DOI: 10.5061/dryad.tx95x6b13
title: Genomic analyses show extremely perilous conservation status of African and Asiatic cheetahs (Acinonyx jubatus)

description: We live in a world characterised by biodiversity loss and global
 environmental change. The extinction of large carnivores can have
 ramifying effects on ecosystems like an uncontrolled increase in wild
 herbivores, which in turn can have knock-on impacts on vegetation
 regeneration and communities. Cheetahs (Acinonyx jubatus) serve important
 ecosystem functions as apex predators; yet, they are quickly heading
 towards an uncertain future. Threatened by habitat loss, human-wildlife
 conflict, and illegal trafficking, there are only approximately 7,100
 individuals remaining in nature. We present the most comprehensive
 genome-wide analysis of cheetah phylogeography and conservation genomics
 to date, assembling samples from nearly the entire current and past
 species' range. We show that their phylogeography is more complex
 than previously thought, and that East African cheetahs (A. j. raineyi)
 are genetically distinct from Southern African individuals (A. j.
 jubatus), warranting their recognition as a distinct subspecies. We found
 strong genetic differentiation between all classically recognised
 subspecies, thus refuting earlier findings that cheetahs show only little
 differentiation. The strongest differentiation was observed between the
 Asiatic and all the African subspecies. We detected high inbreeding in the
 Critically Endangered Iranian (A. j. venaticus) and North-western (A. j.
 hecki) subspecies, and show that overall cheetahs, along with snow
 leopards, have the lowest genome-wide heterozygosity of all the big cats.
 This further emphasizes the cheetah’s perilous conservation status. Our
 results provide novel and important information on cheetah phylogeography
 that can support evidence-based conservation policy decisions to help
 protect this species. This is especially relevant in light of ongoing and
 proposed translocations across subspecies boundaries, and the increasing
 threats of illegal trafficking.
description: <strong>Dataset 2 – Mitochondrial
 DNA</strong>:<strong>
 </strong>We amplified mtDNA
 regions of 929bp for 56 individuals and 681bp for 57 individuals. We
 targeted two mitochondrial genes that included the 14 previously described
 diagnostic SNPs from Charruau et al. (2011), the NADH-dehydrogenase
 subunit 5 (MT-ND5) and the control region (MT-CR). The amplicons were
 Sanger sequenced. We further included the 78 individuals from Charruau et
 al. (2011) in the 681bp dataset. All individuals of the 929bp data set are
 included in the 681bp set, as the 929bp fragment includes the shorter
 681bp fragment. The mtDNA data were used to infer population structure and
 included samples from their present and past distributions. We followed
 the protocol of Rohland, Siedel & Hofreiter (2010) for the DNA
 extraction from museum samples. To avoid DNA contamination, we carried out
 all extractions in a dedicated laboratory for museum samples at the
 Vetmeduni in Vienna, Austria.
 <strong>Dataset 3 –
 mini-barcodes</strong>:<strong> </strong>We designed a
 mini-barcode approach to investigate whether all <em>A. j.
 soemmeringii </em>carry the 3bp deletion in the MT-ND5 gene as
 described in Charruau et al. (2011), and as a quick subspecies assignment
 test. We designed three mini-amplicons that amplify a total of 190bp,
 based on diagnostic sites inferred from our mitochondrial haplotype data
 (dataset 2). Primer sequences can be found in Supplementary Table S5. We
 used the extracts of all <em>A. j. soemmeringii</em> specimens
 from dataset 1 and sequenced the amplicons on an Illumina iSeq 100
 (2x150bp) following the two-step sequencing protocol of Lange et al.
 (2014). <strong>Dataset 4 – immune response
 genes</strong>:<strong>
 </strong>We sequenced the MHC
 class II DRB exon 2 of 46 individuals (belonging to four of the five
 classical subspecies; excluding <em>A. j. hecki</em>) to investigate their immunogenetic diversity.
 We used the Qiagen DNeasy Blood and Tissue kit for DNA extraction from
 hair and tissue samples, and the VWR PeqGold™ Tissue DNA Mini Kit Plus for
 blood samples. We carried out PCR amplifications of the target region as
 described in Castro-Prieto, Wachter & Sommer (2011). Indexing,
 multiplexing, and sequencing were carried out following the Illumina
 Nextera XT DNA Library Prep kit reference guide. Sequencing was performed
 on an Illumina MiSeq (2x250bp).