Two‐step multiplex polymerase chain reaction improves the speed and accuracy of genotyping using DNA from noninvasive and museum samples

Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103 Leipzig, Germany
Molecular Ecology Resources (Impact Factor: 5.63). 12/2008; 9(1):28 - 36. DOI: 10.1111/j.1755-0998.2008.02387.x
Source: OAI

ABSTRACT Many studies in molecular ecology rely upon the genotyping of large numbers of low-quantity DNA extracts derived from noninvasive or museum specimens. To overcome low amplification success rates and avoid genotyping errors such as allelic dropout and false alleles, multiple polymerase chain reaction (PCR) replicates for each sample are typically used. Recently, two-step multiplex procedures have been introduced which drastically increase the success rate and efficiency of genotyping. However, controversy still exists concerning the amount of replication needed for suitable control of error. Here we describe the use of a two-step multiplex PCR procedure that allows rapid genotyping using at least 19 different microsatellite loci. We applied this approach to quantified amounts of noninvasive DNAs from western chimpanzee, western gorilla, mountain gorilla and black and white colobus faecal samples, as well as to DNA from ~100-year-old gorilla teeth from museums. Analysis of over 45 000 PCRs revealed average success rates of > 90% using faecal DNAs and 74% using museum specimen DNAs. Average allelic dropout rates were substantially reduced compared to those obtained using conventional singleplex PCR protocols, and reliable genotyping using low (< 25 pg) amounts of template DNA was possible. However, four to five replicates of apparently homozygous results are needed to avoid allelic dropout when using the lowest concentration DNAs (< 50 pg/reaction), suggesting that use of protocols allowing routine acceptance of homozygous genotypes after as few as three replicates may lead to unanticipated errors when applied to low-concentration DNAs.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Monitoring the population dynamics of endangered species is a critical component of conservation management strategies, but attaining accurate and precise estimates of population sizes using cost and time effective methods can be challenging. Routine censuses of the two populations of critically endangered mountain gorillas (Gorilla beringei beringei) have been conducted over the last decades to monitor populations and evaluate the effectiveness of conservation strategies. A census in 2006 of the mountain gorillas in Bwindi Impenetrable National Park, Uganda, showed the value of genetic analysis of fecal samples collected at nest sites by revealing discrepancies between the numbers of nests and uniquely identified gorillas. In this study, we censused the Bwindi gorilla population using a ‘mark-recapture’ method which involved genetic analysis of fecal samples collected in 2011 during two ‘sweep’ surveys of the entire park. We found that a notable proportion of gorillas were missed in either of the two sweeps (minimum 35% and 31%, respectively). Based on the number of genotyped gorillas and correction factors, we estimated the population to contain a minimum of 400 individuals. Using the mark-recapture approach, we infer possibly as many as 430 gorillas (95% confidence interval: 398–487). As the 2010 census of the Virunga Massif population found 480 gorillas, the total number of mountain gorillas worldwide is at least 880 individuals. Simulations using different mark-recapture models suggest that a future census of Bwindi mountain gorillas would benefit by increasing the number of sweeps in order to achieve accurate and precise results. Finally, based on our results, we recommend a sequential approach incorporating a pilot study and simulations for optimizing time and resources in large mammal genetic census studies.
    Biological Conservation 10/2014; 180. DOI:10.1016/j.biocon.2014.10.011 · 4.04 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The importance of evolutionary conservation – how understanding evolutionary forces can help guide conservation decisions – is widely recognized. However, the historical demography of many endangered species is unknown, despite the fact that this can have important implications for contemporary ecological processes and for extinction risk. Here, we reconstruct the population history of the Seychelles warbler (Acrocephalus sechellensis) – an ecological model species. By the 1960s, this species was on the brink of extinction, but its previous history is unknown. We used DNA samples from contemporary and museum specimens spanning 140 years to reconstruct bottleneck history. We found a 25% reduction in genetic diversity between museum and contemporary populations, and strong genetic structure. Simulations indicate that the Seychelles warbler was bottlenecked from a large population, with an ancestral Ne of several thousands falling to <50 within the last century. Such a rapid decline, due to anthropogenic factors, has important implications for extinction risk in the Seychelles warbler, and our results will inform conservation practices. Reconstructing the population history of this species also allows us to better understand patterns of genetic diversity, inbreeding and promiscuity in the contemporary populations. Our approaches can be applied across species to test ecological hypotheses and inform conservation.
    Evolutionary Applications 08/2014; 7(9). DOI:10.1111/eva.12191 · 4.57 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Like humans, hamadryas baboons (Papio hamadryas) are unusual among primates in having a multilevel social system and stable pair bonds, and are thus a useful model for the evolution of human sociality. While the kinship structure and sex-biased dispersal patterns that underlie human social organization have been extensively elucidated, the impact of these factors on the social system of hamadryas baboons is currently unclear. Here we use genetic analysis of individuals to elucidate the patterns of male and female dispersal across multiple levels of society in a wild population of hamadryas baboons. We characterized 244 members of five hamadryas bands at Filoha, Ethiopia by genotyping one Y-linked and 23 autosomal microsatellite loci and sequencing part of the mitochondrial hypervariable control region I. We found both male and female dispersal to be limited at the level of the band, with more movement of females than males among bands. By integrating long-term behavioral data for Band 1, we also found evidence for male and female philopatry at the clan level. We speculate that male philopatry at the clan level and female dispersal across one-male units and clans may enable both kin-based cooperation among males and the maintenance of kin bonds among females after dispersal. This would mean that, as in humans, kin bonds within both sexes are a core feature of the hamadryas social system, thus contributing to our understanding of the evolution of social organization in humans.
    Journal of Human Evolution 11/2014; DOI:10.1016/j.jhevol.2014.10.019 · 3.87 Impact Factor

Full-text (2 Sources)

Available from
May 28, 2014