Optimizing waterborne eDNA capture from waterholes in savanna systems under remote field conditions.

Environmental DNA (eDNA) is used for biodiversity assessments in a variety of ecosystems across the globe, whereby different eDNA concentration, preservation and extraction methods can outperform others depending on the sampling conditions and environment. Tropical and subtropical ecosystems in Africa are among the less studied systems concerning eDNA-based monitoring. Waterholes in arid parts of southern Africa represent important agglomeration points for terrestrial mammals, and the eDNA shed into such waterbodies provides a powerful source of information for monitoring mammalian biodiversity in the surrounding area. However, the applied methods for eDNA sampling, preservation and filtering in different freshwater systems vary greatly, and rigorous protocol testing in African freshwater systems is still lacking. This study represents the first attempt to examine variations in eDNA concentration, preservation and extraction methods under remote field conditions using waterborne eDNA in a savanna system. Collected samples were heavily affected by microalgal and bacterial growth, impeding eDNA capture and PCR success. We demonstrate clear effects of the methodological choices, which also depend on the state of eDNA. A preliminary metabarcoding run showed little taxonomic overlap in mammal species detection between two metabarcoding primers tested. We recommend water filtering (using filters with pore sizes >1 µm) over centrifugation for eDNA concentration, Longmire's solution for ambient temperature sample preservation and Qiagen's DNeasy PowerSoil Pro Kit for DNA extraction of these inhibitor-prone samples. Furthermore, at least two independent metabarcoding markers should be utilized in order to maximize species detections in metabarcoding studies.

Population genetic structure of the thick-tailed bushbaby (Otolemur crassicaudatus) from the Soutpansberg Mountain range, Northern South Africa, based on four mitochondrial DNA regions.

Greater bushbabies, strepsirrhine primates, that are distributed across central, eastern and southern Africa, with northern and eastern South Africa representing the species' most southerly distribution. Greater bushbabies are habitat specialists whose naturally fragmented habitats are getting even more fragmented due to anthropogenic activities. Currently, there is no population genetic data or study published on the species. The aim of our study was to investigate the genetic variation in a thick-tailed bushbaby, Otolemur crassicaudatus, population in the Soutpansberg mountain range, Limpopo Province, South Africa. Four mitochondrial regions, ranging from highly conserved to highly variable, were sequenced from 47 individuals. The sequences were aligned and genetic diversity, structure, as well as demographic analyses were performed. Low genetic diversity (π = 0.0007-0.0038 in coding regions and π = 0.0127 in non-coding region; Hd = 0.166-0.569 in coding regions and Hd = 0.584 in non-coding region) and sub-structuring (H = 2-3 in coding regions and H = 4 in non-coding region) was observed with two divergent haplogroups (haplotype pairwise distance = 3-5 in coding region and 6-10 in non-coding region) being identified. This suggests the population may have experienced fixation of mitochondrial haplotypes due to limited female immigration, which is consistent with philopatric species, that alternative haplotypes are not native to this population, and that there may be male mobility from adjacent populations. This study provides the first detailed insights into the mitochondrial genetic diversity of a continental African strepsirrhine primate and demonstrates the utility of mitochondrial DNA in intraspecific genetic population analyses of these primates.