Left: Researchers analysing genetic data from Riverine Rabbit DNA samples. Right: Riverine Rabbit in its Karoo habitat at dusk

The value of genetic data is becoming increasingly recognised in the development of conservation management programmes.

Population genetic data can improve our understanding of the genetic relationships between extant populations, and may provide insights into the genetic responses of these populations to habitat fragmentation, range restriction and the potential migratory routes between isolated habitats. Furthermore, it may be useful to understand connectivity between fragmented (sub)populations in order to assess the potential resilience of these (sub)populations to climate change and other threats, and to inform conservation priorities. Small, isolated populations are particularly vulnerable to increased rates of genetic diversity loss over time, and this may act as a contributing factor to local extinction.

One of Africa’s most endangered mammals, the Critically Endangered Riverine Rabbit is characterised by a limited geographic distribution and small population size. The species is known from three populations: one in the Nama Karoo or northern population, where it was first discovered in 1902, and one in the Succulent Karoo or southern population, where it was found in 2003. The third or eastern population was discovered in 2018, approximately 250 kilometres east of the known distribution. A recent population genetic study revealed significant differentiation between the northern and southern Riverine Rabbit populations, as well as differentiation between subpopulations within these. However, only one locality (Uniondale) from the eastern population was included in this study; therefore, the population genetics of this population is still poorly understood.

Over the past few years, the Drylands Conservation team has been collecting rabbit scats for DNA sequence analysis to map distributions and understand genetic connectivity. Our trained scent-detection dog, Delta, helped us distinguish between the scat of different lagomorph species (hares and rabbits) in the field. We extracted DNA from more than 130 lagomorph scat samples across the three Riverine Rabbit populations and conducted DNA barcode analysis to identify the species of origin. Fifty-seven of the samples were identified as Riverine Rabbits (10 in the northern, 32 in the southern and 15 in the eastern population).

The barcode markers we use for species identification are variable among different lagomorph species, but not among individual Riverine Rabbits; therefore, markers with greater variability are required for us to make finer-scale inferences about the genetic relationships among Riverine Rabbit populations. In recent years, SNP (single-nucleotide polymorphism) markers have become the markers of choice in genetic studies, thanks to advances in next-generation sequencing (NGS) technologies that have facilitated the discovery of SNPs across entire genomes. Although each SNP alone might not carry great variability, many SNPs together can yield great power in population genetic analyses.

The raw sequence data produced included an average of 5.4 million sequence reads per individual. These data were quality filtered and assembled, and ultimately used to identify a total of 7,837 genome-wide SNPs that are polymorphic (variable) across the Riverine Rabbit population. These markers can be used for further population genetic studies on Riverine Rabbits, for example:

1. Conducting ddRADseq (using the same restriction enzymes to yield the same set of genome-wide SNP markers) in additional samples could facilitate greater insights into the evolutionary history of the species, gene flow (dispersal) analysis and (finally!) more accurate population size estimation.
2. Developing a marker panel (“SNP-chip”) for Riverine Rabbits, with a relatively small subset of these markers, could facilitate genetic profiling of individual rabbits, which may assist Nature Reserves and private landowners in estimating the number of individuals on their properties, and/or build subpopulation pedigrees through thorough scat sampling at these sites.

We conducted some preliminary population genetic analyses using our small dataset, and the results suggest that the southern population is the youngest, or most derived population of Riverine Rabbits, with the lowest genetic diversity and lowest number of private alleles. The northern and eastern populations appear to be older. Although these preliminary results provide some valuable insights, larger sample sizes will be needed to obtain more robust and fine-scale population genetic estimates and inferences.

So what does all this mean for the conservation of Riverine Rabbits?

From a population genetic perspective, conserving genetic diversity is always a priority, since this is key to a species’ ability to adapt to changing environments and survive. Our study revealed that the species comprises two, rather than three, genetic clusters, and that the northern- and eastern populations may be regarded as a single genetic cluster, or population, with ongoing gene flow between them. This genetic cluster likely represents the ancestral population, with the highest genetic diversity. Therefore, it would be more important to conserve these populations and their associated habitats than those distributed to the south-west (the southern population).

The use of scat samples to monitor Riverine Rabbits, and other elusive or threatened species has opened the door to a new non-invasive and relatively cost-effective approach to monitoring these species. For many years, researchers have been trying to estimate the size of the Riverine Rabbit population. Duthie et al. (1989) speculated that the remaining habitat could potentially support around 1,435 individuals. Collins (2016) subsequently estimated the population size at only 157-207 mature individuals, and the first population genetics study indicated an effective population size in the region of 5000 individuals. These studies were all limited by the challenge of observing these elusive animals in the wild and/or obtaining genetic samples. The use of scat as a source of genetic material can now facilitate larger sample sizes and, therefore, more robust population size estimates.

This project is supported by our longest-standing funder for Riverine Rabbit conservation work, the Zoological Society for the Conservation of Species and Populations (Zoologische Gesellschaft für Arten- und Populationsschutz – ZGAP). Together with the South African National Biodiversity Institute (SANBI) and other partners, the Endangered Wildlife Trust recently completed the regional Red List assessments for mammals, including the Riverine Rabbit. A new population size estimate of between 1,955 and 3,472 mature individuals resulted in the downlisting of the species from Critically Endangered to Endangered. We trust that this is a result, whether direct or indirect, of the ongoing conservation work, research, monitoring, and habitat protection that we, with the support of ZGAP, have contributed to over the past two decades.