Mts. and the mountains bordering with North Macedonia.
Balkan chamois is considered as near threatened (NT) in Greece and is under protection by European and national law. Therefore, a non-invasive genetic sampling was implemented, using DNA extracts mainly from feces and hair samples. The present study investigates the genetic diversity and population structure of the balkan chamois in Greece. For this purpose, five microsatellite markers were amplified and successfully genotyped in 110 individuals from populations across the subspecies distribution.
Non-invasive genetic sampling proved to be a difficult task, especially from material of fecal origin. High levels of amplification and genotyping failure were observed, possibly due to low DNA yield and quality. Samples’ time of collection and their subsequent storage duration might be crucial for the observed failure rates.
The results revealed overall low genetic diversity of the subspecies in Greece when compared with other studies concerning the subspecies in Greece, which could be attributed to the smaller number of used microsatellites or the differences in sample size. Also, the genetic diversity is lower in relation to that of other balkan countries’ natural populations. Among the main sampling sites (Timfi Mt., Smolikas Mt., Olympos Mt., Frakto) the lowest genetic diversity was found in the population from Smolikas while the highest in Frakto region. Indications of genetic differentiation all studied populations apart from Smolikas and Timfi. Based on the data of this study, at least three diverse gene pools could be located in Greece, which correspond to the three different mountainous population blocks.
... Molecular methods incorporating non-invasive sampling (NGS) via the collection of scats or hairs are currently widely used for population monitoring of elusive carnivores (Waits and Paetkau 2005;Schwartz and Monfort 2008;Beja-Pereira et al. 2009). The standard method for monitoring marten populations is scat transect surveys ). ...
... faecal preservation method (Murphy et al. 2002), DNA extraction method (Goossens et al. 2000;Frantz et al. 2003;Wehausen et al. 2004) and amplification method (Goossens et al. 2000;Piggott et al. 2004)], extrinsic field factors [e.g. age of the faecal sample (Piggott 2004, Santini et al. 2007Brinkman et al. 2010), weather conditions (Piggott 2004;Murphy et al. 2007;Brinkman et al. 2010), diet (Murphy et al. 2003) and/or season (Hajkova et al. 2006)] are usually difficult to control in non-invasive field surveys (Beja-Pereira et al. 2009;Monterroso et al. 2012). Also, the considerable remaining variability among studies implies that other unidentified parameters are acting (Broquet et al. 2007;Monterroso et al. 2012). ...
... Effect of sample collector field experience on species identification (mtDNA) and genotyping success (nDNA) of faecal samples Faecal samples could potentially result in highly variable DNA quality and quantity, resulting in different genotyping success and error rates (Beja-Pereira et al. 2009). As already suggested by previous non-invasive genetic studies conducted on carnivore species (Piggott 2004;Murphy et al. 2007;Santini et al. 2007), success rates are greatly improved by the use of very fresh samples. ...
The use of non-invasive genetic sampling (NGS) has become increasingly popular in wildlife research but needs well-planned sampling strategies and reliable laboratory protocols. In this study, we planned to assess the reliability and success of species and individual identifications of sympatric martens (European pine marten Martes martes and stone marten Martes foina) by genotyping non-invasively collected faecal samples. First, we developed a novel and accurate multiplex panel of 15 microsatellite loci, selected by cross species amplification of 41 loci. The application of this panel facilitated species distinction, discarding the presence of pu-tative hybrids. Then, we assessed the impact of sample collector skills on the lab protocol performances. The faecal DNA quality was evaluated by (a) the success of polymerase chain reaction-restriction fragment length polymorphism identification of the two Martes species and (b) the genotyp-ing success and error rates of individual pine marten identifications. The survey was conducted over all the sympatric range of the two species in the Iberian Peninsula by three groups of sample collectors with different experience: expert wildlife biologist, trained volunteers and technical staff from natural parks. Results show that the different expertise between sample collectors significantly influences the success rate of pine marten individual genotyping, but not the species identification success rate. Based on our results, we recommend conducting sampling by experienced field biologist to maximise the quality of NGS and ensure accurate genotyping success. Application of our methods to field collected scats can be used in a cost-effective way to investigate distribution, patterns of genetic diversity and structure as well as to estimate population abundance for sympatric martens.
... It exclusively relies on residual remnants from an animal's life-cycle, such as excreted feces, shed feathers, or similar sources (Faria et al. 2011). This method opens a new pathway for genetic diversity research (Beja-Pereira et al. 2009, Carroll et al. 2018, Baus et al. 2019, Fuwen et al. 2021. However, it is critical to note that even when feathers are directly acquired from live animals or their specimens, it introduces a level of disruption, hence should be classified under NDS, not NIS (Taberlet et al. 1999). ...
... Consequently, this case study on NIS endeavors to surmount the challenge of DNA sampling in endangered butterflies. It will facilitate the research facets within conservation genetics and molecular ecology (Beja-Pereira et al. 2009). ...
The butterfly genus of Teinopalpus, endemic to Asia, embodies a distinct species of mountain-dwelling butterflies with specific habitat requirements. These species are rare in the wild and hold high conservation and research value. Similar to other protected species, the genetic analysis of the rare Teinopalpus aureus poses challenges due to the complexity of sampling. In this study, we successfully extracted DNA and amplified mitochondrial genomic DNA from various noninvasive sources such as larval feces, larval exuviae, larval head capsules, pupal exuviaes, and filamentous gland secretions, all integral parts of butterfly metamorphosis. This was conducted as part of a research initiative focused on the artificial conservation of T. aureus population in Jinggang Shan Nature Reserve. Our findings illustrated the successful extraction of DNA from multiple noninvasive sources, achieved through modified DNA extraction methodologies. Although the DNA concentration obtained from noninvasive samples was lower than that from muscle tissues of newly dead larvae during rearing, all samples met the requirements for PCR amplification and sequencing, yielding complete circular sequences. These sequences are pivotal for both interspecific and intraspecific genetic relationship analysis. Our methods can be extended to other insects, especially scarce species.
... moults). [20][21][22][23] Although non-invasive sampling is ideal in terms of the 3Rs, it is not always achievable as organismal traces may be more difficult to locate than the organism in question, and the sample is most likely of lower quality Tips for tips: Swabbing versus clipping for DNA retrieval Page 2 of 9 than tissue directly removed from a living animal. 24 Although some minimally disruptive techniques -such as extracting DNA from urine 25 or saliva 26 -could offer alternatives to DNA sampling, these methods also have drawbacks. ...
... The quality of these sample types varies widely and alterations to standard protocols are often needed to ensure adequate DNA retrieval. 22,23 One alternative method sometimes proposed for DNA sampling of large to moderately sized lizards is buccal swabbing. 13,14,27 This process includes insertion of a sterile swab into the mouth of the live animal, followed by moderate rotation of the swab to sample epithelial cells from the oral environment. ...
In biodiversity research, the retrieval of genetic material from organisms is a common and essential component for assessing genetic diversity. The welfare of the organism, however, needs to be balanced against the overall goal of the intended research. One sampling technique often applied to retrieve DNA material from small reptiles is the removal of a small portion of the distal end of the tail. While most squamate reptiles have tail autotomy, some species (e.g. many iguanid lizards and snakes) do not regenerate tail tissue. We therefore explored the efficacy of a minimally disruptive technique, buccal swabbing, as an alternative to tissue sampling via tail clipping, particularly for species without tail autotomy, using dwarf chameleons (Bradypodion spp.) as a case study. The two sampling techniques were compared to assess the efficacy of DNA retrieval. We also evaluated the financial implications of each technique. The results indicate that buccal swabs paired with a specialised DNA extraction kit offer a feasible (although expensive), once-off alternative to tissue sampling, but with no material left for biobanking. Deviations in swab type used and the DNA extraction process (i.e. using more affordable extraction procedures) resulted in poor DNA retrieval and unreadable sequences. This finding suggests that buccal swabbing can be a suitable alternative when finances are not constrained, an expensive extraction kit is available, and biobanking is not a concern. For researchers from low- to middle-income economies, this expensive alternative may hamper research progress by placing a financial obstacle in the way, and therefore the next best option is tissue sampling.
... Thus, these markers may be used to compare populations in terms of genetic diversity, since they are not affected by evolutionary pressures (Selkoe and Toonen 2006). Microsatellite regions are generally short and, therefore, their PCR products are also short, which makes them possible to be used with noninvasive samples (Beja-Pereira et al. 2009). Furthermore, with the use of a multilocus panel (Broquet et al. 2006;Beja-Pereira et al. 2009), researchers can identify the individual source of each sample, allowing for the determination of its origin when it is too difficult to obtain such information in the field. ...
... Microsatellite regions are generally short and, therefore, their PCR products are also short, which makes them possible to be used with noninvasive samples (Beja-Pereira et al. 2009). Furthermore, with the use of a multilocus panel (Broquet et al. 2006;Beja-Pereira et al. 2009), researchers can identify the individual source of each sample, allowing for the determination of its origin when it is too difficult to obtain such information in the field. More recently, due to advances in next-generation sequencing (NGS) technologies, the use of other nuclear markers, such as SNPs (Single Nucleotide Polymorphisms), has been made easier. ...
Molecular techniques have emerged as powerful tools to study ecological aspects of biodiversity. As a result, the interdisciplinary field of Molecular Ecology was created, combining a wide range of strategies in order to address ecological questions, which may involve molecular species confirmation, species occurrence and distribution, demography estimates, relatedness among individuals, intra and interspecific interactions, dispersal patterns, sex ratio, as well as other ecological information. In this chapter, we provide an overview of how molecular techniques have been contributing to answering ecological questions about neotropical mammals, using various sources of biological samples (including non-invasive sampling) and the most appropriate molecular markers to achieve each objective. Mammals constitute one of the most threatened taxa due to the direct and indirect environmental effects of human activity, and therefore ecological information must be obtained about this group for the proposal of conservation strategies.KeywordsSpecies monitoringMitochondrial DNAMicrosatellitesSex identificationPhilopatry
... Here, the combination of non-invasive sampling of exuviae and molecular genetic methods offers great potential and is essential when studying rare and endangered species (Beja-Pereira et al. 2009;Lefort et al. 2022). In general, DNA barcoding provides a reliable, inexpensive genetic tool for identification of species. ...
... While invasive genetic sampling could negatively affect populations, especially small ones, non-invasive genetic approaches allow to study wildlife populations without harming them (Lefort et al. 2022). However, despite the tempting advantages of non-invasive genetic approaches and continuous technological advances (Beja-Pereira et al. 2009;De Barba et al. 2017;Carroll et al. 2018;von Thaden et al. 2020), certain limitations regarding the utilization of left-behind animal material (collected in the field) as DNA source remain. DNA isolated from such samples is typically characterized by low DNA quantity and quality (degraded DNA), and the presence of inhibitors (Taberlet et al. 1999), which pose a risk of high sample dropout rates and erroneous results. ...
Monitoring of odonates has become an important instrument for ecological status assessment of (semi-)aquatic habitats. Besides information on presence and abundance, knowledge about a species´ autochthony at the surveyed waterbody is a significant information within the assessment process. Here, the finding of exuviae represents the ultimate proof of successful reproduction. Although feasible for most odonate species, morphological identification of exuviae is often time consuming, as it relies on small, fragile structures. To facilitate species identification of exuviae, a DNA barcoding approach was developed, including (1) non-destructive extraction of DNA using whole exuviae or their tracheal tubes, and (2) primer systems for long (< 600 bp) and short (< 200 bp) CO1 fragments. A total of 85 exuviae from 33 species were analysed and compared to results of morphological identification. Additionally, factors potentially influencing DNA quality and quantity, as well as PCR and sequencing success were investigated. Eighty-two exuviae matched the morphologically identified genus, and 60 matched at species level. Of the 33 species present in the data set, 82% could be identified to species level via DNA barcoding. The results show how DNA-based approaches can support fast and accurate species identification and therefore enhance monitoring of an ecologically important taxonomic group, with high relevance for conservation and habitat restoration. Moreover, the use of exuviae as DNA resource once more shows that non-invasive sampling offers great potential for molecular species identification, which is essential when studying rare and endangered species.
Implications for insect conservation
Our results show how molecular tools, here DNA barcoding of odonate exuviae, can support species monitoring without the need of catching individuals, harming, or even killing them. Obtaining DNA from non-invasive sources can thus be a direct advantage to the conservation of insects, especially when dealing with rare and endangered species and/or populations. Using the example of odonates as bioindicator organisms for aquatic and semi-aquatic habitats, we highlight the importance of non-invasive genetic approaches for population studies and monitoring of insect species and/or species communities for ecosystem assessments and conservation management.
... Many wildlife management and conservation agencies lack robust vital rates of carnivore populations due to low detectability or inadequate funding to conduct large-scale monitoring programs. In response, noninvasive genetic sampling (NGS) has been used for over two decades as a cost-effective method to detect rare species, estimate population vital rates, evaluate landscape connectivity, measure disease dynamics, assess dietary niche, and plot spatial organization (Waits and Paetkau 2005, Long et al. 2008, Beja-Pereira et al. 2009). Traditional capture-mark recapture statistical methods have been applied to NGS samples of individually identifiable animals to obtain some of these critical population measures (Otis et al. 1978, Lukacs andBurnham 2005). ...
... We recognize that there may be errors introduced in our study which is well documented across many studies and emanates from the laboratory genotyping analysis (Bonin et al. 2004, Beja-Pereira et al. 2009, Giangregorio et al. 2019 Further, we acknowledge some possible shortcomings of our OSCR model design given results from other SCR studies. The first occurs in our formulation for the estimation of animal activity centers. ...
Widespread habitat loss and overharvest have been identified as major drivers of population declines and range contractions for terrestrial mammalian species. Protected areas, like national parks, are increasingly important as refuges for rare and exploited species, as well as critical natural laboratories that serve as baseline examples of ecological processes largely free from human intervention. Yellowstone National Park (YNP) sits at the center of a 22-million-acre mosaic of federal, state, and privately managed land in northwest Wyoming known as the Greater Yellowstone Ecosystem. The region boasts one of the last nearly intact temperate-zone ecosystems on Earth, providing vital habitat for seven species of native ungulate and seven large predators. We sought to answer fundamental wildlife conservation and management questions pertaining to large predator distributions and behavior. We snow-tracked cougars (Puma concolor) for four consecutive winters to gather noninvasive genetic samples. We applied Bayesian spatial capture-recapture models to estimate abundance, density, apparent survival and recruitment for this cryptic, low-density species. In addition to these noninvasive studies, we captured and GPS-collared cougars and wolves (Canis lupus), a subset of which contained tri-axial accelerometers to monitor instantaneous movement signatures. Using GPS locations from these animals we searched and identified predation events, allowing us to directly link known predatory behavior to accelerometer readings. We compared patterns in seasonal predatory behavior for these sympatric species that utilize divergent social strategies. Finally, we examined nearly twenty years of wolf GPS locations to understand the impact of growing visitation (>4 million human visitors/year) on habitat selection patterns. We identified behavioral shifts away from the road during peak visitation but found increased tolerance by wolf packs exposed to more human visitation. Findings from our work give managers information to effectively monitor population trends for cryptic species, remotely gain insights on fine scale behavioral patterns and circumvent visitor use management issues that may be modifying wildlife behavior.
... This limitation results from the difficulty to detect, capture, and collect samples in the wild, which hinders the collection of robust data for elusive deer [14]. This is a common scenario in rare or evasive species that has been reverted in recent decades due to non-invasive genetic sampling techniques and the use of dogs trained to detect feces [15][16] [17]. ...
... The search for feces in nature and the analysis of fecal DNA has already allowed extensive genetic sampling and distribution studies of forest deer species in Brazil [18] [19][20] [21]. Despite the advantages of using DNA from feces, some characteristics limit its use for genetic studies, such as the presence of inhibitors, which prevent the amplification of genetic material in PCR, and the degradation of DNA, which hinders the amplification of large fragments [17] [22]. ...
Pampas deer, Ozotoceros bezoarticus (Linnaeus, 1758), is found within a wide geographic distribution in Latin America with five subspecies recognized (O. b. bezoarticus, O. b. celer, O. b. uruguayensis, O. b. arerunguaensis and O. b. leucogaster). The limited access to biological material and the lack of genetic structure information, however, hinder the conservation strategies for the species and the management of free-living populations. Therefore, we selected informative regions and developed primers for amplification of small sequences of mitochondrial DNA (Cytochrome-b, Dloop, ND5 and COI) to provide tools for studies using non-invasive genetic sampling. We evaluated the polymorphisms and phylogenetic signal of each region and successfully amplified it in fecal DNA samples of pampas deer from Pantanal. As results we described a set of primers that allow amplified DNA present in any fecal material to be applied in different approaches of conservation genetics. The methodological approach described here will favor genetic analyses of wild populations and highlights the relevance of non-invasive sampling to assist the decision-making and conservation strategies for this species.
... Non-invasive sampling for wildlife survey and monitoring has evolved almost in parallel with the use of camera traps during the past three decades. Overviews of different DNA sources, applications, and limitations for wildlife studies have been published elsewhere (e.g.,Waits and Paetkau 2005, Beja-Pereira et al. 2009, Carroll et al. 2018, Taberlet et al. 2018, Beng and Corlett 2020, Lefort et al. 2022. When done properly, researchers can use DNA from non-invasive samples and appropriate genetic markers to reliably identify species, as well as individuals within a species and their sex. ...
... invasive samples require particularly rigorous efforts in enriching the DNA fragment or region of interest, which is done by Polymerase Chain Reaction (PCR). PCR from non-invasive samples can be prone to several risks and errors that can result in both false negative and false positive errors(Taberlet et al. 1999, Pompanon et al. 2005, Beja-Pereira et al. 2009, Lampa et al. 2013, Lahoz- Monfort et al. 2016). Thus, conducting pilot surveys before initiating large-scale DNA sampling to optimize the protocols is always recommended to assess cost-efficacy trade-offs(Janečka et al. 2008, Wultsch et al. 2015. ...
● Chapter 1: Background concepts and challenges of surveying and monitoring the manul (E. Moqanaki & G. Samelius)
● Chapter 2: Manul signs and sampling locations (E. Moqanaki, A. Barashkova, S. Ross, C. Augugliaro, I. Smelansky, V. Kirilyuk & G. Samelius)
● Chapter 3: Camera trapping (E. Moqanaki, A. Barashkova, C. Augugliaro, I. Smelansky & G. Samelius)
● Chapter 4: Faecal-DNA sampling (E. Moqanaki, B. Weckworth & H.V. Senn)
● Chapter 5: Snow-tracking surveys (A. Barashkova, I. Smelansky, V. Kirilyuk, S. Ross, G. Samelius & E. Moqanaki)
● Chapter 6: Interview surveys (E. Moqanaki, J.S. Alexander & G. Samelius)
... For example, the monitoring of ecologically and culturally valuable fish species such as salmonids can be difficult and costly but is required to support effective fisheries management decisionmaking. Advances in non-invasive sampling for fisheries and wildlife monitoring have broadened the scope of research possibilities and increased capabilities of management agencies [1,2]. In the past two decades, environmental DNA (eDNA) has emerged on the forefront of these non-invasive monitoring techniques for both rare and abundant aquatic species, including many fish species [3][4][5][6][7][8][9]. ...
... The lower/upper confidence limits for LOD-B are shown. 1 ...
A current challenge for environmental DNA (eDNA) applications is how to account for an environmental (or false-positive) background in surveys. We performed two controlled experiments in the Goldstream Hatchery in British Columbia using a validated coho salmon (Oncorhynchus kisutch) eDNA assay (eONKI4). In the density experiment at high copy number, eDNA in 2 L water samples was measured from four 10 kL tanks containing 1 to 65 juvenile coho salmon. At these densities, we obtained a strong positive 1:1 relationship between predicted copy number/L and coho salmon biomass (g/L). The dilution experiment simulated a situation where fish leave a pool environment, and water from upstream continues to flow through at rates of 141–159 L/min. Here, three coho salmon were placed in four 10 kL tanks, removed after nine days, and the amount of remaining eDNA was measured at times coinciding with dilutions of 20, 40, 80, 160, and 1000 kL. The dilution experiment demonstrates a novel method using Binomial–Poisson distributions to detect target species eDNA at low copy number in the presence of an environmental background. This includes determination of the limit of blank with background (LOB-B) with a controlled false positive rate, and limit of detection with background (LOD-B) with a controlled false negative rate, which provides a statistically robust “Detect” or “No Detect” assessment for eDNA surveys.
... To reveal STR loci profiles, non-invasive samples such as hair or feces can be used and present the advantage of not disturbing the animals [14] and avoiding the use of trapping methods [16]. Nevertheless, some samples may not be amplifiable or may have a higher error genotyping rate than invasive samples (e.g., tissue) because of the DNA degradation caused by field conditions and time [16,17]. In order to avoid genotyping errors, several replicates should be extracted and compared [17][18][19]. ...
... Nevertheless, some samples may not be amplifiable or may have a higher error genotyping rate than invasive samples (e.g., tissue) because of the DNA degradation caused by field conditions and time [16,17]. In order to avoid genotyping errors, several replicates should be extracted and compared [17][18][19]. In two studies using field-collected feces from mammals, one-third of the samples were successfully amplified [20,21]. ...
Landscape fragmentation caused by road infrastructures represents a major threat to the genetic diversity of a region. The resulting genetic isolation between subpopulations may lead to consanguinity, and consequently to population collapse and extinction. However, the construction of wildlife crossings can help maintain connectivity. In the present paper, we evaluated the genetic spatial structuring of populations of wild boars (Sus scrofa) in three areas of the Geneva region connected by an ecological corridor. Those areas are cut off either by a highway that is crossed by a wildlife overpass or by an anthropized sector. Genetic profiling with 9 nuclear microsatellite markers yielded 61 single profiles, which allowed for clustering, parentage, and linkage disequilibrium analyses, uncovering the populations' genetic structure. We also evaluated whether the genetic structure was affected by the sex of individuals. In our analyses, all individuals clustered into a single genetic group, suggesting that no structure limited significantly the gene flow in the region. However, a recent admixture indicated a potential increase in the gene flow between two of the subpopulations due to the wildlife overpass, while the other part of the ecological corridor was not or was only partially functional. Genetic distances between males were significantly higher than between females, although the role of sex remains unclear as to its influence on population genetics. Finally, in order to avoid a subregion becoming fully isolated, urbanization planning should consider this genetic evaluation and proceed with further monitoring, especially by focusing on species more sensitive to landscape fragmentation.
... Noninvasive samples are left behind by an organism and are collected without having to catch, disturb, or sometimes even observe individuals, while minimally invasive samples are collected often through capture but via less invasive methods than blood or tissue extraction (Beja-Pereira et al., 2009;Carroll et al., 2018;Taberlet et al., 1999;Waits & Paetkau, 2005). Due to their tendency to yield low DNA quality and quantity, noninvasive and minimally invasive samples are not the best options for many next-generation sequencing approaches (Andrews et al., 2018; but see Russello et al., 2015). ...
Minimally invasive samples are often the best option for collecting genetic material from species of conservation concern, but they perform poorly in many genomic sequencing methods due to their tendency to yield low DNA quality and quantity. Genotyping‐in‐thousands by sequencing (GT‐seq) is a powerful amplicon sequencing method that can genotype large numbers of variable‐quality samples at a standardized set of single nucleotide polymorphism (SNP) loci. Here, we develop, optimize, and validate a GT‐seq panel for the federally threatened northern Idaho ground squirrel (Urocitellus brunneus) to provide a standardized approach for future genetic monitoring and assessment of recovery goals using minimally invasive samples. The optimized panel consists of 224 neutral and 81 putatively adaptive SNPs. DNA collected from buccal swabs from 2016 to 2020 had 73% genotyping success, while samples collected from hair from 2002 to 2006 had little to no DNA remaining and did not genotype successfully. We evaluated our GT‐seq panel by measuring genotype discordance rates compared to RADseq and whole‐genome sequencing. GT‐seq and other sequencing methods had similar population diversity and FST estimates, but GT‐seq consistently called more heterozygotes than expected, resulting in negative FIS values at the population level. Genetic ancestry assignment was consistent when estimated with different sequencing methods and numbers of loci. Our GT‐seq panel is an effective and efficient genotyping tool that will aid in the monitoring and recovery of this threatened species, and our results provide insights for applying GT‐seq for minimally invasive DNA sampling techniques in other rare animals.
... Luckily, a partnership with two long-term projects focused on monitoring giant armadillo and roadkills (Giant Armadillo Conservation Program and Anteaters and Highways Project, respectively), as well as a third project focused on ecology in a protected area (Parque Nacional das Emas) allowed us to obtain tissue samples of this rare animal for a very first population genetics study of this threatened species. In turn, microsatellites have been used for genetic studies on large number of metazoans worldwide during the two last decades (reviewed in mammals in Túnez et al., 2021), uncovering a wealth of ecological information concerning mammal species (Broquet et al., 2006;Beja-Pereira et al., 2009;Saranholi et al., 2023). Because microsatellites were absent for the giant armadillo, this study describes an initial panel of 15 microsatellites obtained through next-generation sequencing, which can be very helpful in studies of population genetics, as well as for assessing paternity and kinship. ...
The progressive fragmentation and loss of habitats represent the main threats for endangered species, causing genetic consequences that may have potential implications for a population’s long-term persistence. Large mammals are the most affected species among vertebrates. The giant armadillo Priodontes maximus is a large South American mammal threatened species, showing nocturnal, solitary and fossorial behavior, occurring at low population densities, and its population dynamics are still poorly known. In this study, we carried out the first assessment of genetic variability and population genetic structure of the species, using a panel of 15 polymorphic microsatellites developed by high-throughput genome sequencing. The spatial Bayesian clustering, Fst and Dest results indicated the presence of two genetic clusters (K = 2) in the study area. These results suggest a reduction in gene flow between individuals inhabiting the Brazilian savanna (Cerrado) and the Pantanal wetlands, with the increased human-driven habitat modifications possibly contributing for this scenario. A bottleneck signal was detected in both populations, and a subpopulation structuring in the Cerrado may also be reflecting consequences of the extensive habitat modifications. Findings from this study provide important and useful information for the future maintenance of genetic diversity and long-term conservation of this flagship species.
... In spite of that, we believe that it is possible to use this method for DNA sampling. For example, in the analysis of short DNA fragments, there are strategies to deal with low-quality DNA and minimize genotyping errors, including techniques for extracting, preserving, amplifying, and analyzing DNA data (Beja-Pereira et al., 2009;Carroll et al., 2018;Lukacs & Burnham, 2005;Taberlet et al., 1997). ...
The great loss of biodiversity and population declines include mammals as one of the most vulnerable groups worldwide. It is therefore important to monitor species populations, and several methodologies have been designed and improved to obtain information indirectly, with non-invasive or minimally invasive approaches. Our objective was to evaluate the efficiency of two different hair capture methodologies, hair traps and active search, for peccary hair sampling. This survey was conducted on white-lipped (Tayassu pecari) and collared (Dicotyles tajacu) peccaries in two protected areas in the Atlantic Forest of Southeastern Brazil. Between March and October 2019, sixteen hair traps were installed and monitored in a sampling effort of 3,000 days, and active searches were conducted along trails and dirt roads, covering a total of 822 kilometers. When comparing the two methods, hair traps were more efficient for white-lipped peccaries, but the active search was more efficient for collared peccaries. Although each method was more effective for each peccary species, we believe that both methods complement each other. Furthermore, we recommend some adaptations to make future works even more efficient. Finally, we encourage more studies like this to avoid unnecessary spending of resources and improve the collection of biological material of free-living animals, especially for endangered species.
... Owing to its endangered status on the IUCN Red List of threatened species (Wang and Harris 2015), sampling methods applied to FMD are generally non-invasive and limited to those such as feces and urine collection (Beja-Pereira et al. 2009). Transcriptome sequencing is an effective and feasible method for generating large amounts of sequence data, revealing the type and number of genes expressed by different cells, their potential metabolic pathways, and genetic mechanisms (Jia et al. 2018). ...
Forest musk deer (Moschus berezovskii) is one of the most endangered medicinally important wild animals in the world. Forest musk deer farming is the main way of production of musk. However, the single provenance and lack of genetic information lead to reduced genetic diversity of forest musk deer. Therefore, more SSR markers need to be developed to identify forest musk deer germplasm. In this study, bone marrow derived mesenchymal cells were used to construct cDNA library for transcriptome sequencing. The datasets were de novo assembled and annotated. 9 polymorphic simple sequence repeat (SSR) markers were finally identified and used to detect population genetic diversity. 6.07 Gb clean data were generated using Illumina sequencing technology, and de novo assembled into 138,591 transcripts and 81,553 unigenes. 5,777 simple sequence repeats (SSRs) were identified, in which there were 578 repeating motif types, with mono-nucleotide and tri-nucleotides comprising 55.88% and 25.60%, respectively. 100 primer pairs were designed to validate amplification and polymorphism using DNA from fecal samples. 9 polymorphic SSRs were developed and used to detect population genetic diversity of 122 forest musk deer in 2 farms. The average number of alleles per locus varied from 4 to 15 (average = 8.3). The observed heterozygosity (HO) per locus ranged from 0.102 to 0.941, while the expected heterozygosity (HE) per locus was from 0.111 to 0.651. All loci deviated significantly from the Hardy–Weinberg equilibrium (p < 0.001). The polymorphism information content (PIC) of these loci varied from 0.108 to 0.619. 9 polymorphic SSR markers were developed in this research. These sites can be used for breeding planning and conservation of germplasm resources.
... DNA can persist in the environment for varying periods of time, from hours in temperate waters, to hundreds or thousands of years in cold, dry permafrost (Thomsen and Willerslev 2015). Isolation of this DNA from the environment can facilitate detection of organisms in the absence of obvious signs of the organism's presence, and provide genetic information that can be used to identify, study and/or monitor species/individuals across time and space without having to catch, handle, or in some cases, even observe them (Waits and Paetkau 2005;Schwartz et al. 2007;Beja-Pereira et al. 2009). ...
Environmental DNA (eDNA) sampling has attracted worldwide attention over the past few years as an emerging approach to characterising and monitoring biodiversity, and has become particularly important for species that are rare, elusive or endangered. Most animal studies to date have focused on aquatic taxa; studies on other metazoan taxa, particularly wildlife in terrestrial environments, are scarce, with only a handful utilizing soil sources. We aimed to investigate the use of DNA barcoding from soil eDNA in (1) detecting rare/elusive/threatened species and (2) as a tool to investigate and potentially monitor range distributions. Through extensive eDNA sampling along the west coast of South Africa, we aimed to refine the distributions of four golden mole species thought to occur there, and specifically to determine whether De Winton’s golden mole, Cryptochloris wintoni (IUCN Critically Endangered; Possibly Extinct), is in fact extant or extinct. Sequences were generated for three barcode markers (mtDNA cyt b, 12S and nuclear GHR) using next-generation amplicon sequencing. Tissue samples from four specimens were used to generate reference sequences for species identification, along with available GenBank sequences. We were able to (1) successfully detect all four species in our data, and (2) improve records of the distributions of these species. Furthermore, we uncovered cryptic diversity in Eremitalpa granti. Our data conclusively reveal the presence of the elusive Cryptochloris wintoni and suggest that this species may in fact be widespread, but not necessarily abundant, and certainly less so in areas subjected to mining activities, which continue to pose a threat to the species.
... The recent (mainly genetic) assessments are based solely on mtDNA (e.g., Mori et al. 2019;Li et al. 2020;Joshi et al. 2022). Although mtDNA has some known limitations (Wan et al. 2004;Fišer Pečnikar and Buzan 2014;Ritchie et al. 2016;Norman et al. 2019;Kowalczyk et al. 2021), these surveys are often logical in respect of available comparative gene and taxon sampling and availability from degraded samples, in comparison to nuclear or genomic DNA (Wan et al. 2004;Wandeler et al. 2007;Beja-Pereira et al. 2009). Therefore, various genetic assessments of different organisms still utilize mtDNA (Tobe et al. 2010;Gutiérrez et al. 2017;Kowalczyk et al. 2021;Leigh et al. 2021). ...
Gorals represent ungulate mammals of the Palearctic and Indo-Malayan realms that face habitat destruction and intense hunting pressure. Their classification has been the subject of various (mainly genetic) assessments in the last decade, but some results are conflicting, hampering some conservation-based decisions. Genetic sampling of gorals has increased considerably in recent years, at least for mitochondrial (mt) DNA. Results based on two mt genes (cytochrome b and the D-loop) are currently available. Still, the utility of cytochrome oxidase subunit I remains unanalysed, even though it belongs among the gene markers that enable a correct species identification in mammals. This study examines phylogenetic relationships and species delimitation in gorals using all currently available cytochrome oxidase subunit I sequences, including the not yet analysed goral population from Pakistan. Our results of various phylogenetic approaches, such as maximum parsimony, likelihood and Bayesian inference, and exploration of species boundaries via species delimitation support the validity of six species of goral, namely N. baileyi , N. caudatus , N. cranbrooki , N. evansi , N. goral , and N. griseus . This result accords well with results based on other mt genes, especially the cytochrome b from the highly exhaustive data sampling. Our study also summarises common sources of errors in the assessment of goral phylogeny and taxonomy and highlights future priorities in understanding goral diversification.
... The markers discriminated well among individuals with probability of identity (Pi) and probability of identity for siblings (Pisib) lower than the recommended values (10 −2 − 10 −4 ) (Waits et al. 2001). We also genotyped each sample three times to minimize the error rates (Taberlet et al. 1996;Beja-Pereira et al. 2009) and saw only minor differences (mainly a minimized number of failed alleles), indicating that the marker set used is very reliable (Supplementary Table 2). ...
Genetic monitoring has become a popular instrument in the conservation of endangered species, allowing to estimate size and genetic structure of wild populations. Long-term monitoring projects are essential to recognize demographic changes and impact of human activities. Since 2011, an extensive monitoring project on the population size and trends, as well as spatial distribution and survival rates, of two grouse species including the western capercaillie, Tetrao urogallus, has been conducted in Tyrol, in the eastern part of the European Alps, where T. urogallus males are huntable under specific regulations. In this case study, we aimed to compile a set of analyses to be employed in evaluating data from dropping and feather samples for conservation studies. Using eleven microsatellite and two sex markers, we genotyped 251 faeces and feathers of T. urogallus collected in East Tyrol in spring 2019. We analysed population structure and mobility patterns, including sex differences in genetic diversity and mobility. The relationship between habitat parameters and genetic diversity was investigated using multiple linear regressions. We showed that the investigated T. urogallus population is well mixed and likely well connected to neighbouring populations. We also found sex-specific mobility patterns that support female-biased dispersal. As the last step, we demonstrated the general feasibility of a modelling approach using habitat parameters. With this pilot study, further analysis of data is possible for the whole monitoring project, giving a better insight in the grouse populations in Tyrol.
... Humidity and temperature at the site negatively correlated with the quantity of extracted DNA (Lucchini et al. 2002;Fernando et al. 2003;Nsubuga et al. 2004). In addition, fecal samples contained PCR inhibitors such as polysaccharides, urea, bile salts, and bacteria that may have affected the success rate of amplification (Monteiro et al. 1997;Beja-Pereira et al. 2009;Schrader 2012). Feces are known to be a low-quality source of DNA (Taberlet et al. 1996). ...
Pitimol N, Srikosamatara S. 2023. Population ecology and genetic study of gaur (Bos gaurus) in a small protected area in Thailand. Biodiversitas 24: 3369-3377. Gaur population in Khao Phaeng Ma (KPM) restored forest increased from 6 individuals in 1995 to 1996 in 2006 and more afterward. Data on population sizes, distribution and genetic status are needed for effective management. The aim of this study was to obtain data on gaur population size, structure and broad distribution, and on gaur genetic diversity in KPM. For a one-year period from 2014-2015, data were collected in three areas on gaur number, group size, sex, and age using direct observation. Genetic diversity was examined at 15 cross-species microsatellite loci. The total number of gaurs was at least 184, with these individuals being highly concentrated on the western side of the area. The average group size was 13.15±0.98 (n=356). The proportion of adult males to adult females, juveniles, and calves was 35:100:51:65. From the 10 samples (4 tissue, 6 fecal), the number of alleles per locus ranged from 2 to 6 (average 4.07). The expected and observed heterozygosity were 0.594 and 0.649, respectively. The gaur population is increasing and distributes more at the forest edge. Genetic diversity was lower than gaur populations in Vietnam and India. Habitat management should be done to minimize human-gaur conflicts and increase gene flow between populations.
... Оборудование может быть крупногабаритным и сложным по конструкции, а также содержать металлические и пластиковые детали, которые чувствительны к воздействию кислот и окислителей. УФ излучение эффективно только для открытых поверхностей и не подходит для приборов сложной конструкции из-за неполного удаления фрагментов ДНК, а также обладает разрушающей способностью в отношении пластмассовых материалов при многократном использовании [9,10]. Кроме того, большинство распространенных методов деконтаминации недостаточно эффективно для удаления коротких фрагментов ДНК с низкой молекулярной массой (менее 200 п. ...
Важной проблемой при работе в ПЦР-лаборатории является возникновение перекрестного загрязнения, которое приводит к появлению ложноположительных результатов. Существует множество способов решения данной проблемы, однако ни один из них не является универсальным. Для деконтаминации сложных поверхностей большой площади предпочтительнее использовать аэрозольный метод обработки. Цель данного исследования заключалась в определении эффективных режимов применения дезинфицирующих средств для деконтаминации от нуклеиновых кислот аэрозольным методом. Анализ деконтаминирующей активности хлорактивных и кислородактивных соединений проводили, моделируя контаминацию поверхностей нуклеиновыми кислотами и бактериями. В процессе работы установлены эффективные режимы проведения аэрозольной деконтаминации. Показаны различия при удалении нуклеиновых кислот и бактериального загрязнения с лабораторных поверхностей.
... Genetic tools can allow monitoring and inference of population parameters with high levels of accuracy and often overcome limitations of other techniques. For example, noninvasive genetic sampling allows capture-recapture analyses without observing or handling live animals (Waits and Paetkau 2005;Beja-Pereira et al. 2009;Stenglein et al. 2010;Marucco et al. 2011) and close-kin mark recapture analyses allow estimation of demographic parameters even with lethal sampling (e.g. from harvested individuals) (Bravington et al. 2016a, b;Ruzzante et al. 2019;Trenkel et al. 2022). Additionally, genetic parameters such as effective population size are directly informative to managers. ...
Managing and conserving populations of large carnivores, including gray wolves Canis lupus, often involves making decisions that balance conflicting stakeholder desires. To help inform these decisions, we developed a microhaplotype amplicon sequencing panel consisting of 341 loci. Of these, 321 were selected from candidates identified through restriction-site associated DNA sequencing (RADseq) with 62 samples collected across Idaho, United States. These loci were selected for high expected heterozygosity in order to inform individual identification and relationship inference. The mean ± SD expected heterozygosity of these loci in a sample of 733 individuals collected in Idaho during 2018–2020 was 0.57 ± 0.09. The 20 additional loci were composed of ten selected for differentiation of western coyotes and wolves, nine selected for differentiation of wolves and domestic dogs, and one locus designed to amplify sry to identify genetic sex. This panel will facilitate monitoring, conservation, and management of gray wolves in the western United States.
... The use of noninvasive molecular techniques represents a major advance in environmental biology, especially in biodiversity conservation, through analyses conducted in traces of organisms rather than whole organisms [13]. Among these methodologies, analysis through environmental DNA (eDNA) appears to be of recent interest not only because of its noninvasiveness toward organisms but also because of the reduced costs and time required for sampling [14]. ...
Due to the increasing mass mortality of Pinna nobilis, mainly caused by the protozoan Haplosporidium pinnae along the Mediterranean Sea, it is necessary to develop rapid and effective methods to detect the pathogen. The present study describes the development and validation of a species-specific assay based on hydrolysis probe chemistry to detect H. pinnae DNA from faeces and pseudofaeces of P. nobilis. During a study campaign in the Gulf of Trieste (Italy) in the spring and summer of 2022, 18 samples (10 faeces and 8 pseudofaeces) were collected. DNA was isolated from all samples and the presence of H. pinnae was tested by amplifying a small portion of 18S rDNA using qPCR. The newly developed assay detected positive H. pinnae in the faeces of the fan mussel in the spring, while no evidence of an outbreak of H. pinnae was found in the summer. In addition, the method proved to be noninvasive and can be used to monitor suspected H. pinnae infections in the early stages when bivalves are still vital. Furthermore, fecal analysis allows the monitoring of P. nobilis without dissecting tissues. The presented assay can also be used to routinely monitor the progress of mass mortalities caused by H. pinnae and to screen for the pathogen in live fan mussels and other environmental matrices, such as water, sediment, and faeces from other species that can host the protozoan.
... We removed the entirety of each fecal sample from the field, recorded its geographic coordinates, stored approximately 20 g in 50 ml tubes with 20 g of silica isolated at the bottom using a filter paper barrier, and kept it all frozen. The two fecal conservation methods used (silica drying and freezing) were intended to avoid DNA degradation (Beja-Pereira et al. 2009). ...
Habitat use data are key to understanding species ecology and extinction risk. However, such information is lacking for the elusive deer species of Neotropical region. In this context, fecal sampling has emerged as an alternative tool, in which development and evaluation are essential to obtaining unbiased ecological data. We aimed to compare data from GPS-tracked animals and fecal sampling using scat detection dogs to evaluate the noninvasive performance of this method in habitat selection analysis. We carried out the study in the Brazilian Pantanal, where we monitored six free-living Gray Brocket Deer (Mazama gouazoubira) with GPS collars for 1 year (average of 584 GPS locations/animal) and collected fecal samples (n = 649) simultaneously along a set of transects designed for a scat detection dog survey. We evaluated habitat selection using the chi-square test in an availability/utilization analysis and submitted both data to a bootstrap procedure to assess its precision and accuracy with increasing sample size. GPS data indicated habitat selection at a fine utilization scale, in which savanna and cerrado were preferred and open grassland habitat was avoided. Exclusive fecal sampling also indicated habitat selection, revealing the preference for cerrado and avoidance of open grassland. The GPS and fecal habitat utilization estimates did not differ significantly and fecal sampling increased precision and accuracy with increased sample size, reaching minimal values once n = 200 which should be considered a sufficient survey effort. The similarity between the two methods suggested the reliability of fecal sampling, as long as a standardized sampling design is used. This noninvasive sampling framework can provide previously unavailable ecological data for threatened Neotropical deer as well as other elusive species.
... In order to improve monitoring efforts in fisheries, approaches that supplement traditional techniques with the detection of environmental DNA (eDNA), the genetic material an individual releases into their environment as skin cells, gametes, mucus, blood, excrement (feces), etc., are being increasingly studied and implemented (as reviewed in Rourke et al., 2021;Thomsen & Willerslev, 2015;Wang et al., 2021). eDNA approaches can remedy some of the shortcomings of traditional methodologies, as they may be more sensitive for detection of low density and/or elusive species (Furlan et al., 2019;Sard et al., 2019), are less stressful to both the environment and live organisms in comparison to live-capture methods (Beja-Pereira et al., 2009;Lodge et al., 2012), and can be less labor and resource intensive in some settings or applications (Evans et al., 2017;Sigsgaard et al., 2015). eDNA, however, cannot be used to replace certain types of data critical to stock assessments, such as age structure and condition factors, so an approach which compliments traditional methods with eDNA monitoring may be best suited to achieve comprehensive management improvements. ...
Estimating population size and species distribution is essential for fisheries management and conservation. Traditionally, estimates rely on live‐capture and visual surveys; however, these approaches are challenging for low density or elusive species and sensitive habitats. Environmental DNA (eDNA) has shown potential to improve fisheries management, offering a sensitive tool for species detection while reducing some of the unintended harm, uncertainties, and cost of traditional approaches. For eDNA to be incorporated into quantitative population estimates, variability in factors such as DNA shedding and decay must be understood. We assess shedding and decay rates for three developmental stages of muskellunge (Esox masquinongy), a recreational species of social and economic importance and an apex predator of conservation priority in the St. Lawrence River. By housing fish at different biomass levels, we attempt to assess how crowding, and underlying behavioral and/or metabolic responses, affects shedding and decay. Additionally, we collected water from spawning bays and compared eDNA detections to live‐capture data. Total eDNA shedding rates for muskellunge were similar to values reported in previous studies of freshwater fishes and ranged from 9.92 × 103 copies/h/fish for larvae to 1.32 × 106 copies/h/fish for juveniles. Adjusting shedding rates for fish mass revealed no significant difference between larvae and juveniles. eDNA decay rates varied between life stages and experimental aquaria, with coefficients ranging from 0.064 to 0.259. We were unable to detect eDNA from hardened embryos even at high density. Muskellunge DNA was quantified in over 27% of samples from spawning bays, including bays where muskellunge were not captured with traditional approaches. The recovery of muskellunge eDNA in quantifiable levels, often in the absence of live capture, highlights the potential for eDNA approaches to supplement muskellunge monitoring and management. eDNA shedding and decay rates estimated here may also aid in interpretation of future data. Environmental DNA shedding and decay rates were estimated for muskellunge at three distinct developmental stages. Preliminary field trials detected muskellunge eDNA from five spawning bays in the St. Lawrence River, including bays in which live capture attempts were unsuccessful. Our findings indicate the potential for eDNA methods to improve muskellunge monitoring programs and the shedding and decay rates estimated here lay the groundwork for future quantitative interpretations of muskellunge eDNA data." cd_value_code="text
... Оборудование может быть крупногабаритным и сложным по конструкции, а также содержать металлические и пластиковые детали, которые чувствительны к воздействию кислот и окислителей. УФ излучение эффективно только для открытых поверхностей и не подходит для приборов сложной конструкции из-за неполного удаления фрагментов ДНК, а также обладает разрушающей способностью в отношении пластмассовых материалов при многократном использовании [9,10]. Кроме того, большинство распространенных методов деконтаминации недостаточно эффективно для удаления коротких фрагментов ДНК с низкой молекулярной массой (менее 200 п. ...
Cross-contamination that leads to false positive results is a serious problem for laboratories using the PCR method. There are many ways to solve this problem, but none of them could be considered universal. Treatment with aerosols is the preferable method for decontamination of large areas of complex surfaces. The goal of this study was to determine effective aerosol compositions and regimens for the decontamination of nucleic acids on laboratory surfaces. The decontaminating activity of compounds that release active chlorine and active oxygen was studied using model surfaces contaminated with nucleic acids and bacteria. Effective modes of decontamination with aerosols were established by analysis of obtained experimental data. Differences between decontamination of nucleic acids and bacterial disinfection of the laboratory surfaces are shown.
... DNA marker analysis is possible with DNA extracted from feces, feathers, and hairs which can be collected noninvasively from animals (Rudnick et al., 2007;Brinkman and Hundertmark, 2009;Barba et al., 2010;Brinkman et al., 2011). DNA extracted from non-invasively sampled feces or other objects left by individuals allows researchers to collect information on animals without directly observing the individuals of interest (Beja-Pereira et al., 2009). This is particularly useful for studies of animals that are difficult to capture or observe. ...
DNA markers that detect differences in the number of microsatellite repeats can be highly effective for genotyping individuals that lack differences in external morphology. However, isolation of sequences with different microsatellite repeat numbers between individuals has been a time-consuming process in the development of DNA markers. Individual identification of Japanese giant flying squirrels (Petaurista leucogenys) has been challenging because this species is arboreal and nocturnal and exhibits little to no morphological variation between individuals. In this study, we developed DNA markers for sex and individual identification of this species by an efficient method using high-throughput DNA sequence data. Paired-end 5 Gb (2 × 250 bp) and 15 Gb (2 × 150 bp) genome sequences were determined from a female and a male Japanese giant flying squirrel, respectively. We searched SRY and XIST genes located on Y and X chromosomes, respectively, from high-throughput sequence data and designed primers to amplify these genes. Using these primer sets, we succeeded to identify the sex of individuals. In addition, we selected 12 loci containing microsatellites with different numbers of repeats between two individuals from the same data set, and designed primers to amplify these sequences. Twenty individuals from nine different locations were discriminated using these primer sets. Furthermore, both sex and microsatellite markers were amplified from DNA extracted non-invasively from single fecal pellet samples. Based on our results for flying squirrels, we expect our efficient method for developing non-invasive high-resolution individual- and sex-specific genotyping to be applicable to a diversity of mammalian species.
... The obtained results expand the range of application of the biomonitoring of heavy metal contaminations and ecological status of ecosystems in special protected areas where the killing of animals is unacceptable. In this case, the procurement of hair material is performed by a non-invasive method, which is already widely used in the ecological and genetic studies of wildlife (Beja-Pereira et al. 2009, Waits & Paetkau 2005 and especially for examination of carnivores (Patkó et al. 2016). ...
The aim of the study was to check the current load of the hair of red fox (Vulpes vulpes), inhabiting the forest mountain region of northwest Rila Mountains, southwestern Bulgaria, with non-essential metals. We determined the content of trace metals with proven highly toxic effect on living organisms (Cd and Pb) as well as essential metals with concentration-dependent toxic effects (Cu, Zn, Ni, Co and Mn). Further, we explored whether these trace metals could be used for the purpose of environmental biomonitoring in Bulgaria. The concentrations of the elements were expressed as mg/kg of dried weight. The mean values of the average residual content of the studied heavy metals in hair of foxes were: Pb 4.65, Cd 0.20, Cu 13.05, Zn 149.53, Ni 0.87, Co 0.24 and Mn 6.34. The established basic statistical parameters and the ±95 % confidence interval of the residuals of the studied elements in the red fox hair enlarge the knowledge of this species as a zoomonitor to assess the state of the environment. The obtained results expand its range of applications for biomonitoring of the ecological status of ecosystems in specially protected areas, where the killing of the studied individuals is unacceptable.
... Improvements in molecular technology and laboratory methods over the last decade have facilitated the collection and analysis of large-scale genetic data sets [9,[11][12][13] enabling comprehensive research efforts for wide-ranging species. An array of analytical tools exists to examine population genetic structure in wild populations including both population and individualbased methods [14] as well as those that include spatial information and those that do not [15]. ...
Characterizing genetic structure across a species’ range is relevant for management and conservation as it can be used to define population boundaries and quantify connectivity. Wide-ranging species residing in continuously distributed habitat pose substantial challenges for the characterization of genetic structure as many analytical methods used are less effective when isolation by distance is an underlying biological pattern. Here, we illustrate strategies for overcoming these challenges using a species of significant conservation concern, the Greater Sage-grouse ( Centrocercus urophasianus ), providing a new method to identify centers of genetic differentiation and combining multiple methods to help inform management and conservation strategies for this and other such species. Our objectives were to (1) describe large-scale patterns of population genetic structure and gene flow and (2) to characterize genetic subpopulation centers across the range of Greater Sage-grouse. Samples from 2,134 individuals were genotyped at 15 microsatellite loci. Using standard STRUCTURE and spatial principal components analyses, we found evidence for four or six areas of large-scale genetic differentiation and, following our novel method, 12 subpopulation centers of differentiation. Gene flow was greater, and differentiation reduced in areas of contiguous habitat (eastern Montana, most of Wyoming, much of Oregon, Nevada, and parts of Idaho). As expected, areas of fragmented habitat such as in Utah (with 6 subpopulation centers) exhibited the greatest genetic differentiation and lowest effective migration. The subpopulation centers defined here could be monitored to maintain genetic diversity and connectivity with other subpopulation centers. Many areas outside subpopulation centers are contact zones where different genetic groups converge and could be priorities for maintaining overall connectivity. Our novel method and process of leveraging multiple different analyses to find common genetic patterns provides a path forward to characterizing genetic structure in wide-ranging, continuously distributed species.
... While faecal microbiota profiling can be a powerful tool in humans and animals alike (Turjeman & Koren, 2021), trapping and handling animals poses challenges to conversation research. Often, animals are difficult to trap, efforts can be cost-and resourceprohibitive, and many species do not withstand the stress of trapping well (Beja-Pereira et al., 2009). Further, stress associated with trapping and handling can affect the microbiome (Collins & Bercik, 2009), even if it does not seem to visibly harm the target species, thus skewing findings. ...
In ecological and conservation studies, responsible researchers strive to obtain rich data while minimizing disturbance to wildlife and ecosystems. We assessed if samples collected noninvasively can be used for fecal microbiome research, comparing microbiota of noninvasively collected fecal samples to those collected from trapped common cranes at the same sites over the same periods. We found significant differences in fecal microbial composition (alpha and beta diversity), which likely did not result from noninvasive samples’ exposure to soil contaminants, as assessed by comparing bacterial oxygen use profiles. Differences might result from trapped birds’ exposure to sedatives or stress. We conclude that if all samples are collected in the same manner, comparative analyses are valid, and noninvasive sampling may better represent host fecal microbiota because there are no trapping effects. Experiments with fresh and delayed sample collection can elucidate effects of environmental exposures on microbiota. Further, controlled tests of stressing or sedation may unravel how trapping affects wildlife microbiota.
... Ludders et al. (2001) previously validated noninvasive research methods for sandhill cranes and showed that it is feasible. This non-invasive sampling study has solved many problems that cannot be solved due to the limitation of traditional sampling and has been widely used in various fields of ornithological research, including stress physiology (Liu et al., 2018), metabolic physiology (Wasser et al., 2010), genetic diversity (Bejapereira et al., 2009), reproductive physiology (Krepschi et al., 2013;Penfold et al., 2013) and animal immunity studies (Liu et al., 2018). In the present study, using non-invasive methods of stool research, the reproductive physiological characteristics of black-necked cranes were investigated during different reproductive stages to provide a theoretical basis for parameter measurements and procedure timing during the artificial breeding of black-necked cranes. ...
Black-necked cranes (Grus nigricollis) are national first-level protected wild animals in China. Artificial breeding has been adopted by many zoos and reserves to achieve ex-situ conservation of black-necked cranes, but the breeding rate of the species in cages is low. This study used non-invasive methods combined with behavioural observations to investigate changes in sex hormones and glucocorticoid metabolites in the droppings of black-necked cranes during the breeding cycle, with the results showing that (i) levels of estradiol and testosterone in black-necked cranes increased significantly when they entered the breeding period, and these levels could be used as an important physiological indicator to effectively monitor the physiological status of females and males during the reproductive period, thus providing a theoretical basis for the timing of semen collection; (ii) the level of progesterone in the mid-reproduction stage was significantly higher than that in other stages in female black-necked cranes after successful mating, and this level could be an effective indicator of the mating status of female black-necked cranes; (iii) droppings’ glucocorticoid metabolites in the breeding period showed different dynamics between paired and singly caged black-necked cranes, indicating that the physiological phenomenon of reproduction could result in a certain amount of physiological burden on black-necked cranes. These results provide a theoretical basis for the selection of physiological parameters in the artificial breeding of black-necked cranes.
... The samples can then only be processed afterwards, which can be problematic when individuals of a particular sex need to be targeted (e.g. in tracking studies). This technique is also costly as samples need to be analysed in a laboratory by trained professionals (Beja-Pereira et al. 2009;Volodin et al. 2015). Therefore, the use of a more time-efficient and non-invasive technique for sexing seabirds in the field is desired, such as through their call characteristics (Bourgeois et al. 2007;Kriesell et al. 2018). ...
Vocalisations play a vital role in animal communication, as they are involved in many biological functions such as mate selection, individual recognition and care of young. Seabirds often breed in large and dense colonies, making successful recognition between mates or between parents and offspring crucial for reproductive success. Acoustic signals have been shown to play an important role in this regard for several seabird species. Furthermore, most seabird species, including the Cape Gannet Morus capensis, are monomorphic, making sex identification for research challenging. Identifying individual and sexual signatures in their vocal productions could thus facilitate sex identification in the field. This study aimed to better understand the potential use of vocalisations for sex and individual recognition in Cape Gannets by describing the acoustic structure of their display calls at the nest. Vocalisations of nesting Cape Gannets were recorded over a two-week period. Acoustic measurements were extracted from 80 calls (16 individuals) and included 36 variables in both temporal and frequency domains. Twenty acoustic variables showed significant differences in vocalisations between male and female Cape Gannets. However, values of the fundamental frequency and the average of Inter-Onset-Interval (time elapsed between successive sound units) appeared to be the most important acoustic variables for sex determination. Both temporal and frequency parameters showed a potential for individual identity coding, again with the average units’ Inter-Onset- Interval being the most important variable for individual identification for both females and males. This study provides the first evidence of sex-specific and individual vocal signatures in adult breeding Cape Gannets enhancing our understanding of the role of the display calls in mate recognition. From an applied perspective, identified sex-specific differences could potentially be used as a non-invasive method for field-based sex-determination.
... Aussagen zur deren Bestandesdichten können mithilfe verschiedener Methoden getroffen werden. Die Kotgenotypisierung als nicht-invasives Verfahren der Bestandesschätzung hat den Vorteil, dass sie die indirekte Erfassung der Tiere und die Genauigkeit und Präzision der Fang-Wiederfangmethode kombiniert (McKelvey & Schwartz 2004, Beja-Pereira et al. 2009). Für den Damhirsch und Rothirsch sollten nun erstmals belastbare Daten zur Bestandesdichte mithilfe dieser Methode ermittelt werden. ...
Mit dem Ziel einer Bestandesschätzung wurden im Frühjahr 2018 Kotproben entlang
von 101 Transekten vom Damhirsch und Rothirsch im Nationalpark (NLP) Hainich
gesammelt und genetisch analysiert. Hierfür wurde eine Unterstichprobe von insgesamt
1.200 Kotproben gezogen und diese unter Anwendung von acht (Rothirsch) bzw. 16
(Damhirsch) Mikrosatelliten genotypisiert. Im Ergebnis konnten für den Damhirsch
insgesamt 120 unterschiedliche Individuen, von denen 26 männlich und 92 weiblich
waren, identifiziert werden. Bei zwei Individuen war der Geschlechtsmarker nicht auswertbar. Während der genetischen Analysen zeigte sich, dass die genetische Variabilität der Damhirschpopulation im NLP Hainich sehr gering ist. Zudem wurden nur wenige Individuen mehrfach erfasst, sodass eine Bestandesschätzung für den Damhirsch
nicht durchgeführt werden konnte. Beim Rothirsch hingegen wurden 277 Individuen,
davon 104 männlich, 161 weiblich und 12 mit nicht auswertbarem Geschlechtsmarker,
nachgewiesen. Mit einer Mehrfacherfassungsrate von 46 % und der Anwendung eines
räumlich expliziten Fang-Wiederfang-Ansatzes konnte eine Bestandesschätzung durchgeführt werden. Die geschätzte Dichte des Gesamtbestandes beim Rothirsch entspricht etwa 3,4 Individuen/ 100 ha im NLP Hainich. Die Verteilung der mehrfach erfassten Proben beim Rothirsch deuten zudem daraufhin, dass die Art nicht nur den NLP, sondern auch angrenzende Waldbereiche als Lebensraum nutzt.
... This corridor comprises two major forested blocks, the Paranapiacaba (inland region) and Serra do Mar (coastal region) mountain ranges ( Fig. 1), ranging from 0 to 1200 m above sea level (Table S1). We used non-invasive sampling (fecal samples) associated with molecular tools largely employed in mammal genetic studies (Beja-Pereira et al., 2009;Rodgers and Janečka, 2013;Saranholi et al., 2017) to obtain genetic data. A total of 125 fecal samples were collected (Table S2) by actively searching for lowland tapir latrines on pre-existing trails during 2007-2011. ...
Forest corridor has been considered the main strategy for maintaining gene flow between isolated populations, yet their effectivity is poorly tested. Assessing signatures of genetic variation loss, gene flow reduction and inbreeding may be helpful for conservation of the biodiversity that needs large continuous areas. Here we evaluated the genetic structure and diversity of the largest neotropical mammal, the lowland tapir (Tapirus terrestris), living in the largest Atlantic forest corridor in Brazil. We used fecal-derived DNA, genotyped nine polymorphic microsatellite loci of 75 tapirs, and quantified genetic differentiation, genetic diversity, and landscape resistance to gene flow. We found genetic differentiation between the inland and coastal populations, which may be explained by elevation. Expected heterozygosity ranged between 0.64 (inland population) and 0.78 (coastal population), and a small Ne was observed in both populations. We demonstrated that even large continuous rainforests are not totally permeable to the gene flow of large organisms. Our study also changes our perception about the pristine of continuous corridors and their role for long-term survival of large mammals, suggesting that tapir conservation efforts should be taken even for populations in the large protected areas.
Full text available: https://www.sciencedirect.com/science/article/pii/S2530064422000128
Non-invasive genetic analysis allows for monitoring and understanding population dynamics without disturbing wild animals. We present a non-invasive genetic method to identify and characterize the Cantabrian and Pyrenean (of Slovenian origin) brown bear populations. We selected an efficient panel of 61 SNPs to genotype more than 2,000 non-invasive samples from both populations. Results showed successful genotyping of 1,639 bear samples, revealing 400 different individuals. Genetic diversity was similar in both populations and differentiation between populations was highly significant. The Pyrenean population did not show genetic substructuring despite the influence of the breeding male “Pyros”. In contrast, two subpopulations are observed in the Cantabrian population. Moreover, analysis indicated a sex ratio bias in the Cantabrian population, potentially influenced by male dispersal and landscape features. Overall, the study demonstrates the utility of non-invasive genetic methods for monitoring and understanding bear populations, highlighting differences between the Pyrenean and Cantabrian populations, and providing insights into their genetic diversity, structure and demographic trends.
Developing large‐scale monitoring strategies for marine habitats with appropriate temporal and spatial resolution is challenging logistically and economically. We compare potential DNA metabarcoding infaunal identification methods with traditional morphological assessment of marine intertidal biodiversity across two protected sites of contrasting anthropogenic impact on the south coast of England. We show that all the methodologies are effective at distinguishing the distinct communities present at each site. While morphological methods provide community abundance and biomass information, data derived from 18S metabarcoding of sediment scrapes showed the strongest discriminations between sites with contrasting anthropogenic pressures. This difference is due to the inclusion of more taxa from a wider spectrum of biodiversity in this dataset. However, 18S OTU abundance was a poor predictor of morphological taxa abundance and biomass. Examination of the presence or absence of taxa at the more or less impacted sites allows us to identify potential indicator taxa for future surveys, such as families in the phyla Ascidiacea, Cnidaria, Nematoda, and Platyhelminthes; taxa which are not traditionally incorporated into morphology‐based assessments due to the difficulty and expense of identification. DNA metabarcoding tools provide a more comprehensive snapshot of marine biodiversity compared to morphological surveys, and therefore an opportunity to assess the responses of more organisms to environmental stressors and develop novel metrics for habitat assessment. This could greatly benefit future monitoring programs and the assessment of management impacts in marine habitats.
While the utility of environmental DNA (eDNA) metabarcoding surveys for biodiversity monitoring continues to be demonstrated, the spatial and temporal variability of eDNA, and thus the limits of the differentiability of an eDNA signal, remains under‐characterized. In this study, we collected eDNA samples from distinct micro‐habitats (~40 m apart) in a rocky intertidal ecosystem over their exposure period in a tidal cycle. During this period, the micro‐habitats transitioned from being interconnected, to physically isolated, to interconnected again. Using a well‐established eukaryotic (cytochrome oxidase subunit I) metabarcoding assay, we detected 415 species across 28 phyla. Across a variety of univariate and multivariate analyses, using exclusively taxonomically assigned data as well as all detected amplicon sequence variants (ASVs), we identified unique eDNA signals from the different micro‐habitats sampled. This differentiability paralleled expected ecological gradients and increased as the sites became more physically disconnected. Our results demonstrate that eDNA biomonitoring can differentiate micro‐habitats in the rocky intertidal only 40 m apart, that these differences reflect known ecology in the area, and that physical connectivity informs the degree of differentiation possible. These findings showcase the potential power of eDNA biomonitoring to increase the spatial and temporal resolution of marine biodiversity data, aiding research, conservation, and management efforts.
The Arizona Toad (Anaxyrus microscaphus) is restricted to riverine corridors and adjacent uplands in the arid southwestern United States. As with numerous amphibians worldwide, populations are declining and face various known or suspected threats, from disease to habitat modification resulting from climate change. The Arizona Toad has been petitioned to be listed under the U.S. Endangered Species Act and was considered “warranted but precluded” citing the need for additional information – particularly regarding natural history (e.g., connectivity and dispersal ability). The objectives of this study were to characterize population structure and genetic diversity across the species’ range. We used reduced-representation genomic sequencing to genotype 3,601 single nucleotide polymorphisms in 99 Arizona Toads from ten drainages across its range. Multiple analytical methods revealed two distinct genetic groups bisected by the Colorado River; one in the northwestern portion of the range in southwestern Utah and eastern Nevada and the other in the southeastern portion of the range in central and eastern Arizona and New Mexico. We also found subtle substructure within both groups, particularly in central Arizona where toads at lower elevations were less connected than those at higher elevations. The northern and southern parts of the Arizona Toad range are not well connected genetically and could be managed as separate units. Further, these data could be used to identify source populations for assisted migration or translocations to support small or potentially declining populations.
Estimating abundance using capture-recapture methods for animals that move around in a closed population can be difficult. One way of dealing with this, as well as for some stationary populations, is to record the locations or centers of activity of the animals or objects (e.g., animal signs) using so-called spatial capture-recapture (SCR) methods. The probability of capture can then be modeled on the distance of an animal from a trap location.
The “trap” can be any kind of recording device such as a personal observation or camera, or an array of proximity or acoustical detectors. It can also be a single or multicatch trap. Sometimes telemetry can be used at the same time and can be combined with the capture data. SRC can also be combined with occupancy and distance sampling data, as well as with adaptive cluster sampling. Information on activity centers can be used to study animal interactions.
Bayesian models are extensively used and can also be combined with frequency methods. Other extensions to SCR are the use of stratification, presence-absence data only, and, in particular, spatial resight models. Different sightings for marked and unmarked can be allowed for as well as the possible lack of individual recognition.
With technological advances, camera methods are being increasingly used to estimate densities of elusive terrestrial mammals, animals with low densities, and those animals difficult to capture or detect. Along with DNA methods, which are also considered, they have many advantages such as being noninvasive. The design of SCR models is considered. Many examples and applications are given throughout the chapter.
The outcome of species delimitation depends on many factors, including conceptual framework, study design, data availability, methodology employed and subjective decision making. Obtaining sufficient taxon sampling in endangered or rare taxa might be difficult, particularly when non‐lethal tissue collection cannot be utilized. The need to avoid overexploitation of the natural populations may thus limit methodological framework available for downstream data analyses and bias the results. We test species boundaries in rare North American trapdoor spider genus Cyclocosmia Ausserer (1871) inhabiting the Southern Coastal Plain biodiversity hotspot with the use of genomic data and two multispecies coalescent model methods. We evaluate the performance of each methodology within a limited sampling framework . To mitigate the risk of species over splitting, common in taxa with highly structured populations, we subsequently implement a species validation step via genealogical diversification index ( gdi ), which accounts for both genetic isolation and gene flow. We delimited eight geographically restricted lineages within sampled North American Cyclocosmia, suggesting that major river drainages in the region are likely barriers to dispersal. Our results suggest that utilizing BPP in the species discovery step might be a good option for datasets comprising hundreds of loci, but fewer individuals, which may be a common scenario for rare taxa. However, we also show that such results should be validated via gdi , in order to avoid over splitting.
Next-generation sequencing technology has enabled accurate insights into the diet of wildlife species. The protocols for faecal sample collection and DNA extraction for diet analysis have differed from those focusing on target species, even in most studies combining questions on both aspects. We designed an experiment to evaluate two protocols using 11 parameters and select a single one that will generate both target species (Asiatic wild ass, Equus hemionus, in Israel) and diet DNA, as an effective strategy to minimise time, effort, and cost without hampering efficiency. In Protocol A, we swabbed the outer surface of faecal boluses and extracted DNA using a Stool Kit, while for Protocol B, we homogenised faecal matter from inside the bolus followed by extraction using a Powersoil Kit. Protocol A performed significantly better for four parameters , which included, for the target species, microsatellite amplification success and the quantity of the GAPDH gene; and for its diet, the number of exact sequence variants (ESVs) obtained at genus level and plant genus richness. However, there was no significant difference in the amplification success of sex-linked and plant markers, total reads at genus level, number of genera obtained and plant genus composition. Although we chose Protocol A, both protocols yielded results for the target species and its diet, demonstrating that one single protocol can be used for both purposes, although a pilot study is recommended to optimise the protocol for specific systems. This strategy may also be useful for studies combining target species and their gut microbiome and parasitic load. K E Y W O R D S diet of herbivores, DNA extraction, Equus hemionus, metabarcoding, non-invasive genetics, trnL
The world is struggling to solve a devastating biodiversity loss that not only affects the extinction of treasured species and irreplaceable genetic variation, but also jeopardizes the food production, health, and safety of people. All initiatives aimed to conserve biodiversity rely heavily on the monitoring of both species and populations to get accurate spatial patterns and overall population assessments. Conventional monitoring techniques, such as visual surveys and counting individuals, are problematic due to challenges in identifying cryptic species or immature life stages. Environmental DNA (eDNA) is a relatively new technology that has the potential to be a faster, non-invasive, and cost-effective tool for monitoring biodiversity, conservation, and management practices. eDNA has been extracted from materials that are both ancient and present, and its applications range from the identification of individual species to the study of entire ecosystems. In the past few years, there has been a substantial increase in the usage of eDNA in research pertaining to ecological preservation and conservation. However, several technological problems still need to be solved. To reduce the number of false positives and/or false negatives produced by current eDNA technologies, it is necessary to improve and optimize calibration and validation at every stage of the procedure. There is a significant need for greater information about the physical and ecological constraints on eDNA use, as well as its synthesis, current state, expected lifespan, and potential modes of movement. Due to the widespread use of eDNA research, it is also essential to assess the extent and breadth of these studies. In this article, we critically reviewed the primary applications of eDNA in subterranean and aquatic invasive species. Through this review, readers can better understand the challenges and limitations of eDNA metabarcoding.
The emerging field of environmental DNA (eDNA) research lacks universal guidelines for ensuring data produced are FAIR–findable, accessible, interoperable, and reusable–despite growing awareness of the importance of such practices. In order to better understand these data usability challenges, we systematically reviewed 60 peer reviewed articles conducting a specific subset of eDNA research: metabarcoding studies in marine environments. For each article, we characterized approximately 90 features across several categories: general article attributes and topics, methodological choices, types of metadata included, and availability and storage of sequence data. Analyzing these characteristics, we identified several barriers to data accessibility, including a lack of common context and vocabulary across the articles, missing metadata, supplementary information limitations, and a concentration of both sample collection and analysis in the United States. While some of these barriers require significant effort to address, we also found many instances where small choices made by authors and journals could have an outsized influence on the discoverability and reusability of data. Promisingly, articles also showed consistency and creativity in data storage choices as well as a strong trend toward open access publishing. Our analysis underscores the need to think critically about data accessibility and usability as marine eDNA metabarcoding studies, and eDNA projects more broadly, continue to proliferate.
Niche overlap between sympatric species can indicate the extent of interspecific competition. Sympatric competing species can exhibit spatial, temporal, and dietary adjustments to reduce competition. We investigated spatial, temporal, and dietary niche overlap of sympatric Asian palm civet (Paradoxurus hermaphroditus) and small Indian civet (Viverricula indica), in and around Pir Lasura National Park, Pakistan. We used remote cameras to determine the frequency and timing of detections to estimate spatial and temporal overlap, and prey remains from scats to estimate dietary overlap. We collected scat samples of Asian palm civet (n = 108) and small Indian civet (n = 44) for dietary analysis. We found low spatial (O ij = 0.32) and temporal (Δ = 0.39) overlap, but high dietary niche overlap (0.9) between these two civet species. Both civet species were detected at only 11 camera sites and small Indian civets were detected most frequently during 2:00-5:00 h and 8:00-10:00 h, whereas Asian palm civets detections were greatest during 20:00-2:00 h. The overall niche breadth of Asian palm civet was slightly narrower (L = 9.69, Lst = 0.31) than that of the small Indian civet (L = 10, Lst = 0.52). We identified 27 dietary items (15 plant, 12 animal) from scats of Asian palm civet including Himalayan pear (Pyrus pashia; 27%), Indian gerbil (Tatera indica; 10%), Rhesus monkey (Macaca mulatta; 4%), and insects (5%). Scat analysis of small Indian civets revealed 17 prey items (eight plant, nine animal) including Himalayan pear (24%), domestic poultry (15%), Indian gerbil (11%), and house mouse (Mus musculus; 5%). Both civet species consumed fruits of cultivated orchard species. Spatial and temporal partitioning of landscapes containing diverse foods appears to facilitate coexistence between Asian palm civets and small Indian civets.
Non‐invasive genetic sampling (NGS) methods are becoming a mainstay in wildlife monitoring and can be used with spatial capture‐recapture (SCR) methods to estimate population density. Yet SCR based on NGS remains relatively underused for ungulate population monitoring, despite the importance of robust density estimates for this ecologically and economically important group of species. This may be in part attributed to biological characteristics of ungulate species and data collection methods that lead to violations of SCR model assumptions. We conducted a simulation study to evaluate the robustness of SCR methods to spatially heterogeneous density (i.e., configuration of individuals into groups of variable sizes and composition), individual heterogeneity in space‐use patterns, and adaptive sampling (i.e., variation in detectability across space that correlates with density). We evaluated each violation separately and in combination. We parameterized our simulations based on published information and preliminary analyses of NGS data sets of 3 ungulate species: chamois (Rupicapra rupicapra), red deer (Cervus elaphus), and wild boar (Sus scrofa). While SCR estimates were robust to grouping and adaptive sampling, abundance estimates could be negatively biased (up to 10% in our simulations) in the presence of unaccounted individual heterogeneity in space use. The degree to which abundance estimates were underestimated depended mostly on the amount of variation in space use and detectability among age classes. This bias was also accompanied by a reduction in precision and coverage probability of the SCR estimators. We discuss the implications of these findings, possible approaches to identify problematic violations in available data sets (goodness‐of‐fit tests), and potential further developments of SCR models to ensure reliable abundance estimates for ungulate populations from NGS data. In this study, we evaluated the reliability of spatial capture‐recapture (SCR) models to study ungulate populations by simulating 9,600 datasets and scenarios based on published information and preliminary analyses of non‐invasive genetic sampling data of 3 ungulate species: chamois, red deer, and wild boar. While SCR estimates were robust to grouping and adaptive sampling, we found that abundance estimates could be negatively biased (up to 10% in our simulations) in the presence of unaccounted individual heterogeneity. The degree to which abundance estimates were underestimated depended mostly on the amount of variation in space‐use and detectability among age classes. This bias was also accompanied by a reduction in precision and coverage probability of the SCR estimators.
Monitoring the occupancy and abundance of wildlife populations is key to evaluate their conservation status and trends. However, estimating these parameters often involves time and resource-intensive techniques, which are logistically challenging or even unfeasible for rare and elusive species that occur patchily and in small numbers. Hence, surveys based on field identification of signs (e.g. faeces, footprints) have long been considered a cost-effective alternative in wildlife monitoring, provided they produce reliable detectability and meaningful indices of population abundance. We tested the use of sign surveys for monitoring rare and otherwise elusive small mammals, focusing on the Cabrera vole ( Microtus cabrerae ) in Portugal. We asked how sampling intensity affects true positive detection of the species, and whether sign abundance is related to population size. We surveyed Cabrera voles’ latrines in 20 habitat patches known to be occupied, and estimated ‘true’ population size at each patch using DNA-based capture-recapture techniques. We found that a searching rate of ca. 3 min/250m ² of habitat based on adaptive guided transects was sufficient to provide true positive detection probabilities > 0.85. Sign-based abundance indices were at best moderately correlated with estimates of ‘true’ population size, and even so only for searching rates > 12 min/250m ² . Our study suggests that surveys based on field identification of signs should provide a reliable option to estimate occupancy of Cabrera voles, and possibly for other rare or elusive small mammals, but cautions should be exercised when using this approach to infer population size. In case of practical constraints to the use of more accurate methods, a considerable sampling intensity is needed to reliably index Cabrera voles’ abundance from sign surveys.
Collembola are abundant and have significant roles in the soil ecosystem. Therefore, the phenotypic endpoints of Collembola population or community have been used as an effective bioindicator for assessing soil quality. Since the identification and counting the collembolans in the soil is a laborious and costly procedure, environmental DNA (eDNA)‐based biomonitoring was proposed as an analysis tool of collembolan species found in the soil. In this study, standard primer sets for the species‐specific eDNA analysis using Allonychiurus kimi, a soil bioindicator species was selected. Then, the primers were tested for specificity and sensitivity from the soil samples. Two different eDNA samples were tested: (1) eDNA samples were extracted from the soil with A. kimi individuals (intra‐organismal eDNA). (2) The samples from the soil without A. kimi individuals (extra‐organismal eDNA). The two primers were confirmed in their sensitivity and specificity to the two types of eDNA samples selected. Ct‐values from both intra‐ and extra‐organismal eDNA showed the significant correlations to the number of inoculated A. kimi (adj. R2 = 0.7453–0.9489). These results suggest that in excretion, egg, and other exuviae had a significant effect on eDNA analysis from soil samples taken. Furthermore, our results suggest that environmental factors should be considered when analyzing eDNA collected from soil. In this study, standard primer sets for the species‐specific eDNA analysis using Allonychiurus kimi, a soil bioindicator species was selected. Then, the primers were tested for specificity and sensitivity from the soil samples. Two primers were confirmed in their sensitivity and specificity to the two eDNA samples selected. Ct‐values from both intra‐ and extra‐organismal eDNAs showed the significant correlations to the number of inoculated A. kimi. These results suggest that in excretion, egg, and other exuviae had a significant effect on eDNA analysis from soil samples taken.
Background
Once a widespread species across the region of Southeast Europe, the Griffon vulture is now confined to small and isolated populations across the Balkan Peninsula. The population from Serbia represents its biggest and most viable population that can serve as an important reservoir of genetic diversity from which the birds can be used for the region’s reintroduction programmes. The available genetic data for this valuable population are scarce and as a protected species that belongs to the highly endangered vulture group, it needs to be well described so that it can be properly managed and used as a restocking population. Considering the serious recent bottleneck event that the Griffon vulture population from Serbia experienced we estimated the overall relatedness among the birds from this population. Sex ratio, another important parameter that shows the vitality and strength of the population was evaluated as well.
Methods
During the annual monitoring that was performed in the period from 2013–2021, we collected blood samples from individual birds that were marked in the nests. In total, 169 samples were collected and each was used for molecular sexing while 58 presumably unrelated birds from different nests were used for inbreeding and relatedness analyses. The relatedness was estimated using both biparentally (10 microsatellite loci) and uniparentally ( Cytb and D-loop I of mitochondrial DNA) inherited markers.
Results
The level of inbreeding was relatively high and on average it was 8.3% while the mean number of relatives for each bird was close to three. The sex ratio was close to 1:1 and for the analysed period of 9 years, it didn’t demonstrate a statistically significant deviation from the expected ratio of 1:1, suggesting that this is a stable and healthy population. Our data suggest that, even though a relatively high level of inbreeding can be detected among the individual birds, the Griffon vulture population from Serbia can be used as a source population for restocking and reintroduction programmes in the region. These data combined with previously observed genetic differentiation between the populations from the Iberian and Balkan Peninsulas suggest that the introduction of foreign birds should be avoided and that local birds should be used instead.
Los Xenartrhas son un grupo de mamíferos de gran importancia histórica y ecológica originados en sur América. La implementación de técnicas en genética molecular en estos animales está en aumento y para ello los métodos de muestreo mínimamente invasivos son una herramienta exitosa para el monitoreo genético. En esta investigación se comparó la calidad y cantidad de ADN obtenido de sangre, tejidos, saliva, pelos y heces, usando dos kits comerciales: PrepFiler™ y GeneJET™. La concentración pureza e integridad del ADN fueron evaluados usando espectrofotometría y electroforesis. Una ANOVA de dos factores mezclados y una prueba de comparaciones múltiples de Tukey se uso para comparar los diferentes métodos de extracción de ADN y a través de las diferentes muestras biológicas. El mayor rendimiento de ADN fue obtenido para las muestras de tejido con el kit PrepFiler™, con una concentración media 5,25 ng/uL, una pureza de 1,87, y una banda definida en la electroforesis. Sin embargo, no recomendamos el uso de estas muestras en animales vivos. El ADN obtenido de saliva con el kit PrepFiler™ ofrece resultados similares en términos de concentración (media 3,56 ng/uL), pureza de 1,85 e integridad; además, la prueba de comparación de Tukey mostró que no hay diferencias entre las muestras de saliva y sangre (p=0,01028); la obtención de muestras de saliva requiere menos intervención en los animales. Por esta razón, se concluye que la extracción de ADN usando el kit PrepFiler™ en muestras de saliva es la mejor opción para extracción de ADN de calidad en Xenartrhas.
Historical exploitation, and a combination of current anthropogenic impacts, such as climate change and habitat degradation, impact the population dynamics of marine mammalian megafauna. Right whales (Eubalaena spp.) are large cetaceans recovering from hunting, whose reproductive and population growth rate appear to be impacted by climate change. We apply noninvasive genetic methods to monitor southern right whale (E. australis, SRW) and test the application of noninvasive genetics to minimise the observer effects on the population. Our aim is to describe population structure, and interdecadal and interannual changes to assess species status in the Great Acceleration period of Anthropocene. As a basis for population genetic analyses, we collected samples from sloughed skin during post-migration epidermal moult. Considering the exploration-exploitation dilemma, we collaborated with whale watching companies, as part of a citizen science approach and to reduce ad hoc logistic operations and biopsy equipment. We used mitochondrial and microsatellite data and population genetic tools. We report for the first time the genetic composition and differentiation of the Namibian portion of the range. Population genetic parameters suggest that South Africa hosts the largest population. This corresponds with higher estimates of current gene flow from Africa compared to older samples. We have observed considerable interannual variation in population density at the breeding ground and an interdecadal shift in genetic variability, evidenced by an increase in the point estimate inbreeding. Clustering analyses confirmed differentiation between the Atlantic and Indo-Pacific, presumably originating during the ice ages. We show that population monitoring of large whales, essential for their conservation management, is feasible using noninvasive sampling within non-scientific platforms. Observed patterns are concurrent to changes of movement ecology and decline in reproductive success of the South African population, probably reflecting a large-scale restructuring of pelagic marine food webs.
Noninvasive DNA sampling allows studies of natural populations without disturbing the target animals. Unfortunately, high genotyping error rates often make noninvasive studies difficult. We report low error rates (0.0-7.5%/locus) when genotyping 18 microsatellite loci in only 4 multiplex polymerase chain reaction amplifications using fecal DNA from bighorn sheep (Ovis canadensis). The average locus-specific error rates varied significantly between the 2 populations (0.13% vs. 1.6%; P , 0.001), as did multi-locus genotype error rates (2.3% vs. 14.1%; P , 0.007). This illustrates the importance of quantifying error rates in each study population (and for each season and sample preservation method) before initiating a noninvasive study. Our error rates are among the lowest reported for fecal samples collected noninvasively in the field. This and other recent studies suggest that noninvasive fecal samples can be used in species with pellet-form feces for nearly any study (e.g., of population structure, gene flow, dispersal, parentage, and even genome-wide studies to detect local adaptation) that previously required high-quality blood or tissue samples.
The ability to select short DNA oligonucleotide sequences capable of binding solely to their intended target is of great importance in developing nucleic acid based detection technologies. Applications such as multiplex PCR rely on primers binding to unique regions in a genome. Competing side reactions with other primer pairs or template DNA decrease PCR efficiency. Freely available primer design software such as Primer3 screens for potential hairpin and primer-dimer interactions while selecting a single primer pair. The development of multiplex PCR assays (in the range of 5 to 20 loci) requires the screening of all primer pairs for potential cross-reactivity. However, a logistical problem results due to the number of total number of comparisons required. Comparing the primer set for a 10-plex assay (20 total primer sequences) results in 210 primer-primer combinations that must be screened. The ability to screen sets of candidate oligomers rapidly for potential cross-reactivity reduces overall assay development time. Here we report the application of a familiar sliding algorithm for comparing two strands of DNA in an overlapping fashion. The algorithm has been employed in a software package wherein the user can compare multiple sequences in a single computational run. After the screening is completed, a score is assigned to potential duplex interactions exceeding a user-defined threshold. Additional criteria of predicted melting temperature (T m ) and free energy of melting (ΔG) are included for further ranking. Sodium counterion and total stand concentrations can be adjusted for the T m and ΔG calculations. The predicted interactions are saved in a text file for further evaluation.
Population genomics has the potential to improve studies of evolutionary genetics, molecular ecology and conservation biology, by facilitating the identification of adaptive molecular variation and by improving the estimation of important parameters such as population size, migration rates and phylogenetic relationships. There has been much excitement in the recent literature about the identification of adaptive molecular variation using the population-genomic approach. However, the most useful contribution of the genomics model to population genetics will be improving inferences about population demography and evolutionary history.
Demographic isolation of populations due to habitat fragmentation results in an accumulation of relatives as a consequence of reduced dispersal. This has profound potential implications for mating and social systems – and ultimately population persistence – since the chance for inbred matings and kin interactions are amplified. Despite its conservation impact, rarely have studies addressed human-induced within-fragment changes to population processes. With this aim, we examined spatial relatedness structure in two candidate isolated populations (Kulpara and Scrubby Peak) of southern hairy-nosed wombats (Lasiorhinus latifrons). Wombats were sampled by remote hair-trapping for highly-resolving microsatellite genotyping. In the longest-isolated population (Kulpara), habitat fragmentation was associated with inhibited female dispersal and high population density, which profoundly altered kin relationships. There were two primary responses. First, female relatives preferred to coexist, a radical departure from the active avoidance in continuous habitat. Females under the constraints of high wombat density and inhibited dispersal interact with female relatives rather than less-related females, perhaps as a means to minimize the costs of sociality and/or maximize indirect fitness. Second, inbreeding avoidance appeared to be stronger at Kulpara than in other populations of the species, which may be due to an increased effort to avoid inbreeding under inhibited dispersal.
We used polymerase chain reaction (PCR)-based RFLP (restriction fragment length polymorphism) and microsatellite analyses to identify canid species, gender, and individual genotype in samples containing a large excess of domestic sheep DNA. These methods were then used to investigate the feasibility of identifying predators from saliva on predation wounds. We analyzed predation wound samples from 19 sheep carcasses. Coyote DNA was identified in 18 samples (95%), of which 17 contained male coyote DNA (94%) and 11 (61%) yielded heterozygous microsatellite genotypes at ≥1 locus. These methods have promise for genetic identification of individual predators.
We tested non-invasive genetic methods for estimating the abundance of marten Martes americana using baited glue-patch traps to pull hair samples from individual animals. We divided our 800-km2 study area into 3 × 3 km cells and put one hair trap in each cell. We trapped 309 sites for an average of 15 days each between 15 January and 14 March 1997. Based on tracks in snow and hair morphology, we captured hair from marten, red squirrels Tamiasciurus hudsonicus, flying squirrels Glaucomys sabrinus, short or long-tailed weasels Mustela erminea and M. frenata, and several unidentified mouse and vole species. Of 309 sites, 58% collected a marten hair sample while 8% of sites moved weasel hair. When roots were embedded in adhesive, a xylene wash was used to remove them before extracting DNA. All marten samples were genotyped at six microsatellite loci to identify individuals. Xylene-washed samples yielded similar genotyping success to samples that had never been exposed to xylene, and genotyping success increased with the number of hairs in the sample. Genetic data allowed 139 samples to be assigned to 88 individual marten, constituting 124 capture events during the four trapping sessions. The population estimate for our study area was 213 (95% CI: 148-348) and the average capture probability was 0.15. The density of marten in our study area was 0.33/km2 when inhospitable habitat was removed from the calculation. We believe hair sampling and genetic analysis could be used to measure population distribution, trend and size for marten, and perhaps also for other carnivores.
We evaluated and compared sixteen combinations of commonly used storage and extraction methods for faecal DNA from two Australian marsupial herbivores, two marsupial carnivores and an introduced carnivorous mammal. For all species the highest amplification and lowest genotyping error rates were achieved using dried faeces extracted via a surface wash followed by spin column purification. The highest error rates were seen in the two Dasyurus spp. and the lowest in Vulpes vulpes. The rates observed for each species were incorporated into computer simulations to identify the number of PCR replicates required to achieve accurate genotyping of DNA isolated via the optimised protocol. Three replicates per sample were sufficient for V. vulpes, Thylogale billardierii and Petrogale penicillata. However, further replicates may be required for marsupial carnivores, as their faeces yielded DNA that amplified substantially less often and less reliably, for all preservation and extraction methods tested, than did the other species. Although pilot studies remain vital for evaluating the feasibility of non-invasive sampling prior to undertaking any in-depth study the availability of a thoroughly tested storage and DNA extraction combination protocol known to be optimal for five different species should make that process much simpler.
DNA was extracted from mucus secreted by snails that had been allowed to crawl over glass microscope slides. The mucus contained many epithelial cells and a few blood cells. Microsatellite DNA regions were amplified using template DNA from the mucus and clear bands obtained showing the same positions as when using template DNA from the foot. Pedal mucus is therefore a reliable source of DNA, which can be extracted by a simple methodology that is readily applied in the field. The technique has considerable potential for conservation- and behavioural ecology.
Resource managers lack an inexpensive and quantifiable method to detect lynx presence across large landscapes. We tested efficacy of a protocol based on hair snagging to detect presence of lynx (Lynx canadensis). We tested 2 key elements of the protocol: 1) a hair snaring device and 2) commercial lures used to attract and elicit rubbing behavior in lynx. The commercial lures we tested included: 1) beaver (Castor canadensis) castoreum and catnip oil, 2) Cat Passionv: 3) Pacific CaIlT ") 4) Hawbacker's Cat lure #I T '''; and 5) BB1T~ To compare detection rates among lures, we randomly placed lures at scent stations along 78 transects; each transect contained all 5 lures. We detected lynx at 45% of tran sects, and detections varied significantly among lures (xl= 13.4, P=0.009). Hair snares baited with castoreum and catnip oil were used significantly more than expected (P= 0.002). The relatively high overall detection rate demonstrated that deploying an effec tive lure along transects is an effective method to detect presence or absence.
Genetic studies of native herpetofauna populations are important for the conservation of European biodiversity, but previous studies have been largely dependent on invasive sample collection. Here we explore the efficiency of noninvasive sampling (NIS) for molecular studies and review the various potential sources of such samples. Snakes produce a multitude of by-products, such as sloughed skin, faeces and eggs or embryos, that, along with road kills, predated specimens and museum samples, could potentially be used in molecular studies. We describe a new method for obtaining snake faeces in the field and, using mitochondrial cytochrome b primers, we successfully amplified 500 and 758 bp sequences from a variety of tissues collected by NIS. The availability and degradation of such material differed greatly, and both DNA extraction and PCR success appeared dependent upon sample origin and storage. Nevertheless, for the first time we demonstrate that faecal, egg and foetal tissues, as well as sloughed skin and carcasses, represent valuable NIS source material permitting genetic studies with minimal disturbance to the individual and its population.
Non-invasive genetic sampling has become a favored tool to enumerate wildlife. Genetic errors, caused by poor quality samples, can lead to substantial biases in numerical estimates of individuals. We demonstrate how the computer program DROPOUT can detect amplification errors (false alleles and allelic dropout) in a black bear (Ursus americanus) dataset collected in 2003 from northern Idaho, USA, and detect scoring and other database errors (misreads, shifts in scoring, and transcription errors). Removing errors from our sample via computer techniques reduced our minimum number alive index from 187 to 146 bears and was less expensive than commonly used multi-tube approaches. We subsequently estimated gene flow between our 2 study areas (Purcell and Selkirk Mountains), which are separated by a large, open, agricultural valley. Gene flow data suggested that, although this valley was not a complete barrier to movement, its effects on population substructure were not inconsequential. We documented a low level of substructure (G′ST = 0.097) between study areas. Assignment tests confirmed this, as assignment to the population where the animal was captured was 74% for the Purcell Mountains and 89% for the Selkirk Mountains.
Amplification of a mitochondrial DNA fragment was used to compare the efficiency of five methods for extracting DNA from bat droppings. The Qiagen DNA Stool Kit, which yielded > 90% mtDNA amplification success, was chosen to extract DNA from 586 samples taken over two years in three French colonies of the lesser horseshoe bat (Rhinolophus hipposideros). Samples, for which mtDNA amplification was successful, were subject to the multiplex amplification of eight microsatellite loci. This resulted in > 95% amplification success over 12,592 PCRs. Allelic dropout (ADO) and false allele (FA) rates were low, and consequently, sample and locus quality indexes (QI) were high. These results demonstrate that large scale noninvasive studies of bat colonies are possible.
Adult Greater Flamingos (Phoenicopterus roseus) are sexually dimorphic, with males being on average larger and heavier than females. However, there is no practical way to sex the chicks by their morphology. Here we describe a method relying on quick and easy DNA extraction from feathers. A PCR test employing primers to amplify introns whose lengths usually differ between the CHD-W and the CHD-Z genes allow sex discrimination. This method is thus a fast, accurate and inexpensive protocol to sex flamingo chicks from feathers bulbs sampled in the field.
In order to complement ecological information with genetic data we isolated and characterized 14 polymorphic microsatellite markers from raccoons (Procyon lotor). Three multiplexed panels comprising the loci were developed and 29 individuals from a contiguous habitat patch in northern Indiana, USA were genotyped. The number of alleles per locus ranged from four to 18, and overall heterozygosities ranged from 0.31 to 1.00. One locus was identified as possibly being X-linked, since males appeared to be hemizygous. Data generated using these markers will be used to further our understanding of small-scale raccoon population dynamics in a highly fragmented landscape.
Noninvasive samples are of increasing importance to study wild populations. In this study, we investigate the applicability of urine samples as the sole source of DNA for routine noninvasive genetic monitoring of wildlife using wolves (Canis lupus) as an example. Within the scope of a long-term wolf population survey, we collected during winter snow tracking in Bieszczady Mountains, Poland 41 urine samples considered as utilizable for genetic analyses. DNA concentration was determined by quantitative real-time polymerase chain reaction (qPCR) and six microsatellite loci were genotyped in threefold repeated genotyping experiments to assess the reliability of genetic analyses of urine. DNA concentration of 33 urine samples was successfully quantified and of 14 samples, we obtained congruent results for all analysed loci and all repeated genotyping experiments. The gender of urine samples was identified with a Y-chromosome-linked marker. Considering the high discovery rate of urine in conjunction with its genotype reliability, our study confirms that urine is a valuable source in noninvasive genetic monitoring. Additionally, preselection of samples via qPCR proved to be a powerful tool contributing to a beneficial cost-value ratio of genetic analyses by minimizing genotyping errors.
We describe here a procedure for the purification of high molecular weight genomic DNA that combines the economies of ‘do-it-yourself’, single-tube protocols with the sample throughput and DNA quality of microplate-based DNA extraction and purification kits from commercial suppliers. The procedure allows the preparation of genomic DNA of a quality suitable for polymerase chain reaction-based studies of large populations at around one-tenth of the cost of commercially available kits. Furthermore, 96 samples can be purified from crude tissue digests in around 30 min and are produced in microtitre plate format to allow efficient downstream processing of samples.
There are large museum collections of mammalian skins and we wished to determine their usefulness for DNA-based evolutionary and conservation studies. Methods derived from the ancient DNA approach were used to process samples from skins of the stoat (Mustela erminea) from 18 museums in 11 countries. Successful polymerase chain reaction (PCR) amplification was achieved in 56%, 46% and 40% of all samples for 0.65-, 0.70- and 0.78-kb PCR products of mitochondrial DNA, respectively. Some of the best-preserved skin samples were those in tight-fitting containers in a dry and cold environment. With care in their preservation, mammalian skin collections may be a good source of DNA.
Life history data for three unexploited populations of brook trout, Salvelinus fontinalis, were used to test the predictions of life history theory that, relative to juveniles, (1) high adult survival favors low reproductive effort and delayed reproduction, and (2) increased juvenile growth rate favors high effort and early reproduction. Field data supported both predictions. The population having the highest adult-to-juvenile survival ratio expended the least effort, reproduced latest in life, and experienced the lowest survival cost of reproduction. Among populations a high juvenile-to-adult growth rate was associated with early reproduction, high reproductive effort, and high reproductive cost. Early reproduction was also associated with increased growth within populations. The adaptive significance of interpopulation variation in life history was assessed by comparing the fitness, r, of observed life histories with those of potentially alternative strategies. Empirically derived fitness functions supported the hypothesis that population differences in life history were adaptive. Observed combinations of age-specific survival and fecundity were those that maximized fitness. Within populations the fitness advantages associated with reproducing early in life favored reduced age at reproduction for the fastest-growing individuals. The results are consistent with the predictions of life history theory and demonstrate empirically how survival and growth rate can independently and interactively influence life history evolution.
Likelihood methods have been developed to partition individuals in a sample into full-sib and half-sib families using genetic marker data without parental information. They invariably make the critical assumption that marker data are free of genotyping errors and mutations and are thus completely reliable in inferring sibships. Unfortunately, however, this assumption is rarely tenable for virtually all kinds of genetic markers in practical use and, if violated, can severely bias sibship estimates as shown by simulations in this article. I propose a new likelihood method with simple and robust models of typing error incorporated into it. Simulations show that the new method can be used to infer full- and half-sibships accurately from marker data with a high error rate and to identify typing errors at each locus in each reconstructed sib family. The new method also improves previous ones by adopting a fresh iterative procedure for updating allele frequencies with reconstructed sibships taken into account, by allowing for the use of parental information, and by using efficient algorithms for calculating the likelihood function and searching for the maximum-likelihood configuration. It is tested extensively on simulated data with a varying number of marker loci, different rates of typing errors, and various sample sizes and family structures and applied to two empirical data sets to demonstrate its usefulness.
Over the past two decades, new molecular genetic tech- niques have had substantial impacts on the fields of ecology, evolution and conservation. However, our cur- rent toolbox of genetic methodologies remains inadequate for answering many questions and there are significant technological and analytical limitations. We review the possible uses of single nucleotide poly- morphisms (SNPs) as novel genetic markers for com- mon questions in population genetics. Furthermore, we evaluate the potential of SNPs relative to frequently used genetic markers, such as microsatellite loci and mitochondrial DNA (mtDNA) sequences, and we dis- cuss statistical power, analytical approaches, and tech- nological improvements and limitations. Although ascertainment bias is a problem for some applications, SNPs can often generate equivalent statistical power whilst providing broader genome coverage and higher quality data than can either microsatellites or mtDNA, suggesting that SNPs could become an efficient and cost-effective genetic tool.
We evaluated the feasibility and efficiency of using swabs to collect buccal epithelial cells from small (2- to 13-g) birds as a source of DNA for genetic studies. We used commercially available buccal swab kits to collect samples from 42 adult and 39 nestling (4- to 8-day-old) black-capped chickadees (Poecile atricapillus) and from 6 4-day-old nestling boreal chickadees (P. hudsonica). We compared DNA from buccal epithelial samples to that from blood samples from the same individuals. We extracted sufficient quantities of DNA for analysis from all buccal samples, and samples remained viable even after being stored in original plastic sampling tubes at room temperature for up to 18 months. Yields were equivalent whether extracted using the proprietary quick-extraction solution provided with buccal swab kits or using a salt-extraction process with inexpensive reagents. Yields of DNA from buccal samples were consistently lower than those from blood samples, but quantities were sufficient for all analyses. Assignment of sex, based on DNA extracted from paired buccal and blood samples, was identical for all 87 birds. We found no difference in the genotypes obtained from buccal and blood samples for 12 individuals tested using 5 microsatellite loci and found perfect concordance in sequencing of an 823-base-pair segment within the control region of mitochondrial DNA for 7 individuals tested. Use of buccal swabs is highly recommended as a rapid, noninvasive technique for sampling avian genomic DNA, especially for extremely young altricial nestlings or small-bodied adults, or for any birds for which blood sampling may be impossible or stressful.
Multiplex polymerase chain reaction (PCR) refers to the simultaneous amplification of multiple regions of deoxyribonucleic acid (DNA) using PCR. Commercial short tandem repeat (STR) assays that can coamplify as many as 16 different loci have become widely used in forensic DNA typing. This chapter will focus on some of the aspects of constructing robust STR multiplex assays, including careful design and quality control of PCR primers. Examples from the development of a cat STR 12plex and a human Y chromosome STR 20plex are used to illustrate the importance of various parts of the protocol. Primer design parameters and Internet-accessible resources are discussed, as are solutions to problems with residual dye artifacts that result from impure primers.
Multiplex quantitative PCR based on novel design of fluorescent primers is described. Fluorogenic primers are labeled with
a single fluorophore on a base close to the 3′ end with no quencher required. A tail of 5–7 nt is added to the 5′ end of the
primer to form a blunt-end hairpin when the primer is not incorporated into a PCR product. This design provides a low initial
fluorescence of the primers that increases up to 8-fold upon formation of the PCR product. The hairpin oligonucleotides (ΔG from –1.6 to –5.8 kcal/mol) may be as efficient as linear primers and provide additional specificity to the PCR by preventing primer-dimers
and mispriming. Multiple fluorogenic primers were designed by specialized software and used for real-time quantitation of
c-myc and IL-4 cDNAs in the presence of reference genes such as β‐actin, GAPDH and 18S rRNA. Targets of 10–107 copies were detected with precision in PCR using FAM-labeled primers for variable genes and JOE-labeled primers for the reference
genes. This method was also used to detect single nucleotide polymorphism of the human retinal degeneration gene by allele-specific
PCR with end-point detection using a fluorescent plate reader or a UV-transilluminator. We conclude that fluorogenic mono-labeled
primers are an efficient and cost-effective alternative to FRET-labeled oligonucleotides.
With the increasing demand for higher throughput single nucleotide polymorphism (SNP) genotyping, the quantity of genomic
DNA often falls short of the number of assays required. We investigated the use of degenerate oligonucleotide primed polymerase
chain reaction (DOP‐PCR) to generate a template for our SNP genotyping methodology of fluorescence polarization template‐directed
dye‐terminator incorporation detection. DOP‐PCR employs a degenerate primer (5′‐CCGACTCGAGNNNNNNATGTGG‐3′) to produce non‐specific
uniform amplification of DNA. This approach has been successfully applied to microsatellite genotyping. We compared genotyping
of DOP‐PCR‐amplified genomic DNA to genomic DNA as a template. Results were analyzed with respect to feasibility, allele loss
of alleles, genotyping accuracy and storage conditions in a high‐throughput genotyping environment. DOP‐PCR yielded overall
satisfactory results, with a certain loss in accuracy and quality of the genotype assignments. Accuracy and quality of genotypes
generated from the DOP‐PCR template also depended on storage conditions. Adding carrier DNA to a final concentration of 10
ng/µl improved results. In conclusion, we have successfully used DOP‐PCR to amplify our genomic DNA collection for subsequent
SNP genotyping as a standard process.
Detection and visualisation of nucleic acids is integral to genome analyses. Exponential amplification procedures have provided
the means for the manipulation of nucleic acid sequences, which were otherwise inaccessible. We describe the development and
application of a universal method for the labelling of any PCR product using a single end-labelled primer. Amplification was
performed in a single reaction with the resulting amplicon labelled to a high specific activity. The method was adapted to
a wide range of PCRs and significantly reduced the expense of such analyses.
Delineating a species' geographic range using the spatial distribution of museum specimens or even contemporary detection-non-detection data can be difficult. This is particularly true at the periphery of a species range where species' distributions are often disjunct. Wolverines (Gulo gulo) are wide-ranging mammals with discontinuous and potentially isolated populations at the periphery of their range. One potentially disjunct population occurred in the Sierra Nevada Mountains, California, USA, and appears to have been extirpated by the 1930s. Many early 20th century naturalists believed that this population was connected to other populations occurring in the Cascade Range of northern California, Oregon, and Washington, USA, but a recent analysis of historical records suggests that California wolverines were isolated from other populations in North America. We used DNA extracted from museum specimens to examine whether California wolverines were isolated. Both nuclear and mitochondrial DNA data indicate that California wolverines were genetically distinct from extant populations, suggesting long-term isolation. We identified 2 new control region (mitochondrial DNA) haplotypes located only within California. We used these data and referenced sequences from the Rocky Mountains, USA, to make inferences regarding potential wolverine translocations into California. In addition, we used these genetic data to make inferences about wolverine conservation throughout western North America.
We assessed the effect of DNA extraction and sample preservation methods on the DNA yield and quality obtained from earthworm (Aporrectodea caliginosa Savigny) gut samples and on the results obtained by bacterial and fungal automated ribosomal intergenic spacer analysis (ARISA) of DNA extracts. Methods based on a hexadecyltrimethylammonium bromide dithiotreitol (CTAB-DTT) extraction buffer yielded more favourable results than those based on a sodium dodecyl sulphate (SDS) buffer. For both of these buffers, incorporation of a bead-beating during the lysis step increased the ARISA-derived bacterial ribotype numbers and diversity estimates, as determined for gut wall samples (P b 0.01). Although spectrophotometric analysis indicated that DNA extracted by the CTAB-DTT and SDS-based methods were of comparable quality (P z 0.05), the former method yielded N1.5 times more DNA from both gut contents and gut walls of earthworms than the latter method (both incorporating the bead beating step) (P b 0.01). ARISA analysis detected more reproducible ribotypes and more microbial diversity in DNA extracted by the CTAB-DTT-as compared to the SDS-based method (P b 0.01). Significant difference between bacterial communities of gut contents and gut walls were detected within DNA extracted by the CTAB-DTT (but not by the SDS-based) method (Global R = 0.76, P b 0.001, analysis of similarity). Using the CTAB-DTT-based method, we showed that earthworm preservation in ethanol yielded higher quality DNA from gut contents than preservation in either chloroform or liquid N, as determined by spectrophotometry, PCR inhibition analysis and bacterial and fungal ARISA (P b 0.05). Bacterial or fungal communities in the gut contents of fresh and ethanol-preserved earthworms were more similar and were significantly different from those of earthworms preserved in chloroform or liquid N (Global R = 0.79 and 0.83 for bacteria and fungi, respectively; P b 0.001, analysis of similarity). We propose that ethanol preservation and the CTAB-DTT-based DNA extraction method described herein are also suitable for the analysis of gut-associated microbiota in other soil and sediment feeding invertebrates.
Estimating black bear (Ursus americanus) population size is a difficult but important requirement when justifying harvest quotas and managing populations. Advancements in genetic techniques provide a means to identify individual bears using DNA contained in tissue and hair samples, thereby permitting estimates of population abundance based on established mark-capture-recapture methodology. We expand on previous noninvasive population-estimation work by geographically extending sampling areas (36,848 km(2)) to include the entire Northern Lower Peninsula (NLP) of Michigan, USA. We selected sampling locations randomly within biologically relevant bear habitat and used barbed wire hair snares to collect hair samples. Unlike previous noninvasive studies, we used tissue samples from harvested bears as an additional sampling occasion to increase recapture probabilities. We developed subsampling protocols to account for both spatial and temporal variance in sample distribution and variation in sample quality using recently published quality control protocols using 5 microsatellite loci. We quantified genotyping errors using samples from harvested bears and estimated abundance using statistical models that accounted for genotyping error. We estimated the population of yearling and adult black bears in the NLP to be 1,882 bears (95% CI = 1,389-2,551 bears). The derived population estimate with a 15% coefficient of variation was used by wildlife managers to examine the sustainability of harvest over a large geographic area.
Abstract We investigated the effect of multiple variables on the amplification success rate of microsatellite DNA extracted from faeces of wild Eurasian otters. The success rate was affected by (i) type of sample, with higher success rates in anal jelly samples than faeces, and (ii) temperature, with a negative effect of increased temperature at time of collection. To increase the effectiveness of microsatellite genotyping of otter faeces, we recommend collecting samples in cold months and early in the morning, preferably in a frozen state, and the collection of anal jelly samples, or the jelly part from faeces, whenever possible.
Abstract Improvements in the determination of individual genotypes from samples with low DNA quantity and quality are of prime importance in molecular ecology and conservation for reliable genetic individual identification (molecular tagging using microsatllites loci). Thus, errors (e.g. allelic dropout and false allele) appearing during samples genotyping must be monitored and eliminated as far as possible. The multitubes approach is a very effective but a costly and time-consuming solution. In this paper, we present a simulation software that allows evaluation of the effect of genotyping errors on genetic identification of individuals and the effectiveness of a multitubes approach to correct these errors.
Track plates and cameras are proven methods for detecting and identifying fishers (Martes pennanti) and other mesocarnivores. But these methods are inadequate to achieve demographic and population-monitoring objectives that require identifying sex and individuals. Although noninvasive collection of biological material for genetic analysis (i.e., hair-snaring methods) may help achieve these objectives, they have yet to be evaluated. We incorporated wire- and glue-snares into track-plate enclosures in bait stations deployed at 3 locations in California, USA, in 2002 and 2003 to compare their efficacy. We detected 5 species of carnivores via their tracks, fisher and marten (M. americana) most frequently. We collected 96 hair samples, 71 (80%) of which had sufficient DNA to yield a species identification. The wire-snares were more permeable to all species, but both snares were permeable to fishers. Glue-snares were more effective at collecting hair than the wire-snare configuration used. Small species, such as the marten, did not readily leave hair on the wire-snare. Glue was more cumbersome to handle than wire, but, given that it collected more hair and more reliably, we favor use of a glue-snare for fisher surveys. Glue also was effective at collecting hair that was correctly identified as marten on 72% of visits by martens. The hair-snaring method resulted in a 58% and 75% rate of successful identification of fishers that entered the enclosure for wire and glue, respectively. Most failures were due to either insufficient DNA in the sample or no hair snared during a visit to the bait. The success of verifying the presence of a species also was affected when a second species visited the station between check intervals. We believe that changes in laboratory technique can mitigate this problem. Although only 75% of the visits by fishers to glue-snares resulted in hair that yielded sufficient DNA for confirmation, we believe this success rate guarantees a high probability that the presence of a fisher would be confirmed by at least one of their visits to a multiple-station sample unit in which each station is checked several times. A sample of hair from fishers and martens were selected for individual identification, and 9 of the 12 fisher samples had sufficient DNA to identify a minimum of 6 different individuals. Track and genetic methods need not compete for use by carnivore surveyors; each is inexpensive enough when deployed in the integrated unit we tested here to justify their use as companion methods. However, additional innovations will be necessary to increase the number of species that can be detected by track and genetic means at the same location.
Noninvasive genetic sampling provides great potential for research and management applications in wildlife biology. Researchers can obtain DNA from a variety of sources including hair, feces, urine, feathers, shed skin, saliva, and egg shells without handling or observing animals. These samples can then be used to identify the presence of rare or elusive species, count and identify individuals, determine gender, and identify diet items, or samples can be used to evaluate genetic diversity, population structure, and mating system. We review the recent advancements and techniques used for identifying species, individuals, and gender. We also address the potential pitfalls of noninvasive genetic sampling and provide recommendations for laboratory- and field-based methods to improve the reliability and accuracy of data collected from noninvasive genetic samples.
Resolving conflicts between predators and livestock producers depends on obtaining reliable information about the predators that kill livestock. We used salivary DNA obtained from attack wounds on domestic sheep carcasses to identify the species of predator responsible for the kill, as well as the sex and individual identity of coyotes (Canis latrans) that killed sheep. Coyotes killed 36 of 37 depredated sheep. Breeding pairs whose territories overlapped sheep grazing areas were the primary predators on domestic sheep, and only breeding pairs killed multiple sheep. Breeding males, acting alone or with their mate, were involved in 21 of 25 kills. Breeding females participated in 13 kills, but only 1 breeding female killed sheep on her own. Transient females did not kill sheep, and both kills by transient males occurred in territories with a breeding vacancy. Our results suggest that predator control should be targeted at breeding male coyotes. Salivary DNA is a potentially powerful means of both investigating predation patterns and evaluating the effectiveness of control at targeting individuals that kill livestock.
Noninvasive sampling of mammalian hairs for surveying their populations and for providing density estimations is widely applicable in wildlife ecology and management. However, the efficiency of the method may differ depending on the species or local circumstances. We modified a method of hair trapping from free-ranging Eurasian lynx (Lynx lynx) to collect DNA samples to work in a low-density population. We constructed hair traps based on a device developed for Canada lynx and assessed their effectiveness in Białowieża Forest, Poland. We set 153 hair traps baited with beaver (Castor canadensis) castoreum and catnip oil at points previously used by lynx for scent-marking. We conducted the study in 2 consecutive winter and summer seasons during 2003–2004. Lynx rubbed 22–46% of the hair traps in 5 different trapping sessions. Lynx were more likely to rub hair traps set directly at scent-marking points on conspicuous marked objects than when they were set some distance (1–3 m) from the marked objects. Efficiency of hair-trapping sessions increased from 30.1 to 46.4% after selecting the most likely points. The percentage of traps visited and rubbed by lynx was higher in winter than summer in 2 consecutive years (30.1 vs. 22.2% and 46.4 vs. 23.3%, respectively), which may be related to mating behavior. This method proved efficient for monitoring low-density Eurasian lynx populations.
Noninvasive hair and fecal DNA sampling provides a means of collecting information on elusive species, while causing little or no disturbance. However, current methods of hair collection do not preclude multiple sampling, thus risking sample contamination. We developed a hair snare that prevents multiple sampling, is cost-effective, easy to construct, and safe for target and nontarget species. Our initial field tests on endangered San Joaquin kit foxes (Vulpes macrotis mutica) and swift foxes (Vulpes velox) suggest that this hair snare may be effective in collecting uncontaminated samples for DNA analysis.
Abstract We developed a snare for collection of black bear (Ursus americanus) hair that obtained a unique hair sample at each snare site, improved the quantity of collected hair compared to barbed-wire corrals, and was easy to deploy over a wide range of topographical features and habitat conditions. This device allowed us to implement intensive sampling methodology needed in mark-recapture experiments with minimal effort. By improving the quantity of hair collected, we also lowered the potential for bear identification errors at the lab. During 2003–2004, bears in 2 study areas triggered snares 1,104 times, which resulted in the collection of 981 hair samples. Of the samples we collected, 79% (775) produced valid genetic data. In 2003, 454 samples identified 79 genetically distinct individuals, and 321 samples identified 86 genetically distinct individuals in 2004. Analysis of capture-recapture data indicated that capture probabilities were affected by heterogeneity among individuals and behavioral responses, but showed little evidence of time effects. Consequently, we used the Pollock and Otto (1983) estimator for model Mbh to estimate abundance with reasonably good precision (CV: 12–14%). Density on the Steamboat and Toketee, Oregon, USA, study areas over the 2-year period averaged 19 bears/100 km2 and 22 bears/100 km2, respectively. Average capture and recapture probabilities over the 2 years of the study were 30% and 63%, respectively, indicating a trap-prone behavioral response. Knowledge of bear densities on the Steamboat and Toketee study areas will enable managers to set hunting quotas, advise land management agencies on habitat issues, and create a baseline database to assist in the long-term monitoring of bear trends in a changing landscape.
The ability to rapidly and reliably determine the sex of birds is very important for successful captive-bird breeding programs, as well as for field research. Visual inspection of adult birds is sufficient for sexually dimorphic species, but nestlings and monomorphic species are difficult, if not impossible, to sex by sight only. A method for rapid extraction of gDNA from blood, shell-membrane blood vessels, and fully grown feathers, using Chelex, and the PCR conditions for determination of sex-specific bands in 47 species (39 genera, 21 families, and 10 orders) are described. The PCR primers used amplify a length of DNA spanning an intron in the CHD-1 gene, which is present on both the W and Z chromosomes. The intron differs in size between the two sex chromosomes, resulting in PCR products that separate into two bands for females and a single band for males in most avian species (except ratites). Because this simple technique uses Chelex, a rapid gDNA isolation protocol, and sets of PCR primers independent of restriction enzyme digestion, birds can be accurately sexed within 5 hr of sample collection. Zoo Biol 22:561–571, 2003. © 2003 Wiley-Liss, Inc.
Hair samples are an increasingly important DNA source for wildlife studies, yet optimal storage methods and DNA degradation rates have not been rigorously evaluated. We tested amplification success rates over a one-year storage period for DNA extracted from brown bear (Ursus arctos) hair samples preserved using silica desiccation and −20 °C freezing. For three nuclear DNA microsatellites, success rates decreased significantly after a six-month time point, regardless of storage method. For a 1000 bp mitochondrial fragment, a similar decrease occurred after a two-week time point. Minimizing delays between collection and DNA extraction will maximize success rates for hair-based noninvasive genetic sampling projects.
Naturally shed hairs are an important source of genetic material for both conservation and forensics but are notoriously poor sources of DNA. DNA degradation in hair roots is caused by apoptosis as part of the cycle of hair growth and by autolysis in decomposing animals. Shed hairs are additionally exposed to degenerative environmental processes. However, genetic studies rarely examine hair root morphologies or refer to root growth phases prior to analysis, and detailed knowledge of the rapidity of DNA degradation amongst shed hairs is lacking. We examined the effects of biological and environmental processes on western lowland gorilla (Gorilla gorilla gorilla Savage and Wyman) hair roots with respect to morphological characteristics and polymerase chain reaction (PCR) success at eight nuclear loci. Root type frequencies indicate that gorilla body hairs may exhibit a longer telogen phase than human head hairs. All plucked hair root types amplified more efficiently than shed hairs, and only 41% of shed hairs had root types considered suitable for genotyping. Telogen hairs from fresh nests were four-fold more useful for genotyping if the roots were associated with translucent epithelial tissue, and preselection of these root types doubled the overall data-yield to 58%. Nest age correlated with root morphology and PCR success, and PCR success was almost halved after 3 days of exposure. Finally, an association between postmortem interval, root morphology, and PCR success was observed that was consistent with postmortem changes reported in human head hairs. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 281–294.
The endemic mountain pygmy-possum, Burramys parvus, is an endangered Australian marsupial restricted to mainland alpine regions. Population structure, breeding system and gene flow are crucial for the development of effective conservation strategies, but have not been investigated in B. parvus. Here we have isolated eight polymorphic microsatellite markers from B. parvus to investigate these parameters. We found two to 12 alleles with observed heterozygosity values ranging from 0.321 to 0.878 for these loci in initial estimates from a single population.
Individual genotypes determined from noninvasive DNA samples (typically extracted from shed hairs or scats) are used to estimate population size in monitoring projects of elusive species. However, polymerase chain reaction (PCR) success rates usually are lower, and genotyping errors higher than in standard population genetic surveys, due to DNA degradation or contamination in aged field samples. In this study, we evaluate the results of common garden experiments showing that DNA degradation is significant in wolf (Canis lupus) scats older than 3 days, and it is enhanced in scats in direct contact with soil. A storage test showed that samples kept frozen in 95% ethanol performed better compared to other methods. However, variance of PCR success among samples was high, independent on sample age or storage condition. The detrimental consequences of DNA degradation can be avoided by collecting scat samples as fresh as possible, and implementing efficient multitube procedures and stringent quality control of the laboratory results. Efficient multitube procedures can produce reliable data, like in this study, which showed that the consensus genotypes obtained from excremental DNA exactly matched distinct reference genotypes obtained from wolf blood samples.
The use of historical and ancient tissue samples for genetic analysis is increasing, with ever greater numbers of samples proving to contain sufficient mitochondrial and even nuclear DNA for multilocus analysis. DNA yield, however, remains highly variable and largely unpredictable based solely on sample morphology or age. Quantification of DNA from his- torical and degraded samples can greatly improve efficiency of screening DNA extracts prior to attempting sequencing or genotyping, but requires sequence-specific quantitative polymerase chain reaction (qPCR) based assays to detect such minute quantities of degraded DNA. We present two qPCR assays for marine mammal DNA quantification, and results from analysis of DNA extracted from preserved soft tissues, bone, baleen, and tooth from several cetacean species. These two assays have been shown to amplify DNA from 26 marine mammal species representing 12 families of pinnipeds and cetaceans. Our results indicate that different tissues retain different ratios of mitochondrial to nuclear DNA, and may be more or less suitable for analysis of nuclear loci. Specifically, historical bone and tooth samples average 60-fold higher ratio of mitochondrial DNA to nuclear DNA than pre- served fresh soft tissue, and the ratio is almost 8000-fold higher in baleen.
Biologists need a variety of tools to determine the population and genetic status of the ocelot (Leopardus pardalis), an elusive Neotropical cat that favors dense habitats. We developed and tested a technique that entices ocelots to rub on scented hair snares and uses DNA analysis of the hair to determine species, gender, and individual identity. Twenty-seven (84%) of 32 captive ocelots rubbed against the scented pads. In field tests at Laguna Atascosa National Wildlife Refuge in south Texas, we detected a minimum of 6 ocelots, including at least 3 of 4 radiocollared animals. Using a 6-locus microsatellite analysis, we made individual identification for 10 of 20 samples. Scented hair snares can provide useful information on the population and genetic status of ocelots and identifi- cation of key areas and connecting linkages. We suggest that surveys for ocelots deploy 1 station per 25-50 ha and check them every 1-2 weeks.