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Abstract The lion Panthera leo is one of the world's most charismatic carnivores and is one of Africa's key predators. Here, we used a large dataset from 357 lions comprehending 1.13 megabases of sequence data and genotypes from 22 microsatellite loci to characterize its recent evolutionary history. Patterns of molecular genetic variation in multiple maternal (mtDNA), paternal (Y-chromosome), and biparental nuclear (nDNA) genetic markers were compared with patterns of sequence and subtype variation of the lion feline immunodeficiency virus (FIV Ple), a lentivirus analogous to human immunodeficiency virus (HIV).
In spite of the ability of lions to disperse long distances, patterns of lion genetic diversity suggest substantial population subdivision (mtDNA Φ ST = 0.92; nDNA F ST = 0.18), and reduced gene flow, which, along with large differences in sero-prevalence of six distinct FIV Ple subtypes among lion populations, refute the hypothesis that African lions consist of a single panmictic population. Our results suggest that extant lion populations derive from several Pleistocene refugia in East and Southern Africa (∼324,000–169,000 years ago), which expanded during the Late Pleistocene (∼100,000 years ago) into Central and North Africa and into Asia. During the Pleistocene/Holocene transition (∼14,000–7,000 years), another expansion occurred from southern refugia northwards towards East Africa, causing population interbreeding. In particular, lion and FIV Ple variation affirms that the large, well-studied lion population occupying the greater Serengeti Ecosystem is derived from three distinct populations that admixed recently. Author Summary 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.
Citation: Antunes A, Troyer JL, Roelke ME, Pecon-Slattery J, Packer C, Winterbach C, et al. (2008) The Evolutionary Dynamics of the Lion Panthera leo Revealed by Host and Viral Population Genomics. PLoS Genet 4(11): e1000251. Editor: Arnaud Estoup, INRA, France Received: July 27, 2007; Accepted: October 2, 2008; Published: November 7, 2008 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This research has been funded in part by federal funds from the National Cancer Institute, National Institutes of Health (N01-CO-12400), by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, by the National Science Foundation (No. 0343960) and by the Portuguese Foundation for Science and Technology (PTDC/BIA-BDE/).
AA received a grant from the Portuguese Foundation for Science and Technology (SFRH/BPD/5700/2001). Competing interests: The authors have declared that no competing interests exist. Introduction 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. 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. 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 that occur only in sub-Saharan Africa and the Gir Forest, India. Expected and observed coalescent times for the different markers studied in lions according to their pattern of inheritance.
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). Population Structure of Lion 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; π = 0.0066) (;; and ). 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 (; ). 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 and three-dimensional factorial correspondence analyses (; ). Most clusters represented geographically circumscribed populations: Namibia (N am), Kruger National Park (K ru), Ngorongoro Crater (N gc), Kenya (K en), Uganda (U ga), and Gir (G ir).
Two distinct clusters were found in Botswana, B ot-I that included lions from southern Botswana and Kalahari (South Africa) ( F k = 0.24) and B ot-II found exclusively in northern Botswana ( F k = 0.18). Surprisingly, three distinct clusters were found in a single geographical locale (approximately 60×40 km square) in the large panmyctic population of the Serengeti National Park (S er-I/S er-II/S er-III) ( F k = 0.18, 0.21, and 0.15, respectively). Population structure analyses in lions.
(A) Bayesian population assignment test of the 357 lions using 24 nDNA loci ( ADA, TF, and 22 microsatellites) and mtDNA data, and considering K = 11 (11 populations). (B) Three-dimensional factorial correspondence analysis (FCA) based on the 24 nDNA loci genotypes in the 357 lions. Axe 1, 2, and 3 represent 49.90% of the genetic variation observed. (C) FCA representation excluding the GIR lions.
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Axe 1, 2, and 3 represent 51.35% of the genetic variation observed. (D) FCA representation considering only the SER lions supportive of a three distinct population clusters subdivision (SER-I, SER-II, and SER-III). Two captive lions from Angola (A ng), one from Zimbabwe (Z bw) and four Morocco Zoo Atlas lions (A tl; presently extinct from the wild) were included in the analyses to explore the relationship of lions from more isolated, endangered, or depleted areas. A ng and Z bw lions were assigned to B ot-II ( q = 0.90 and 0.87; 90%CI: 0.47–1.00) and K ru ( q = 0.85; 90%CI: 0.52–1.00) (Bayesian analyses ) populations, respectively, as expected based on their geographical proximity. However, these lions differed from B ot-II and K ru by up to 8 mtDNA mutations, sharing haplotypes with the A tl lions (H5 in A ng and H6 in Z bw).
The A tl 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). The significant positive and monotonic correlation across all the scatterplot pairwise comparisons for the nDNA markers (bi-parental) 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 G ir 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 plotted both against the geographic distance (model assuming habitat to be arrayed in an infinite one-dimensional 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 isolation-by-distance model would provide the best fit for the nDNA data ( R 2 = 0.25; Mantel's test, P = 0.0022), but instead the one-dimensional isolation-by-distance model performed better ( R 2 = 0.71; Mantel's test, P = 0.0476). Genetic differentiation of host and viral genetic markers with geographic distance.
Regression of lion pairwise F ST (nDNA and mtDNA) and FIV Ple ( pol-RT) on geographic distance. 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.
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 K en population. The removal of the K en 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 (π = 0.0066). A similar process has been suggested in whales (π = 0.0007) and African savannah elephants (π = 0.0200), 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. 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 (; ).
Lineage I consisted of a divergent haplotype H4 from K en, 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. Evolutionary relationships of the host and viral genetic markers among lion populations. (A) Unrooted neighbour-joining (NJ) tree from nDNA genotypes of 24 loci ( ADA, TF, and 22 microsatellites) in the 11 lion populations (left), and rooted NJ tree for the distinct mtDNA ( 12S– 16S, 1,882 bp) haplotypes in lion (right). The distinct mtDNA lineages were labelled I to IV.
Bootstrap support (BPS) values 50 are indicated. (B) NJ tree of the 301 bp FIV Ple pol-RT sequences.
The distinct FIV Ple subtypes were labelled A to F. BPS values are placed at each branchpoint and in parenthesis are the BPS values obtained for a tree established with 520 bp of FIV Ple pol-RT sequence for a representative subset of individuals. (C) Distinctiveness of host and viral molecular genetics in lion populations. Population Structure of FIV Ple Seroprevalence studies indicate that FIV Ple is endemic in eight of the 11 populations but absent from the Asian G ir lions in India and in Namibia and southern Botswana/Kalahari regions (N am/B ot-I) in Southwest Africa. Phylogenetic analysis of the conserved pol-RT region in 117 FIV-infected lions depicted monophyletic lineages, that affirm six distinct subtypes (A–F) that are distributed geographically in three distinct patterns (; ). 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 (S er-I, S er-II and S er-III) and subtypes A and E within lions of Botswana (B ot-II) ( and ). Second, unique FIV Ple subtypes may be restricted to one location as subtype F in Kenya, subtype E in Botswana (B ot-II), subtype C in Serengeti, and subtype D in Krugar Park ( and ).
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 ( and ). Not unexpectedly, FIV Ple pairwise genetic distances, represented as population F ST among the eight lion FIV-infected populations, were not significantly correlated with geographic distance ( R 2 = 0.08; Mantel's test, P = 0.165) , and affirms that patterns of viral dissemination do not conform to a strict isolation-by-distance model. Rather, the two distinct clusters observed reflect the complex distribution of FIV Ple among African lions. Indeed, despite the low geographic distance within East-African lion populations, the FIV Ple genetic divergence showed a broader range in F ST (0.03 to 0.79 for most of first cluster; ). By contrast, approximately half of the range in F ST (0.26 to 0.69 for the second cluster; ) was observed among East and Southern Africa in spite of its large geographic separation.
In contrast with the patterns observed in lions, linearized F ST estimates for FIV Ple were better correlated with log geographic distance (two-dimensional lattice model) ( R 2 = 0.15) than with geographic distance (one-dimensional model) ( R 2 = 0.02), although in both cases the Mantel's test was not significant ( P0.2474). Natural History of Lions as Inferred from Lion and FIV Ple Markers The mtDNA coalescence dating suggested that the East African lineage I (K en haplotype H4) had an old origin of ∼324,000 years (95% CI: 145,000–502,000).
Extant East African populations (K en/N gc/S er-I/S er-II/S er-III) also showed a slightly significant higher nDNA allelic richness and genetic diversity relative to populations to the south (K ru/N am/B ot-I/B ot-II) and north (U ga/G ir) ( A = 2.43, 2.39, and 1.62, P = 0.021; H O = 0.64, 0.62, and 0.34, P = 0.019; respectively). Moreover, the FIV Ple subtype diversity was higher in East African clades (exhibiting four out of the six known viral-strains), including the most divergent FIV Ple subtype C. These genetic data from lions and FIV Ple is consistent with the older origin of extant East African lions, which is further supported by the oldest lion fossils discovered in East Africa. Relative to East Africa, Southern lions have a slightly more recent mtDNA coalescence. Lineage II, found in N am, B ot-II and K ru has an estimated coalescence of 169,000 years (95% CI: 34,000–304,000) and the more widespread lineage IV found in the Southern populations of B ot-I, B ot-II and K ru as well as the Eastern populations of S er (I, II, and III), N gc and U ga, coalesces ∼101,000 years ago (95% CI: 11,000–191,000). However, the similar levels of nDNA genetic diversity, the occurrence of an exclusively Southern mtDNA lineage II and highly divergent FIV Ple subtypes, FIV Ple subtype D found only in K ru and subtype E exclusive to B ot-II, suggests that both East and Southern Africa were important refugia for lions during the Pleistocene. Therefore, the co-occurrence of divergent mtDNA haplotypes (6 to 10 mutations; ) in southern populations may be the consequence of further isolation within refugia during colder climatic periods.
Contemporary fragmentation of lion populations could further explain the results of nested-clade phylogeographical analysis (NCPA ) , which inferred restricted gene flow with isolation-by-distance between mtDNA haplotypes H9 (B ot-II) and H10 (K ru) (χ 2 = 10.00, P = 0.0200), between haplotypes H1 (B ot-II/N am) and H2 (K ru) (χ 2 = 71.00, P≤0.0001), and between haplotypes H9–H10 (B ot-II/K ru) and haplotypes H11–H12 (B ot-I/K ru/S er/N gc/U ga) (χ 2 = 187.83, P≤0.0001). Further isolation within refugia (sub-refugia) may also have occurred in East Africa. This is suggested by the distinctive mtDNA haplotype H4 and the unique FIV Ple subtype F found in the Kenya population, which may have resulted from reduced gene flow across the Rift valley, a scenario that has been suggested for several bovid and carnivore populations (see and references therein).
Sentou yousei yukikaze ost rar. The best example of concordance between host genome markers and viral transmission patterns is observed in the Serengeti National Park in Tanzania. Our previous findings described markedly high levels of FIV Ple subtype A, B and C circulating within the Serengeti lion population to such an extent that 43% of the lions sampled were multiply-infected with two or three subtypes, and were hypothesized to represent recent admixture of three formerly separated populations. Such result is confirmed here by lion genomic markers. Further, although lions within the Serengeti can be assigned to one of three populations (S er-I, S er-II or S er-III) by host genomic markers, FIV Ple subtypes are distributed ubiquitously in all three, characteristic of rapid horizontal retroviral transmission subsequent to host population admixture. The possible isolating mechanism remains to be elucidated as there is no apparent barrier to gene flow in this ecosystem. Genomic Signatures Left by Migration Based on patterns of genetic diversity and phylogenetic analysis of lion nDNA/mtDNA and FIV Ple markers, we propose a scenario of a period of refugia/isolation in the Late Pleistocene followed by two major lion expansions across Africa and Asia.
The first expansion, supported by the mtDNA NCPA (χ 2 = 690.00, P≤0.0001; ), was a long-distance colonization of mtDNA lineage-III (G ir/A tl/A ng/Z bw) around 118,000 years ago (95% CI: 28,000–208,000), with subsequent fragmentation of haplotypes H5–H6 into Central and North Africa and haplotypes H7–H8 into West Asia (M1- ). Support for this initial expansion is also found in nDNA. The ADA haplotype A5 fixed in G ir in also present in K en, S er-II, and S er-III, suggesting that lions likely colonized West Asia from the East Africa refugia. Such an expansion may have been favored by the start of a warmer and less arid period in Africa 130,000–70,000 years ago.
This “out-of-Africa event” would have occurred much later than the initial lion expansion through Eurasia based on fossils (∼500,000 years ago). It is likely that multiple lion expansions occurred in the Pleistocene, as occurred with humans. A second, more recent lion expansion probably occurred at the Pleistocene/Holocene transition, this one from Southern Africa toward East Africa (M2-, ).
This is reflected in the mtDNA linage IV, where haplotypes present in Southern lions are basal (older) to those found in the East. Overall, mtDNA population nucleotide diversity decreases from Southern to East Africa , a finding supported by pairwise mismatch analysis (raggedness, r = 0.086; P. Utility of Population Genomic Datasets If we had restricted our inferences to mtDNA, we might have concluded that East African lion populations, which are fixed or nearly fixed for haplotype H11, went extinct during the Pleistocene/Holocene transition (similar to the well known mega-fauna extinctions of the Late Pleistocene ) and were then colonized by Southern populations. However, our population genomics data better fit a scenario of lion population expansion and interbreeding rather than simple replacement. First, genetic diversity and allelic richness at nDNA are slightly higher in East Africa populations relatively to those in Southern Africa. This is contrary to the expected pattern of population expansion in which there is usually a progressive decline in genetic diversity and allelic richness.
Second, S er lions carry two diverse FIV Ple subtypes found only in East Africa (B–C), and not only FIV Ple subtype A, which was presumably introduced in East Africa coincidently with the mtDNA expansion event northwards from South. Third, the East African FIV Ple subtype B found in U ga/S er-I/S er-II/S er-III/N gc showed evidence of a population expansion (raggedness, r = 0.004; P. Conservation Implications Accurate interpretation of past and contemporary population demographic scenarios is a primary goal for the effective conservation of endangered species. In this study, we found substantial population subdivision, reduced gene flow, and large differences in FIV Ple sequence and sero-prevalence among lion populations, as well as evidence of historic secondary contact between populations (; to ).
The very low population level of mtDNA nucleotide diversity, the number of haplotypes private to a single population , and probably also the lack of SRY genetic variation across all male lions (haplotype S1, n = 183) suggests that lion numbers diminished considerably following the Late Pleistocene. The last century reduction in lion distribution further eroded its genomic diversity, and microsatellite variation suggested recent population bottlenecks in seven out of the 11 populations (standardized differences test, P. Study Site, Sampling, and Molecular Genetic Analyses of Lions A total of 357 individuals were obtained across most of the lion range in Africa and Asia (; ). Genetic variation among lion specimens was assessed using maternal ( 12S and 16S genes), paternal ( SRY gene) and bi-parental autosomal (22 microsatelite loci, and the ADA and TF genes) markers (GenBank accession numbers: FJ151632–FJ151652). Analyses of mtDNA in Panthera species are complicated by the presence of a 12.5 kb mtDNA integration into chromosome F2. Accordingly, mtDNA specific primers were designed for the 12S and 16S genes and we used long-range PCR amplification.
We designed primers to amplify segments of the ADA (exon 10 and intron 10) and the TF (intron 3) genes , two of the most variable protein loci in lion populations, localized on the domestic cat Felis catus chromosome A3p and C2q, respectively. The Y-chromosome SRY-3′UTR gene was also amplified. PCR products were amplified from 50 ng of genomic DNA in a 25 µL reaction system containing 1.5 mM MgCl 2, 1.0 mM dNTPs, 0.25 units of AmpliTaq Gold DNA polymerase (Applied Biosystems), and 1× PCR buffer II; the amplification protocol was: denaturation 10 min at 95°C, a touch-down cycle of 95°C for 30 s, 52°C for 60 s decreased by 1°C in the next cycle for 10 cycles, 72°C for 120 s, then 35 amplification cycles of 95°C for 30 s, 52°C for 60 s, and 72°C for 120 s, followed by an extension of 10 min at 72°C.
PCR products were sequenced on an ABI 377. Sequences were aligned and cleaned using SEQUENCHER (Gene Codes). Twenty two polymorphic microsatellite loci (20 dinucleotide repeats: FCA006, FCA008, FCA014, FCA069, FCA077, FCA085, FCA091, FCA098, FCA105, FCA126, FCA129, FCA139, FCA205, FCA208, FCA211, FCA224, FCA229, FCA230, FCA247, and FCA281; and two tetranucleotide repeats: FCA391 and FCA441) were amplified. Microsatellites were scored in an ABI 377 and analyzed using G enescan 2.1 and G enotyper 2.5. These loci are located on 11 of the 19 F. Catus chromosomes, occurring in different linkage groups or at least 12 centimorgans apart.
Sero-Prevalence and Molecular Genetics of FIV Ple Western blots using domestic cat and lion FIV as antigen were performed as previously described. The supernatant from virus-infected cells was centrifuged at 200 g for 10 min at 5°C. The resultant supernatant was centrifuged at 150,000 g at 4°C for 2 hours.
Pelleted viral proteins were resuspended in 1/20 th of the original volume and total protein content was assayed using the Biorad Protein Assay. Twenty mg of viral protein were run on 4–20% Tris-Glycine gels and transferred to PDVF membranes (BioRad). Membrane strips were exposed 2–12 h to a 1∶25 or 1∶200 dilution of serum or plasma. After washing, samples were labeled with goat anti-cat HRP or phosphate conjugated antibody (KPL laboratories) at a 1∶2000 dilution, washed, and incubated in ECL Western Blotting detection reagents (Amersham Biosciences) for 2 min, then exposed to Lumi-Film Chemiluminescent Detection Film (Boehringer Mannheim) or incubated in BCIP/NBP phosphatase substrate (KPL laboratories) for 15 min –. Results were visualized and scored manually based on the presence and intensity of antibody binding to the p24 gag capsid protein. Yamaha ex5 keyboard.
Nested PCR amplification of partial FIV Ple pol-RT was performed. Briefly, first round PCR reactions used 100 ng of genomic DNA, 2.5 mM MgCl 2 and an annealing temperature of 52°C. Second round PCR reactions used identical conditions with 1–5 µl of first-round product as template. All PCR products were sequenced as described above for lion genetic analyses (GenBank accession numbers: AY549217–AY552683; AY878208–AY878235; FJ225347–FJ225382). Statistical Analyses We used the G enetix 4.02, G enepop 3.3, M icrosat, and D naSP 4.10 to calculate the following descriptive statistics: (i) percentage of polymorphic loci ( P 95), number of alleles per locus ( A), observed and expected heterozygosity ( H E and H O), and number of unique alleles ( A U); (ii) assess deviations from HWE; (iii) estimate the coefficient of differentiation ( F ST), and (iv) nucleotide (π) and haplotype ( h) diversity.
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We tested the hypothesis that all loci are evolving under neutrality for both the lion and the FIV Ple data. For frequency data, we used the method described by Beaumont and Nichols and implemented in F dist. The F ST values estimated from microsatellite loci plotted against heterozygosity showed that all values fall within the expected 95% confidence limit and consequently no outlier locus were identified. For sequence data (lion nDNA/mtDNA and FIV Ple pol-RT), we ruled out any significant evidence for genetic hitchhiking and background selection by assessing Fu and Li's D. and F.
tests and Fu's F S statistics. A Bayesian clustering method implemented in the program S tructure was used to infer number of populations and assign individual lions to populations based on multilocus genotype (microsatellites) and sequence data ( ADA, TF, and mtDNA genes) and without incorporating sample origin. For haploid mtDNA data, each observed haplotype was coded with a unique integer (e.g. 100, 110) for the first allele and missing data for the second (S tructure analyses with or without the mtDNA data were essentially identical). For K population clusters, the program estimates the probability of the data, Pr( X K), and the probability of individual membership in each cluster using a Markov chain Monte Carlo method under the assumption of Hardy-Weinberg equilibrium (HWE) within each cluster. Initial testing of the HWE in each of the populations defined by the geographic origin of sampling revealed no significant deviation from HW expectations with the exception of S er and B ot population (later subdivided by S tructure in 3 and 2 clusters, respectively; such deviations from HW expectations were interpreted as evidence of further population structuring).
We conducted six independent runs with K = 1–20 to guide an empirical estimate of the number of identifiable populations, assuming an admixture model with correlated allele frequencies and with burn-in and replication values set at 30,000 and 10 6, respectively. S tructure also estimates allele frequencies of a hypothetical ancestral population and an alpha value that measures admixed individuals in the data set. The assignment of admixed individuals to populations using S tructure has been considered in subsequent population analyses.
For each population cluster k, the program estimates F k, a quantity analogous to Wright's F ST, but describing the degree of genetic differentiation of population k from the ancestral population. Patterns of gene flow and divergence among populations were described using a variety of tests. First, to visualize subtle relationships among individual autosomal genotypes, three-dimensional factorial correspondence analyses (FCA) were performed in G enetix, which graphically projects the individuals on the factor space defined by the similarity of their allelic states. Second, neighbor-joining (NJ) analyses implementing the Cavalli-Sforza & Edwards' chord genetic distance ( D CE) were estimated in P hylip 3.6, and the tree topology support was assessed by 100 bootstraps. Third, the difference in average H O and A was compared among population groups using a two-sided test in F stat 2.9.3.2, which allows to assess the significance of the statistic OS x using 1,000 randomizations. Four, the equilibrium between drift and gene flow was tested using a regression of pairwise F ST on geographic distance matrix among all populations for host nDNA(microsatellites)/mtDNA and FIV Ple data.
A Mantel test was used to estimate the 95% upper probability for each matrix correlation. Assuming a stepping stone model of migration where gene flow is more likely between adjacent populations, one can reject the null hypothesis that populations in a region are at equilibrium if (1) there is a non-significant association between genetic and geographic distances, and/or (2) a scatterplot of the genetic and geographic distances fails to reveal a positive and monotonic relationship over all distance values of a region. We also evaluated linearized F ST i.e.
F ST/(1− F ST) among populations. We tested two competing models of isolation-by-distance, one assuming the habitat to be arrayed in an infinite one-dimensional lattice and another assuming an infinite two-dimensional lattice. Both models showed that genetic differentiation increased with raw and log-transformed Euclidean distances, respectively. We determined the confidence interval value of the slope of the regression for the nDNA data using a non parametric ABC bootstrap in G enepop 4.0. The demographic history of populations was compared using a variety of estimators based on the coalescence theory. First, signatures of old demographic population expansion were investigated for mtDNA and FIV Ple pol-RT haplotypes using pairwise mismatch distributions in D naSP.
The goodness-of-fit of the observed data to a simulated model of expansion was tested with the raggedness ( r) index. Second, the occurrence of recent bottlenecks was evaluated for microsatellite data using the method of Cornuet & Luikart in B ottleneck and using 10,000 iterations.
This approach, which exploits the fact that rare alleles are generally lost first through genetic drift after reduction in population size, employs the standardize differences test, which is the most appropriate and powerful when using 20 or more polymorphic loci. Tests were carried out using the stepwise mutation model (SMM), which is a conservative mutation model for the detection of bottleneck signatures with microsatellites. Third, to discriminate between recurrent gene flow and historical events we used the nested-clade phylogeographical analysis, (NCPA) for the mtDNA data. When the null-hypothesis of no correlation between genealogy and geography is rejected, biological inferences are drawn using a priori criteria. The NCPA started with the estimation of a 95% statistical parsimony mtDNA network in T cs 1.20.
Tree ambiguities were further resolved using a coalescence criteria. The network was converted into a series of nested branches (clades), which were then tested against their geographical locations through a permutational contingency analysis in G eoD is 2.2. The inferences obtained were also corroborated with the automated implementation of the NCPA in AN eCA. To address potential weaknesses in some aspects of the NCPA analysis, we further validated the NCPA inferences with independent methods for detecting restricted gene-flow/isolation-by-distance (using matrix correlation of pairwise F ST and geographic distance) and population expansion (using pairwise mismatch distributions). Four, to test the significance of the total mtDNA genetic variance, we conducted hierarchical analyses of molecular variance (AMOVA) using A rlequin 2.0.
Total genetic variation was partitioned to compare the amount of difference among population groups, among populations within each groups, and within populations. Phylogenetic relationships among mtDNA and FIV Ple pol-RT sequences were assessed using Minimum evolution (ME), Maximum parsimony (MP), and Maximum likelihood (ML) approaches implemented in P aup. The ME analysis for mtDNA consisted of NJ trees constructed from Kimura two-parameter distances followed by a branch-swapping procedure and for FIV Ple data employed the same parameter estimates as were used in the ML analysis. The MP analysis was conducted using a heuristic search, with random additions of taxa and tree-bisection-reconnection branch swapping.
The ML analysis was done after selecting the best evolutionary model fitting the data using M odeltest 3.7. Tree topologies reliability was assessed by 100 bootstraps. For the FIV Ple data, the reliability of the tree topology was further assessed through additional analyses using 520 bp of FIV Ple pol-RT sequences in a representative subset of individuals. The time to the most recent common ancestor (TMRCA) for the ADA and TF haplotypes was estimated following Takahata et al., where we calculate the ratio of the average nucleotide differences within the lion sample to one-half the average nucleotide difference between leopards ( P. Pardus) and lions and multiplying the ratio by an estimate of the divergence time between lions and leopards (2 million years based on undisputed lion fossils in Africa).
The mtDNA TMRCA was estimated with a linearized tree method in L intree and using the equation H = 2μT, where H was the branch height (correlated to the average pairwise distance among haplotypes), μ the substitution rate, and T the divergence time. Leopard and snow leopard ( P. Uncia) sequences were used as outgroups. Inference of the TMRCA for microsatellite loci followed Driscoll et al. Where the estimate of microsatellite variance in average allele repeat-size was used as a surrogate for evolutionary time based on the rate of allele range reconstitution subsequent to a severe founder effect. Microsatellite allele variance has been shown to be a reliable estimator for microsatellite evolution and demographic inference in felid species. Genetic variation of 12S–16S (mtDNA) and ADA and TF (nDNA) genes in lions.
(A) Haplotypes and variable sites for the 12S–16S mtDNA region surveyed in lions (total length 1,882 bp). Position 1 corresponds to position 1441 of the domestic cat ( Felis catus) mtDNA genome (GenBank U20753). The “-” represents a gap and “.” matches the nucleotide in the first sequence.
Shading indicates a fixed difference in the mtDNA lineage. (B) Haplotypes and variable sites for the ADA gene segment surveyed in lions (total length 427 bp). (C) Haplotypes and variable sites for the TF gene segment surveyed in lions (total length 427 bp). (0.12 MB PDF).
Linearized genetic differentiation of host and viral genetic markers with geographic distance. Regression of linearized F ST estimates for lion (nDNA and mtDNA) and FIV Ple ( pol-RT) genetic data plotted both against the geographic distance (model assuming habitat to be arrayed in an infinite one-dimensional lattice; one-dimension isolation-by-distance IBD) and the log geographic distance (model assuming an infinite two-dimensional lattice; two-dimension isolation-by-distance) on geographic distance. (0.13 MB PDF).
Phylogenetic relationships of the 12S–16S mtDNA lion haplotypes. Neighbour-joining tree of the 1,882 bp 12S–16S mtDNA sequences. Bootstrap values are placed at each branchpoint for the minimum evolution/maximum parsimony/maximum likelihood analyses, respectively (ME/MP/ML). Outgroups: Ppa – leopard, Panthera pardus; Pun – snow-leopard, Panthera uncia. The symbol (.) represents nodes with bootstrap support. Acknowledgments Tissues were obtained in full compliance with specific Federal Fish and Wildlife Permits.
The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. We would like to thank A. Beja-Pereira, M.
Branco and J. Martenson for suggestions and technical assistance. Comments made by the Associate Editor A. Estoup and three anonymous referees improved a previous version of this manuscript.
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