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Paragraph: Text: Lion fossils trace to the Late Pliocene in Eastern Africa and the Early Pleistocene in Eastern and Southern Africa coincident with the flourishing of grasslands ,2-1.5 million years ago [1, 2] . By Mid Pleistocene (,500,000 years ago) , lions occupied Europe and by the Late Pleistocene (,130,000-10,000 years ago) lions had the greatest intercontinental distribution for a large land mammal (excluding man), ranging from Africa into Eurasia and the Americas [3] . Lions were extirpated from Europe 2,000 years ago and within the last 150 years from the Middle East and North Africa. Today, there are less than 50,000 free-ranging lions [4] that occur only in sub-Saharan Africa and the Gir Forest, India ( Figure 1A) . Understanding the broader aspects of lion evolutionary history has been hindered by a lack of comprehensive sampling and appropriately informative genetic markers [5] [6] [7] [8] [9] , which in species of modern felids requires large, multigenic data sets due to its generally rapid and very recent speciation [10, 11] . Nevertheless, the unique social ecology of lions [12] [13] [14] and the fact that lions have experienced well-documented infectious disease outbreaks, including canine distemper virus, feline parvovirus, calicivirus, coronavirus, and lion feline immunodeficiency virus (FIV Ple ) [15] [16] [17] [18] provide a good opportunity to study lion evolutionary history using both host and virus genetic information. Indeed, population genetics of transmitted pathogens can accurately reflect the demographic history of their hosts [19, 20] . Unlike other of the 36 cat species, lions have a cooperative social system (prides of 2-18 adult females and 1-9 males) and their populations can have high frequencies of FIV Ple , a lentivirus analogous to human immunodeficiency virus (HIV), which causes AIDS-like immunodeficiency disease in domestic cats. FIV Ple is a retrovirus that integrates into the host genome and is transmitted by cell-to-cell contact, which in felids occurs during mating, fighting and motherto-offspring interactions. Thus, viral dissemination is a function in part of the frequency of contact between infected and naïve lions within and among populations. The virus is quite genetically diverse in lions [15, 18] , offering a unique marker for assessing ongoing lion demographic processes. To unravel lion population demographic history we used a large multigenic dataset. Distinct sets of markers may not necessarily

The lion Panthera leo, a formidable carnivore with a complex cooperative social system, has fascinated humanity since pre-historical times, inspiring hundreds of religious and cultural allusions. Here, we use a comprehensive sample of 357 individuals from most of the major lion populations in Africa and Asia. We assayed appropriately informative autosomal, Y-chromosome, and mitochondrial genetic markers, and assessed the prevalence and genetic variation of the lion-specific feline immunodeficiency virus (FIV Ple ), a lentivirus analogous to human immunodeficiency virus (HIV) that causes AIDS-like immunodeficiency disease in domestic cats. We compare the large multigenic dataset from lions with patterns of genetic variation of the FIV Ple to characterize the population-genomic legacy of lions. We refute the hypothesis that African lions consist of a single panmictic population, highlighting the importance of preserving populations in decline rather than prioritizing larger-scale conservation efforts. Interestingly, lion and FIV Ple variation revealed evidence of unsuspected genetic diversity even in the well-studied lion population of the Serengeti Ecosystem, which consists of recently admixed animals derived from three distinct genetic groups. Historical and current geographic distribution of lion, Panthera leo. A three-letter code pointing to a white dotted circle represents the geographic location of the 11 lion populations determined by Bayesian analyses [22] and factorial correspondence analyses [23] of the genetic distinctiveness of 357 lion samples (see text): GIR, Gir Forest, India; UGA, Uganda (Queen Elizabeth National Park); KEN, Kenya (Laikipia), SER, Serengeti National Park, Tanzania; NGC, Ngorongoro Crater, Tanzania; KRU, Kruger National Park, South Africa; BOT-I, southern Botswana and Kalahari, South Africa; BOT-II, northern Botswana; and NAM, Namibia. Green squares represent captive individual samples to explore the relationship of lions from more isolated/ endangered/depleted areas: ATL, Morocco Atlas lions (n = 4); ANG, Angola (n = 2); and ZBW, Zimbabwe (n = 1). Deduced historical expansions (M1 and M2) are represented by red arrows (see text). (B) Haplotype frequencies observed in the 11 lion populations for nDNA (ADA and TF), and mtDNA (12S-16S) genes, paralleled with the FIV Ple serum-prevalence frequencies (black -sero-positive; gray -indeterminate; white -sero-negative). Population sample sizes are indicated within parenthesis. (C) Statistical parsimony networks of lion ADA, TF, and 12S-16S haplotypes. Circle size is proportional to the haplotype frequency and crossbars represent the number of step mutations connecting haplotypes. The mtDNA haplotypes H5 and H6 are shaded gray as they were detected only in the individual samples from ANG, ATL, and ZBW, which do not group in unique population clusters (see text). doi:10.1371/journal.pgen.1000251.g001 yield similar inferences of population history, as coalescent times vary as a function of their pattern of inheritance [21] . There is also a large variance in coalescent times across loci sharing a common pattern of inheritance especially in complex demographical histories (Table 1) . However, the accurate interpretation of the differences among loci can provide a more resolved and coherent population history, affording more-nuanced insights on past demographic processes, levels of admixture, taxonomic issues, and on the most appropriate steps for effective conservation and management of remaining populations. The goal of this study was to assess the evolutionary history of lion by (1) characterizing lion population structure relative to patterns of FIV Ple genetic variation, (2) detect signatures of migration using both host and viral population genomics, and (3) reconstruct lion demographic history and discuss its implication for lion conservation. We assess genetic variation from 357 lions from most of its current distribution, including mitochondrial (mtDNA; 12S-16S, 1,882 bp), nuclear (nDNA) Y-chromosome (SRY, 1,322 bp) and autosomal (ADA, 427 bp; TF, 169 bp) sequences, and 22 microsatellites markers. We further document patterns of FIV Ple variation in lions (FIV Ple pol-RT gene, up to 520 bp). Genetic analyses of 357 lions from throughout the extant species range showed that paternally inherited nDNA (SRY) and maternal inherited (mtDNA) sequence variation was generally low (only one paternal SRY-haplotype and 12 mtDNA haplotypes; p = 0.0066) ( Figure 1 ; Figure S1 ; Tables S1 and S2). The most common mtDNA haplotype H11 was ubiquitous in Uganda/Tanzania and parts of Botswana/South Africa, H1 was common in Southern Africa, and H7 and H8 were unique to Asian lions. The autosomal nDNA sequences showed fairly distinct patterns of variation ( Figure 1 ; Figure S1 ). Of the five ADA haplotypes, A2 was the most common and most-widely distributed. The other four haplotypes, which are derived and much less common, included one (A5) that was fixed in Asian lions. The three TF haplotypes were more widely and evenly distributed. Levels of population subdivision among lions were assessed using microsatellite and sequencing data. Eleven groups were identified using Bayesian analyses [22] and three-dimensional factorial correspondence analyses [23] (Figure 2 ; Table S3 ). Most clusters represented geographically circumscribed populations: Namibia (NAM), Kruger National Park (KRU), Ngorongoro Crater (NGC), Kenya (KEN), Uganda (UGA), and Gir (GIR). Two distinct clusters were found in Botswana, BOT-I that included lions from southern Botswana and Kalahari (South Africa) (F k = 0.24) and BOT-II found exclusively in northern Botswana (F k = 0.18). Surprisingly, three distinct clusters were found in a single geographical locale (approximately 60640 km square) in the large panmyctic population of the Serengeti National Park (SER-I/SER-II/SER-III) (F k = 0.18, 0.21, and 0.15, respectively). Two captive lions from Angola (ANG), one from Zimbabwe (ZBW) and four Morocco Zoo Atlas lions (ATL; presently extinct from the wild) ( Figure 1A) were included in the analyses to explore the relationship of lions from more isolated, endangered, or depleted areas. ANG and ZBW lions were assigned to BOT-II (q = 0.90 and 0.87; 90%CI: 0.47-1.00) and KRU (q = 0.85; 90%CI: 0.52-1.00) (Bayesian analyses [22] ) populations, respectively, as expected based on their geographical proximity. However, these lions differed from BOT-II and KRU by up to 8 mtDNA mutations, sharing haplotypes with the ATL lions (H5 in ANG and H6 in ZBW) ( Figure 1B and 1C). The ATL lions did not group in a unique cluster. Both nDNA and mtDNA pairwise genetic distances among the 11 lion populations showed a significant relationship with geographic distance (R 2 = 0.75; Mantel's test, P = 0.0097; and R 2 = 0.15; Mantel's test, P = 0.0369; respectively) ( Figure 3 ). The significant positive and monotonic correlation across all the scatterplot pairwise comparisons for the nDNA markers (biparental) was consistent with isolation-by-distance across the sampled region. However, the correlation between nDNA F ST and geographic distance considerably decreased when the Asian GIR population was removed (R 2 = 0.19; Mantel's test, P = 0.0065) suggesting that caution should be taken in interpreting the pattern of isolation-by-distance in lions. We further compared linearized F ST estimates [24] plotted both against the geographic distance (model assuming habitat to be arrayed in an infinite onedimensional lattice) and the log geographic distance (model assuming an infinite two-dimensional lattice). The broad distribution of lions might suggest a priori that a two-dimensional isolationby-distance model would provide the best fit for the nDNA data (R 2 = 0.25; Mantel's test, P = 0.0022), but instead the onedimensional isolation-by-distance model performed better (R 2 = 0.71; Mantel's test, P = 0.0476) ( Figure S2 ). The pattern observed for the mtDNA (maternal) was more complex. While there was a significant relationship between mtDNA F ST and geographic distance, there was an inconsistent pattern across broader geographic distances ( Figure 3 ). This is partly due to the fixation or near fixation of haplotype H11 in six populations and the fixation of a very divergent haplotype H4 in KEN population ( Figure 1B and 1C). The removal of the KEN population considerably increased the correlation between mtDNA F ST and geographic distance (R 2 = 0.27; Mantel's test, P = 0.0035). Thus, the null hypothesis of regional equilibrium for mtDNA across the entire sampled region is rejected despite the possibility that isolation-by-distance may occur regionally. These contrasting nDNA and mtDNA results may be indicative of differences in dispersal patterns between males and females, which would be consistent with evidence that females are more phylopatric than males. Alternatively, selection for matrilineally transmitted traits upon which neutral mtDNA alleles hitchhike is possible, given the low values of nucleotide diversity of the mtDNA (p = 0.0066). A similar process has been suggested in whales (p = 0.0007) [25] and African savannah elephants (p = 0.0200) [26] , where both species have female phylopatry and like lions, a matriarchal social structure. However, genetic drift tends to overwhelm selection in small isolated populations, predominantly affecting haploid elements due to its lower effective population size (Table 1) . Therefore, we suggest that the contrasting results obtained for nDNA and mtDNA are more likely further evidence that lion populations underwent severe bottlenecks. The highly structured lion matrilines comprise four monophyletic mtDNA haplo-groups ( Figure 4A ; Figure S3 ). Lineage I consisted of a divergent haplotype H4 from KEN, lineage II was observed in most Southern Africa populations, lineage III was widely distributed from Central and Northern Africa to Asia, and lineage IV occurred in Southern and Eastern Africa. Seroprevalence studies indicate that FIV Ple is endemic in eight of the 11 populations but absent from the Asian GIR lions in India and in Namibia and southern Botswana/Kalahari regions (NAM/ BOT-I) in Southwest Africa ( Figure 1B ). Phylogenetic analysis of the conserved pol-RT region in 117 FIV-infected lions depicted monophyletic lineages [15, 18] that affirm six distinct subtypes (A-F) that are distributed geographically in three distinct patterns ( Figure 4B ; Figure S4 ). First, multiple subtypes may circulate within the same population as exemplified by subtypes A, B and C all ubiquitous within the three Serengeti lion populations (SER-I, SER-II and SER-III) and subtypes A and E within lions of Botswana (BOT-II) ( Figure 4B and 4C and Figure S4 ). Second, unique FIV Ple subtypes may be restricted to one location as subtype F in Kenya, subtype E in Botswana (BOT-II), subtype C in Serengeti, and subtype D in Krugar Park ( Figure 4B and 4C and Figure S4 ). Third, intra-subtype strains cluster geographically, as shown by distinct clades within subtype A that were restricted to lions within Krugar Park, Botswana and Serengeti and within subtype B that corresponded to Uganda, Serengeti and Ngorongoro Crater lions ( Figure 4B and 4C and Figure S4 ).

Question: When did lions first occupy Europe?
By Mid Pleistocene (,500,000 years ago)