Final Report Summary - SIMBA (Search for Innate Markers of Barbary Affinity) The objectives of Project SIMBA were fourfold.Our first task was to identify, extract, and build into genomic libraries, ancient DNA from extinct and endangered lion populations throughout the natural range of the species. To this end we sourced over one hundred lion samples and extracted them in the sterile clean rooms of the Centre for GeoGenetics(CGG). After initial tests for DNA content, 75 were deemed well-preserved enough for building into Illumina compatible adaptor-ligated libraries.The second task was to amplify, index, and sequence the resultant libraries to assess endogenous content and potentially generate informative sequence data. From this initial data we have also been able to bioinformatically assemble complete mitogenomes for a number of specimens with a high level of coverage (including North African, Iranian, Cape, Senegalese, Sudanese, and Gabonese). Our third task was to target enrich our best libraries for nuclear DNA. To this end we created RNA baits from fresh modern lion tissue and also generated additional baits of exomic regions using the recently published lion genome1. We used a bead-capture method2 with these baits to hybridise the felid portion of the libraries and concentrate it beyond the background level of exogenous contamination. This has been achieved for 9 voucher specimens from key regions of the natural range of Panthera leo (including the extinct North African, Iranian, and Cape populations and the Pleistocene cave lion) from which megabases of nuclear DNA have been generated to high coverage. The final objective was to analyse the data. Our preliminary data analysis, using data from cytochrome b has already been published3. We are currently preparing a number of manuscripts to disseminate our results. One will consist of the results of a complete mitogenome from a Pleistocene cave lion (Panthera spelaea) with a focus on phylogenetics within the Panthera genus. Another will consist of an intraspecific lion phylogeny using our high coverage mitogenomes, with identification of useful diagnostic SNPs for separation of regional populations (e.g. Barbary). Our third paper will consist of the exomic data and will compare and contrast regional SNP differences, Y-chromosome phylogeography, and population demographics (e.g. PSMC4).Our initial results have resulted in much discussion of lion conservation units and the necessity of influencing the IUCN to modify its conservation listing for lions. We are currently involved with a consortium of other lion researchers through the medium of the African Lion Working Group, to petition the IUCN for recognition of certain regional populations as deserving of special protection. 1 Cho, Y. S. The tiger genome and comparative analysis with lion and snow leopard genomes. Nature Communications (2013).2 Carpenter, M. L. et al. Pulling out the 1%: whole-genome capture for the targeted enrichment of ancient DNA sequencing libraries. Am J Hum Genet 93, 852-864, doi:10.1016/j.ajhg.2013.10.002 (2013).3 Barnett, R. et al. Revealing the maternal demographic history of Panthera leo using ancient DNA and a spatially explicit genealogical analysis. BMC Evolutionary Biology 14, 70, doi:10.1186/1471-2148-14-70 (2014).4 Orlando, L. et al. Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse. Nature 499, 74-78, doi:10.1038/nature12323http://www.nature.com/nature/journal/v499/n7456/abs/nature12323.html - supplementary-information (2013).
