Statistical Analyses
On the basis of the levels of diversity observed in the CD209/CD209L genomic region, we calculated the average number of pairwise differences (π) and the Watterson's estimator (θw) (Watterson 1975). Under the standard neutral model of a randomly mating population of constant size, these are unbiased estimators of the population mutation rate θ=4Neμ, where N e is the diploid effective population size and μ is the mutation rate per generation per site. To test whether the frequency spectrum of mutations conformed to the expectations of this standard neutral model, we calculated Tajima's D (Tajima 1989) and Fay and Wu's H tests (Fay and Wu 2000). P values for the different tests were estimated from 104 coalescent simulations under an infinite-site model, with use of a fixed number of segregating sites and the assumption of no recombination, which has been shown to be a conservative assumption (Gilad et al. 2002). In parallel, we estimated P values for all these tests, using the empirical distribution obtained from sequencing data of 132 genes in a panel of 24 African Americans and 23 European Americans (Akey et al. 2004). All these analyses, together with the interspecies McDonald-Kreitman (McDonald and Kreitman 1991) and K A/K S (Kimura 1968) tests, were performed using the DnaSP package (Rozas et al. 2003). Genetic distances between populations (F ST) and heterozygosity values were estimated using the Arlequin package (Schneider et al. 2000). F ST statistical significance was assessed using 10,000 bootstrap replications. To bear out a deficit or an excess of heterozygosity in the neck region of CD209 and CD209L, we used BOTTLENECK (Cornuet and Luikart 1996) to compute for each geographic region, the distribution of the heterozygosity expected from the observed number of alleles, given the sample size (n) under the assumption of mutational-drift equilibrium. This distribution was obtained through simulation of the coalescent process of n genes under two mutational models, the infinite-site model and the stepwise mutation model. In addition, to obtain information on the fraction of genetic variance in the neck region that is due to intra- and interpopulation differences, we performed an analysis of molecular variance (AMOVA), using the Arlequin package (Schneider et al. 2000). The AMOVA results were compared with those of 377 microsatellites analyzed in the same population panel (Rosenberg et al. 2002).
Haplotype reconstruction was performed by use of the Bayesian statistical method implemented in Phase (v.2.1.1) (Stephens and Donnelly 2003). We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs. After haplotype reconstruction, linkage disequilibrium (LD) between pairs of SNPs was computed using Lewontin's D′ index (Lewontin 1964). For this analysis, only markers presenting a minimum allele frequency (MAF) of 10% were considered, since rare alleles have been shown to present a higher probability of being in significant LD than do common ones (Reich et al. 2001). The graphic display of the LD plots was constructed using GOLD (Abecasis and Cookson 2000; Center for Statistical Genetics). To support the existence of a recombination hotspot in the region under study, we used the hotspot-recombination model implemented in Phase (v.2.1.1). Under this model, we assumed that there was, at most, one hotspot of unknown position. We then estimated the background population-recombination rate (ρ) and the relative intensity of any recombination hotspot. To obtain better estimates, we increased 10 times the number of iterations of the final run of the algorithm. All our estimations were obtained by averaging results of five independent runs with use of different seed numbers. Since the model used is Bayesian, we could also estimate, for each population, the posterior probability of a hotspot of intensity >1 (λ>1) and >10 (λ>10).
We obtained the gene tree and estimated the time of the most recent common ancestor (T MRCA) for CD209, using the maximum-likelihood coalescent method implemented in GENETREE (Griffiths and Tavare 1994). The mutation rate μ for each gene was estimated on the basis of the net divergence between humans and chimpanzees and under the assumption both that the species separation occurred 5 million years ago (MYA) and of a generation time of 20 years. Using this μ and θ maximum likelihood (θML), we estimated the effective population size parameter (N e). With the assumption of a generation time of 20 years and the estimated N e, the coalescence time, scaled in 2Ne units, was converted into years. The coalescence process implemented in SIMCOAL2 (Laval and Excoffier 2004) allowed us to estimate the probability of the T MRCA for CD209, through 2×104 simulations, with use of both the number of observed segregating sites and the estimated N e .