Isolation and characterization of microsatellite loci in the western pearlshell mussel, Margaritifera falcata (Gould)

Ten microsatellite loci were isolated from the western pearlshell, Margaritifera falcata (Gould, 1850) and characterized in populations from Washington and Montana, USA. We also assessed eight microsatellite loci developed in M. margaritifera, two of which showed utility. Both of our test populations showed significant heterozygote deficiencies at most loci, consistent with a hermaphroditic life history. Populations differed markedly with respect to allelic richness, allele frequencies and numbers of identical multilocus genotypes. This panel of loci should prove useful in describing gene flow and genetic diversity patterns among M. falcata populations, information that should aid future conservation efforts.     

Quantitative polymerase chain reaction analysis of DNA from noninvasive samples for accurate microsatellite genotyping of wild chimpanzees (Pan troglodytes verus)

Noninvasive samples are useful for molecular genetic analyses of wild animal populations. However, the low DNA content of such samples makes DNA amplification difficult, and there is the potential for erroneous results when one of two alleles at heterozygous microsatellite loci fails to be amplified. In this study we describe an assay designed to measure the amount of amplifiable nuclear DNA in low DNA concentration extracts from noninvasive samples. We describe the range of DNA amounts obtained from chimpanzee faeces and shed hair samples and formulate a new efficient approach for accurate microsatellite genotyping. Prescreening of extracts for DNA quantity is recommended for sorting of samples for likely success and reliability. Repetition of results remains extensive for analysis of microsatellite amplifications beginning from low starting amounts of DNA, but is reduced for those with higher DNA content.     

An evaluation of long-term preservation methods for brown bear (Ursus arctos) faecal DNA samples

Relatively few large-scale faecal DNA studieshave been initiated due to difficulties inamplifying low quality and quantity DNAtemplate. To improve brown bear faecal DNA PCRamplification success rates and to determinepost collection sample longevity, fivepreservation methods were evaluated: 90%ethanol, DETs buffer, silica-dried, oven-driedstored at room temperature, and oven-driedstored at –20 °C. Preservationeffectiveness was evaluated for 50 faecalsamples by PCR amplification of a mitochondrialDNA (mtDNA) locus (146 bp) and a nuclear DNA(nDNA) locus (200 bp) at time points of oneweek, one month, three months and six months. Preservation method and storage timesignificantly impacted mtDNA and nDNAamplification success rates. For mtDNA, allpreservation methods had 75% success atone week, but storage time had a significantimpact on the effectiveness of the silicapreservation method. Ethanol preserved sampleshad the highest success rates for both mtDNA(86.5%) and nDNA (84%). Nuclear DNAamplification success rates ranged from 26–88%, and storage time had a significant impacton all methods but ethanol. Preservationmethod and storage time should be importantconsiderations for researchers planningprojects utilizing faecal DNA. We recommendpreservation of faecal samples in 90% ethanolwhen feasible, although when collecting inremote field conditions or for both DNA andhormone assays a dry collection method may beadvantageous.     

Noninvasive Saliva Collection for DNA Analyses From Free‐Ranging Tibetan Macaques (Macaca thibetana)

Cryptic and endangered fauna, including many primate taxa, pose challenges for noninvasive collection of biomaterials. As a result, application of noninvasive genotyping to primates has been limited to the use of samples such as feces and hair for the extraction of PCR‐amplifiable DNA. We present a method for noninvasive collection of saliva from habituated, free‐ranging monkeys. The method utilizes a low‐cost apparatus that controls for contamination and is usable with individual, free‐ranging primates. Saliva samples were collected from 18 individuals in a population of Tibetan macaques (Macaca thibetana) in the Valley of Wild Monkeys in Huangshan, People's Republic of China. DNA was extracted from these samples and PCR‐amplified for both mitochondrial and nuclear genes, Cytochrome B and MHC‐DR Beta 1, respectively. These results indicate this is an effective technique for the noninvasive collection of saliva across age and sex class, and dominance rank in a free‐ranging, terrestrial primate species. This device could have wide application for obtaining high‐quality saliva samples from free‐ranging primate populations for use in epidemiological studies, hormonal analyses of HPA axis function, pathogen screening, noninvasive genotyping, and behavioral genetics. Am. J. Primatol. 74:1064‐1070, 2012. © 2012 Wiley Periodicals, Inc.     

The influence of diet on faecal DNA amplification and sex identification in brown bears (Ursus arctos)

To evaluate the influence of diet on faecal DNA amplification, 11 captive brown bears (Ursus arctos) were placed on six restricted diets: grass (Trifolium spp., Haplopappus hirtus and Poa pratensis), alfalfa (Lupinus spp.), carrots (Daucus spp.), white-tailed deer (Odocoileus virginianus), blueberries (Vaccinium spp.) and salmon (Salmo spp.). DNA was extracted from 50 faecal samples of each restricted diet, and amplification of brown bear DNA was attempted for a mitochondrial DNA (mtDNA) locus and nuclear DNA (nDNA) locus. For mtDNA, no significant differences were observed in amplification success rates across diets. For nDNA, amplification success rates for salmon diet extracts were significantly lower than all other diet extracts (P < 0.001). To evaluate the accuracy of faecal DNA sex identification when female carnivores consume male mammalian prey, female bears were fed male white-tailed deer. Four of 10 extracts amplified, and all extracts were incorrectly scored as male due to amplification of X and Y-chromosome fragments. The potential biases highlighted in this study have broad implications for researchers using faecal DNA for individual and sex identification, and should be evaluated in other species.     

Sampling large geographic areas for rare species using environmental DNA: a study of bull trout Salvelinus confluentus occupancy in western Montana

This study tested the efficacy of environmental DNA (eDNA) sampling to delineate the distribution of bull trout Salvelinus confluentus in headwater streams in western Montana, U.S.A. Surveys proved fast, reliable and sensitive: 124 samples were collected across five basins by a single crew in c. 8 days. Results were largely consistent with past electrofishing, but, in a basin where S. confluentus were known to be scarce, eDNA samples indicated that S. confluentus were more broadly distributed than previously thought.     

Carrion fly‐derived DNA as a tool for comprehensive and cost‐effective assessment of mammalian biodiversity

Large‐scale monitoring schemes are essential in assessing global mammalian biodiversity, and in this framework, leeches have recently been promoted as an indirect source of DNA from terrestrial mammal species. Carrion feeding flies are ubiquitous and can be expected to feed on many vertebrate carcasses. Hence, we tested whether fly‐derived DNA analysis may also serve as a novel tool for mammalian diversity surveys. We screened DNA extracted from 201 carrion flies collected in tropical habitats of Côte d'Ivoire and Madagascar for mammal DNA using multiple PCR systems and retrieved DNA sequences from a diverse set of species (22 in Côte d'Ivoire, four in Madagascar) exploiting distinct forest strata and displaying a broad range of body sizes. Deep sequencing of amplicons generated from pools of flies performed equally well as individual sequencing approaches. We conclude that the analysis of fly‐derived DNA can be implemented in a very rapid and cost‐effective manner and will give a relatively unbiased picture of local mammal diversity. Carrion flies therefore represent an extraordinary and thus far unexploited resource of mammal DNA, which will probably prove useful for future inventories of wild mammal communities.     

Urine as another potential source for template DNA in polymerase chain reaction (PCR)

This technical note examines the potential for preparing template DNA in polymerase chain reactions (PCR) from urine in Japanese macaques (Macaca fuscata). Microsatellite band patterns from urine samples showed close agreement with those of blood and fecal samples, and only a few hundred μl of urine yielded a template DNA for PCR. This research will increase the opportunity for scientists to examine the genetic backgrounds of their target animals by using non‐invasive sample collection in the wild. Am. J. Primatol. 48:299–304, 1999. © 1999 Wiley‐Liss, Inc.     

A new method for DNA extraction from feces and hair shafts of the South China tiger (Panthera tigris amoyensis)

It is commonly known that tigers (Panthera tigris) groom themselves by licking their coats, which leads to an abundance of hairs in their feces. These hairs are designated specially as “fecal hairs”. In our study, in order to explore fecal hairs potential as a DNA source for genetic analysis, 55 fecal hair samples were collected from 23 captive South China tigers (P. t. amoyensis). According to the amplification of mitochondrial primers loop F and loop R, DNA quality of noninvasive samples were grouped into three grades: grade I—the highest-quality DNA, grade II—high-quality DNA, and grade III—poor-quality DNA. No failed amplifications on microsatellite primers and only 0.27% genotyping errors occurred with grade I fecal hair DNA, as compared with 9.4% failed amplifications on microsatellite primers and 9.5% genotyping errors with grade II fecal hair DNA. It was found that 25.45% of fecal hair DNA was grade I and 65.45 and 10.00% of fecal hair DNA were grades II and III, respectively, as compared with 4.35% grade I fecal DNA and 34.78 and 60.87% grades II and III fecal DNA, respectively. Thus, higher-quality DNA can be extracted from fecal hairs than feces. In addition, DNA could be extracted from hair shafts of tigers and a minimum of 2000 hair shafts were required for visible DNA bands on a 1% agarose gel. These findings demonstrate that fecal hairs may serve as a convenient and reliable genomic DNA source for genotype analysis. Zoo Biol 28:49–58, 2009. © 2008 Wiley-Liss, Inc.     

Modern pollen-vegetation relationships within a small Mediterranean temporary pool (western Morocco)

Morocco is rich in temporary pools which harbour numerous rare plant species. Long-term conservation of such threatened plant communities should be based on the understanding of their past dynamics. Despite conditions unfavourable to pollen preservation, surface sediments of acidic temporary pools are shown to contain pollen assemblages likely to allow vegetation reconstruction. Knowledge of the modern relationships between pollen and vegetation is, however, necessary for interpreting fossil data in terms of past vegetation. Surface pollen assemblages and floristic surveys of a temporary pool in Benslimane forest, western Morocco, are compared in order to evaluate the pollen record of the local hydrophytic vegetation. Floristic surveys were carried out for 12 years (1996-2008) along two crossing permanent transects. A set of 21 surface-sediment samples, taken along the same transects in 2007, were analysed for pollen. The spatial relationships between vegetation and pollen assemblages are explored by means of multivariate analyses, statistical tests and linear regressions. The calculation of representation indices moreover allows proposing quantitative ways for pollen-based plant-abundance reconstruction.Results reveal that the vegetation structure along the hydrological gradient is well recorded in the pollen assemblages, with: (1) a marginal zone characterised by terrestrial taxa and rare amphibious taxa (Elatine, Pilularia), (2) an intermediate zone of amphibious taxa (Alisma-type, Illecebrum/Paronychia, Isoetes velata-type), and (3) a central zone of aquatics (Myriophyllum alterniflorum, Ranunculus-type). The best correlation between the pollen record and total pool vegetation was found in the centre of the pool, which supports the reliability of the study of a single core from the centre of the pool for the reconstruction of the past dynamics of the local hydrophytic vegetation. Both the qualitative ‘community’ approach (representation indices and indicator pollen taxa) and the quantitative ‘taxa’ approach (correction factors) suggest that reconstructions of past populations can be achieved from a few taxa, namely Isoetes velata-type, Myriophyllum alterniflorum and Ranunculus-type. For these taxa, regression parameters (slope and y-intercept) have been calculated between pollen percentages and plant percentages in present vegetation, and between pollen influxes and plant abundances, respectively. These parameters can be extended to interpret fossil data from other temporary pools within the same region to reconstruct their relative and absolute past plant abundances.     

Factors affecting the amount of genomic DNA extracted from ape faeces and the identification of an improved sample storage method

Abstract Genetic analysis using noninvasively collected samples such as faeces continues to pose a formidable challenge because of unpredictable variation in the extent to which usable DNA is obtained. We investigated the influence of multiple variables on the quantity of DNA extracted from faecal samples from wild mountain gorillas and chimpanzees. There was a small negative correlation between temperature at time of collection and the amount of DNA obtained. Storage of samples either in RNAlater solution or dried using silica gel beads produced similar results, but significantly higher amounts of DNA were obtained using a novel protocol that combines a short period of storage in ethanol with subsequent desiccation using silica.     

Abundance,genetic diversity and conservation of Louisiana black bears (<Emphasis Type="Italic">Ursus americanus luteolus</Emphasis>) as detected through noninvasive sampling

Although the Louisiana black bear (Ursus americanus luteolus) is currently listed as threatened under the Endangered Species Act, there have been no attempts to estimate range-wide abundance. This subspecies was thought to occupy a near contiguous range across southern Mississippi, Louisiana and east Texas but is now restricted to three isolated areas in Louisiana. In 1964, Louisiana initiated a restocking program in which black bears from Minnesota were introduced into two of these areas. It is not clear how the additions affected population structure or if substantial breeding occurred between native and introduced bears. Using baited sites to snare hair samples, and microsatellite DNA analysis to distinguish individuals, we estimated abundance of two geographically isolated bear populations in south central Louisiana: Inland and Coastal. Additionally, we examined genetic variation both within and between the two populations. Mark recapture analysis of the distribution of individual captures during two primary sampling periods resulted in population estimates of 77 ± 9 for Coastal and 41 ± 6 for Inland. Genetic analysis revealed significant population differentiation (F _ST = 0.206) between the two populations. The apparently smaller Inland population exhibited more diversity than the Coastal, which suggests that the genetic structure of the Inland population has been influenced by the reintroduction. Both of these populations are isolated and face considerable demographic and genetic threats, thus conservation measures to protect both are warranted. However, the Coastal population is more representative of Louisiana black bears prior to reintroduction and special consideration should be given to insure its integrity.     

Population structure of brush-tailed rock-wallaby (Petrogale penicillata) colonies inferred from analysis of faecal DNA

Genetic data obtained using faecal DNA were used to elucidate the population structure of four brush-tailed rock-wallaby (Petrogale penicillata) colonies located in Wollemi National Park, New South Wales. The results suggested that the four sampled colonies are genetically differentiated and do not form a panmictic unit. Based on assignment tests, approximately 5% of sampled individuals were inferred to be dispersers and both male and female migrants were detected. Multilocus spatial autocorrelation analyses provided evidence for increased philopatry among females compared to males within the largest colony in the valley. Females in close spatial proximity were more genetically similar than expected under a random distribution of females, and females separated by more than 400 m were less genetically similar than expected. In contrast, there was no evidence of a significant clustering of related males. This suggests that within-colony dispersal is male biased. We also investigated the best strategies for conserving genetic diversity in this population. All of the four sampled colonies were found to contain distinct components of the genetic diversity of the Wolgan Valley P. penicillata population and loss of any colony is likely to result in the loss of unique alleles. Conservation and management plans should take into account that these colonies represent genetically differentiated discrete subpopulations. This approach is also the best strategy for maintaining the genetic diversity of the populations in this valley.     

Increased DNA typing success for feces and feathers of capercaillie (Tetrao urogallus) and black grouse (Tetrao tetrix)

Noninvasive sampling, for example, of droppings or feathers, is a promising approach for molecular genetic studies on endangered and elusive animal species. Yet, such specimens are known for containing only minute amounts of DNA, resulting in lower typing success rates relative to analyses on fresh tissues such as muscle or blood. Furthermore, artefactual signals as well as contamination are more likely to occur when DNA is limited. To increase the reliability of DNA typing from noninvasive samples, optimized DNA extraction and polymerase chain reaction protocols were developed, taking advantage of developments in the forensic field aiming at successful molecular genetic analysis of DNA templates being low in quality and quantity. In the framework of an extensive monitoring project on population dynamics of capercaillie and black grouse in the Tyrolean Alps, feces samples and molted feathers from both species were collected. On a subset comprising about 200 specimens of either species, eight polymorphic short tandem repeat (STR) markers were analyzed to test these improved protocols. Besides optimizing DNA yields, both lowered sample consumption and reduced hands‐on time were achieved, and the rates of informative profiles amounted to 90.7% for capercaillie and 92.4% for black grouse. Similarly, high success rates had not been achieved in earlier studies and demonstrate the benefit of the improved methodology, which should be easily adaptable for use on animal species other than those studied here. The STR genotypes were not only powerful enough to discriminate among unrelated birds but also appeared fit for telling apart closely related animals, as indicated by Pi and Pi_sib values. The software package allelematch aided analysis of genotypes featuring possible dropout and drop‐in effects. Finally, a comparison between molecular genetic and morphology‐based species‐of‐origin determination revealed a high degree of concordance.     

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

Targeted species‐specific and community‐wide molecular diagnostics tools are being used with increasing frequency to detect invasive or rare species. Few studies have compared the sensitivity and specificity of these approaches. In the present study environmental DNA from 90 filtered seawater and 120 biofouling samples was analyzed with quantitative PCR (qPCR), droplet digital PCR (ddPCR) and metabarcoding targeting the cytochrome c oxidase I (COI) and 18S rRNA genes for the Mediterranean fanworm Sabella spallanzanii. The qPCR analyses detected S. spallanzanii in 53% of water and 85% of biofouling samples. Using ddPCR S. spallanzanii was detected in 61% of water of water and 95% of biofouling samples. There were strong relationships between COI copy numbers determined via qPCR and ddPCR (water R² = 0.81, p < .001, biofouling R² = 0.68, p < .001); however, qPCR copy numbers were on average 125‐fold lower than those measured using ddPCR. Using metabarcoding there was higher detection in water samples when targeting the COI (40%) compared to 18S rRNA (5.4%). The difference was less pronounced in biofouling samples (25% COI, 29% 18S rRNA). Occupancy modelling showed that although the occupancy estimate was higher for biofouling samples (ψ = 1.0), higher probabilities of detection were derived for water samples. Detection probabilities of ddPCR (1.0) and qPCR (0.93) were nearly double metabarcoding (0.57 to 0.27 marker dependent). Studies that aim to detect specific invasive or rare species in environmental samples should consider using targeted approaches until a detailed understanding of how community and matrix complexity, and primer biases affect metabarcoding data.     

Identification of the endangered small red brocket deer (Mazama bororo) using noninvasive genetic techniques (Mammalia; Cervidae)

The small red brocket deer Mazama bororo is one of the most endangered deer in the Neotropics. The great morphological similarities with three other sympatric brocket deer species, coupled with the fact that they inhabit densely forested habitats complicate detection and prevent the use of traditional methodologies for accurate identification of species. The ability to determine the presence of this endangered species in an area is crucial for estimating its distribution range, and is critical for establishing conservation management strategies. Here we describe a fast and reliable noninvasive genetic method for species identification of Mazama species from faeces. We designed a primer set that amplifies a short 224-bp fragment of the cytochrome b and demonstrate its effectiveness in successful amplification of DNA isolated from both tissue and faecal samples. This fragment contains a BSTNI/ECORII digestion site that is unique to the endangered M. bororo. The digested polymerase chain reaction products yielded a 160-bp fragment that is clearly visible in a 2% agarose gel. Two other diagnostic sites were identified to differentiate the other three sympatric species, SspI (M. gouazoubira) and AflIII (M. americana, and M. nana).     

Stool DNA test targeting methylated syndecan-2 (SDC2) as a noninvasive screening method for colorectal cancer

Despite the steadily increasing worldwide incidence of colorectal cancer (CRC), an effective noninvasive approach for early detection of CRC is still under investigation. The guaiac-based fecal occult blood test (FOBT) and fecal immunochemical test (FIT) have gained popularity as noninvasive CRC screening tests owing to their convenience and relatively low costs. However, the FOBT and FIT have limited sensitivity and specificity. To develop a noninvasive tool for the detection of CRC, we investigated the sensitivity, specificity, and accuracy of a stool DNA test targeting methylated syndecan-2 (SDC2), which is frequently methylated in patients with CRC. The present study enrolled 62 patients diagnosed as having stage 0-IV CRC and 76 healthy participants between July 2018 and June 2019 from two institutions. Approximately 4.5 g of stool sample was collected from each participant for detection of human methylated SDC2 gene. In total, 48 of 62 (77.4%) patients with CRC showed positive results, whereas 67 out of 76 (88.2%) healthy participants showed negative results. The area under the curve of the receiver operating characteristic curve constructed was 0.872 for discrimination between patients with CRC and healthy individuals. The present study highlights the potential of the fecal methylated SDC2 test as a noninvasive detection method for CRC screening with a relatively favorable sensitivity of 77.4%, a specificity of 88.2% and a positive predictive value of 84.2% compared with other available fecal tests. Further multicenter clinical trials comprising subjects of varied ethnicities are required to validate this test for the mass screening of patients with CRC.