Abstract
Despite the critical need for non-invasive tools to improve monitoring of wildlife populations, especially for endangered and elusive species, faecal genetic sampling has not been adopted as regular practice, largely because of the associated technical challenges and cost. Substantial work needs to be undertaken to refine sample collection and preparation methods in order to improve sample set quality and provide cost-efficient tools that can effectively support wildlife management. In this study, we collected an extensive set of forest elephant (Loxodonta cyclotis) faecal samples throughout Gabon, Central Africa, and prepared them for genotyping using 107 single-nucleotide polymorphism assays. We developed a new quantitative polymerase chain reaction (PCR) assay targeting a 130-bp nuclear DNA fragment and demonstrated its suitability for degraded samples in all three elephant species. Using this assay to compare the efficacy of two sampling methods for faecal DNA recovery, we found that sampling the whole surface of a dung pile with a swab stored in a small tube of lysis buffer was a convenient method producing high extraction success and DNA yield. We modelled the influence of faecal quality and storage time on DNA concentration in order to provide recommendations for optimized collection and storage. The maximum storage time to ensure 75% success was two months for samples collected within 24 hours after defecation and extended to four months for samples collected within one hour. Lastly, the real-time quantitative PCR assay allowed us to predict genotyping success and pre-screen DNA samples, thus further increasing the cost-efficiency of our approach. We recommend combining the validation of an efficient sampling method, the build of in-country DNA extraction capacity for reduced storage time and the development of species-specific quantitative PCR assays in order to increase the cost-efficiency of routine non-invasive DNA analyses and expand the use of next-generation markers to non-invasive samples.
Highlights
Since the early 1990’s, the use of non-invasive DNA analysis has evolved rapidly, allowing the study of species, individuals, gender, kinship and genetic variation [1,2], with clear ethical and practical advantages in endangered or elusive species [3]
Following DNA extraction, the colour of 76 DNA eluates was brown and failure of quantitative polymerase chain reaction (PCR) reactions indicated the presence of inhibitors
By simulating two different reduced panels of 15 and 40 single-nucleotide polymorphism (SNP) using high quality loci and numbers of loci commonly used for individual identification or parentage analyses [77,78], we showed that the relationship between target DNA concentration and genotyping success varied across number of markers and individual loci
Summary
Since the early 1990’s, the use of non-invasive DNA analysis has evolved rapidly, allowing the study of species, individuals, gender, kinship and genetic variation [1,2], with clear ethical and practical advantages in endangered or elusive species [3]. Cost-effectiveness of non-invasive DNA surveys has been demonstrated [9], but strongly relies on the ability to overcome technical challenges inherent in the use of faecal DNA samples. Faecal samples often contain polymerase chain reaction (PCR) inhibitors and low quantities of target DNA, and are prone to DNA degradation and co-recovery of non-target DNA. All of these parameters are strongly influenced by the diet of the sampled individual [13,14] and the environmental conditions affecting the faecal sample in the field. DNA degrades rapidly in tropical environments due to heat, humidity and a high diversity of microorganisms [15,16]
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