African forest elephants are considered a keystone species due to their positive impact on the forest ecosystem. As the largest frugivores on earth, they have unique ecological properties including their ability to carry large seeds intact over distances which is particularly beneficial for trees such as Balanitis Wilsoniana1. The signature of elephant seed dispersal is evident in the spatial distribution of trees, suggesting that elephants maintain tree diversity2. Large numbers of forest elephants ranging over large areas may be essential for ecosystem function.
In 2021 the International Union for Conservation of Nature (IUCN) declared forest elephants as critically endangered. The species are faced with the immediate threat of poaching and the long-term threat of habitat loss. The loss of elephants will have important negative consequences for the ecological trajectories of some plant species and whole ecological communities2.
The forest elephants (Loxodonta cyclotis) of Gabon are estimated to account for 60% to 70% of the global total of the species. They are the smaller than their cousins the savanna elephants (Loxodonta Africana Africana) and have a slower reproduction rate. Living in dense forests in west and central Africa makes them elusive, which in turn makes it difficult to accurately estimate population size using aerial spotting.
The research team at Agence Nationale des Parcs Nationaux (ANPN), Libreville, Gabon successfully tagged several elephants with GPS collars. This gave the researchers data to further understand the elephants’ roaming patterns, but it did not provide herd details.
Unlocking herd data through dung
The ANPN scientists turned to genetic analysis of elephant dung as genotyping could identify individual herd members to give a more accurate count of numbers in a herd, the composition of the herds social grouping, the sex of herd members and their plant consumption in type and volume.
The use of dung samples is non-invasive, providing DNA without affecting the physical integrity of the animal3. Dung genotyping has been used for over 30 years, with one of the earliest examples being a study on European brown bears in the Brenta region of Italy4.
The scientific team set out to collect elephant dung while tracking 10 males and 10 females across 20 herds. As herds defecate synchronously, the researchers covered a radius of 35 m from the tracked elephant and then a further 35 m radius from each dung pile they found.
KASP offers a straightforward, cost-effective solution 
Dung samples were analysed using competitive allele specific PCR (KASP) targeting 41 single nucleotide polymorphic loci (SNP) markers selected from 107 validated assays based on high minor allele frequencies (MAF) in forest elephants in Gabon5. An additional sexing assay targeting the orthologous sexual chromosome zinc finger protein genes ZFX/ZFY was run alongside the SNP panel5.
“Performing genotyping in Gabon was crucial for us, and KASP technology proved to be a cost-effective solution that we could implement in-house. Moreover, it was compatible with a variety of real-time PCR machines, and we initially started with a 48-well setup before transitioning to a more advanced 96-well model” said Stephanie Bourgeois, Scientific Researcher at ANPN.
“During the design phase of our study, conducting SNP genotyping on wildlife fecal samples was quite new and challenging. Engaging in discussions with scientists from LGC was very useful as they were helpful and willing to explore genotyping with fecal samples. Following a successful pilot study, we adopted KASP technology for the entire project."
“Working with KASP is generally straightforward; the genotyping plots are usually clear and easy to interpret. However, challenges arise when dealing with fecal samples, which exhibit varying DNA quantities and qualities. As anticipated, low-quality samples lead to less defined clusters, requiring additional PCR cycles. To address this, we implemented quality control measures. Initially, we quantify the amount of elephant DNA and exclude samples with insufficient quantities before genotyping. Subsequently, we replicate all samples with moderate amplification success. Finally, we replicate any ambiguous or failed datapoints from the initial run.”
New insights to improve conservation efforts
585 samples were extracted, of which 333 qualified for the minimal threshold, which identified 172 unique individuals. Average group sizes where a male elephant was the focal elephant were estimated at 4.1 (+ 2.3). Where the focal elephant was female, group sizes were estimated at 5.2 (+ 2.3)5. 
With this data, the researchers found an interesting lack of correlation between abundancy of fruit and an increase in group size, which runs counter to expectations of optimal group size and ecological constraints theory5. One theory for this is that the elephants spread out during fruiting season, as one heavily fruited tree won’t meet the calorific expectations of an elephant.
The scientists also concluded that they now have a more robust method of calculating group sizes and in turn the overall population estimates of forest elephants in Gabon. These important conservation statistics can now be used to effectively monitor the elephant population and help ensure their survival.
The future of wildlife genotyping
"Moving forward, our goal is to create SNP panels that have applicability across the entire habitat range of forest elephants, extending beyond just Gabon. Additionally, we aim to enhance our capacity for studying relatedness and population structure. Achieving this will necessitate incorporating a greater number of SNPs and optimizing our workflow for increased efficiency,” said Stephanie.
“Targeted NGS enabling the targeting of thousands of genetic markers per sample would provide invaluable insights into relatedness and fine-scale population structure. This capability would offer unprecedented precision and depth in understanding the genetic diversity, evolutionary history and adaptive potential of forest elephants. Consequently, it would significantly contribute to their conservation and management.”
Non-invasive genetic sampling for genotyping has become a highly useful tool for wildlife conservation research, with research projects successfully using a variety of samples including dung, hair, eggshells, feathers and blowhole swabs. The reducing costs of NGS and targeted NGS open up new opportunities for scientists in their goal to conserve the world’s wildlife.
To find out more about the work of Agence Nationale des Parcs Nationaux (ANPN), and to read their research, please visit https://www.facebook.com/PARCSGABON/ To find out more about the laboratories work in Gabon please visit https://www.labo.parcsgabon.com/
To find out more about KASP please visit KASP genotyping assays, PCR-based genotyping | LGC Biosearch Technologies.
Further reading
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Fruit availability and human disturbance influence forest elephant group size.
- KASP Celebrating 21 years of game changing genotyping
References
- Fred Babweteera, Peter Savill, Nick Brown, Balanites wilsoniana: Regeneration with and without elephants, Biological Conservation, Volume 134, Issue 1, 2007, Pages 40-47, ISSN 0006-3207, https://doi.org/10.1016/j.biocon.2006.08.002.
- Ahimsa Campos-Arceiz, Steve Blake, Megagardeners of the forest – the role of elephants in seed dispersal, Acta Oecologica,Volume 37, Issue 6, 2011, Pages 542-553, ISSN 1146-609X, https://www.sciencedirect.com/science/article/abs/pii/S1146609X11000154
- Zemanova MA. Non-invasive Genetic Assessment Is an Effective Wildlife Research Tool When Compared with Other Approaches. Genes (Basel). 2021 Oct 23;12(11):1672. doi: 10.3390/genes12111672
- Höss M, Kohn M, Pääbo S, Knauer F, Schröder W. Excrement analysis by PCR. Nature. 1992 Sep 17;359(6392):199. doi: 10.1038/359199a0. PMID: 1528260.
- Amelia C. Meier, Stephanie Bourgeois, Evan Adams, Hugues Bikang, Liam Jasperse-Sjolander, Matthew Lewis, Juliana Masseloux, Dana J. Morin, John R. Poulsen, Fruit availability and human disturbance influence forest elephant group size, Animal Behaviour, Volume 203, 2023, Pages 171-182, ISSN 0003-3472, https://doi.org/10.1016/j.anbehav.2023.07.002.
