Cell (Sun et al., 2012). High humidity, especially in the summers, may traits such as hair phenotypes and breast size. This creates have provided a seasonally selective advantage for individuals conditions in which biases in mate preference could rapidly better able to functionally activate more eccrine glands and evolve and reinforce more direct competitive advantages. Is sweat more effectively(Kuno, 1956). To explore this hypoth- Consequently, the cumulative selective force acting over time esis, greater precision on when and where the allele was under on diverse traits caused by a single pleiotropic mutation could selection-perhaps using ancient DNA sources-in conjunction have driven the rise and spread of 370A with more detailed archaeological and climatic data are needed er E branching or fat pad size could have been adaptive. The increased branching of 370A mouse mammary glands and th Haplotype Analysis importance of mammary tissue in evolutionary fitness(Anderson DNA from1, 064 individuals from 52 global populations was acquired from the et al., 1983; Oftedal, 2002)make this organ an interesting candi- uman Genome Diversity Panel(HGDP-CEPH)(Cann et aL, 2002). We used MassARRAY iPLEX Gold to pe 48 SNPs in a 0.83cM date. Alterations in gland structure have been reported to disrupt (1, 400 kb) region surrounding the 370A allele and combined our data with lactation in mice(Ramanathan et al., 2007), suggesting a func- published HGDP-CEPH data(Jakobsson et al., 2008; Li et al, 2008). After tional consequence for this change Unfortunately it is not excluding monomorphic SNPs and SNPs with inconsistent genotypes, we highlighting the importance of animal models. Reports of smaller from 51 populations. we interred haplotype data by phasing with fastPHASE breast size in East Asian women(Maskarinec et al., 2001; Chen in the region revealed a 139 kb block surrounding 370A(Figure S1).We al.,2004)are notable in light of the effects of 370A on fat pad counted the number of chromosomes of each haplotype in each population size and the importance of breast morphology in human mate and plotted the frequencies on a world map. preference(Furnham et al., 1998, 2006: Dixson et aL, 2011). Further analysis of the functional implications of 370A in the Forward simulation mouse and development of methods to assay these phenotypes The spatially explicit model takes into account evolutionary processes such as in humans are critical to evaluate such hypotheses and also to population structure, drift, and natural selection(tan et al,2009),imple- demographic processes, including population growth, sporadic long-range gated, such as those linked to differential susceptibility to respi- migration, cultural diffusion of farming technology gene flow between demes ratory disease(Clarke et aL., 1987; Mauldin et aL., 2009) and between cultural groups, and the effects of the spread of farming on In light of 370A's pleiotropy it is possible that selection acted carrying capacities (see Modeling the origins and Sprea on multiple traits. The tendency to seek a single driving character Approximate Bayesian Computation Framework and Figure S2 for details) is underlain by the perception that pleiotropic changes are inherently disadvantageous. Evolution is believed to proceed We applied an ABC inference framework to estimate p of interest primarily through mutations in gene regulatory regions rather (Bertorelle et al, 2010: Csillery et al, 2010). We compared summary statistics han exons because this reduces pleiotropic effects(King and (370A allele frequency in 29 populations) reco Wilson, 1975; Stern, 2000; Carroll, 2008 ) From the perspective observed of this model, a specific effect of 370A,s pleiotropic conse- the differences were sufficiently small. We calculated the Euclidean distance quences was favored under the conditions present in East Asia (8)between the simulated and observed statistics for each simulated data and conferred an advantage with other neutral or deleterious set and retained those with the smallest values. Parameter sets were obtained according to the associated 8. See Modeling the origins and spread of 370A in traits hitchhiking along with the selected trait. However, the large an Approximate Bayesian Computation Framework for details on posterior coefficient of selection on 370A contrasts with the relatively density and choice of simulation cut-off modest magnitude of structural changes on any one affected trait and suggests alternative interpretations. One possibility is Mouse strains that the effects of 370A were magnified by coselection on o construct mice carrying the 3/A allele, a targeting vector conta another variant. For example, a coding variant of the related 326C point mutation was injected into J1 ES cells(Brigham and Women's EDA2R gene affects human hair and has swept to fixation Hospital Transgenic Mouse Facility, Boston USA). Chimeric mice that trans- hitted the knockin allele to the germline were recovered from a correctly East Asia ( Sabeti et al., 2007; Prodi et al., 2008) rgeted clone after injection into C57BL/6 blastocysts. The chimera was Alternatively, it could be precisely the pleiotropic nature of bred to a ubiquitously expressing B-Actin Cre line(gift from Susan Dymecki 370A that allowed multiple distinct selective forces to act on Harvard Medical School, Boston USA. Mice subsequently bred onto this variant over its long history, when many of the postulated an PVB (Charles River Laboratories)background for five generations, by which selective pressures such as temperature and humidity changed 370A Knockin Mouse for details of knockin construction). We obtained df mice dramatically. The fact that EDAR acts mostly on ectodermal on a C3HeB/FeJ background from Jackson Laboratories and crossed them ppendages and that the phenotypic effects of the 370A allele onto an FVB background for one generation. Mouse work was performed in are not extreme reduces the costs of pleiotropy and would facil- accordance with protocols approved by the Harvard M ate this process. Thus, what were initially neutral changes in Committee on Anima some appendages driven by 370A would gain adaptive signifi- cance in the face of new selective pressures. It is worth noting Mouse Hair Size lair from the back of pl9-P21 that largely invisible structural changes resulting from the 370A nted on slides in Gelvatol and allele that might confer functional advantage, such as increased analyzed on a Nikon eclipse cope to score medulla cells. A minimum of 700 hairs were sc mouse. 370V (n= 12), 370V/ eccrine gland number, are directly linked to visually obvious 370A(n=11), and 370A(n Cell 152, 691-702, February 14, 2013(2013 Elsevier Inc. 699(Sun et al., 2012). High humidity, especially in the summers, may have provided a seasonally selective advantage for individuals better able to functionally activate more eccrine glands and thus sweat more effectively (Kuno, 1956). To explore this hypoth￾esis, greater precision on when and where the allele was under selection—perhaps using ancient DNA sources—in conjunction with more detailed archaeological and climatic data are needed. Alternatively, another phenotype, such as mammary gland branching or fat pad size could have been adaptive. The increased branching of 370A mouse mammary glands and the importance of mammary tissue in evolutionary fitness (Anderson et al., 1983; Oftedal, 2002) make this organ an interesting candi￾date. Alterations in gland structure have been reported to disrupt lactation in mice (Ramanathan et al., 2007), suggesting a func￾tional consequence for this change. Unfortunately, it is not possible to assess mammary gland branching in living humans, highlighting the importance of animal models. Reports of smaller breast size in East Asian women (Maskarinec et al., 2001; Chen et al., 2004) are notable in light of the effects of 370A on fat pad size and the importance of breast morphology in human mate preference (Furnham et al., 1998, 2006; Dixson et al., 2011). Further analysis of the functional implications of 370A in the mouse and development of methods to assay these phenotypes in humans are critical to evaluate such hypotheses and also to analyze additional potential 370A phenotypes yet to be investi￾gated, such as those linked to differential susceptibility to respi￾ratory disease (Clarke et al., 1987; Mauldin et al., 2009). In light of 370A’s pleiotropy, it is possible that selection acted on multiple traits. The tendency to seek a single driving character is underlain by the perception that pleiotropic changes are inherently disadvantageous. Evolution is believed to proceed primarily through mutations in gene regulatory regions rather than exons because this reduces pleiotropic effects (King and Wilson, 1975; Stern, 2000; Carroll, 2008). From the perspective of this model, a specific effect of 370A’s pleiotropic conse￾quences was favored under the conditions present in East Asia and conferred an advantage with other neutral or deleterious traits hitchhiking along with the selected trait. However, the large coefficient of selection on 370A contrasts with the relatively modest magnitude of structural changes on any one affected trait and suggests alternative interpretations. One possibility is that the effects of 370A were magnified by coselection on another variant. For example, a coding variant of the related EDA2R gene affects human hair and has swept to fixation in East Asia (Sabeti et al., 2007; Prodi et al., 2008). Alternatively, it could be precisely the pleiotropic nature of 370A that allowed multiple distinct selective forces to act on this variant over its long history, when many of the postulated selective pressures such as temperature and humidity changed dramatically. The fact that EDAR acts mostly on ectodermal appendages and that the phenotypic effects of the 370A allele are not extreme reduces the costs of pleiotropy and would facil￾itate this process. Thus, what were initially neutral changes in some appendages driven by 370A would gain adaptive signifi- cance in the face of new selective pressures. It is worth noting that largely invisible structural changes resulting from the 370A allele that might confer functional advantage, such as increased eccrine gland number, are directly linked to visually obvious traits such as hair phenotypes and breast size. This creates conditions in which biases in mate preference could rapidly evolve and reinforce more direct competitive advantages. Consequently, the cumulative selective force acting over time on diverse traits caused by a single pleiotropic mutation could have driven the rise and spread of 370A. EXPERIMENTAL PROCEDURES Haplotype Analysis DNA from1,064 individuals from 52 global populations was acquired from the Human Genome Diversity Panel (HGDP-CEPH) (Cann et al., 2002). We used Sequenom MassARRAY iPLEX Gold to genotype 48 SNPs in a 0.83cM (
1,400 kb) region surrounding the 370A allele and combined our data with published HGDP-CEPH data (Jakobsson et al., 2008; Li et al., 2008). After excluding monomorphic SNPs and SNPs with inconsistent genotypes, we obtained a final data set of 280 SNPs in 984 samples (1,968 chromosomes) from 51 populations. We inferred haplotype data by phasing with fastPHASE (Scheet and Stephens, 2006). Examination of linkage disequilibrium patterns in the region revealed a
139 kb block surrounding 370A (Figure S1). We counted the number of chromosomes of each haplotype in each population and plotted the frequencies on a world map. Forward Simulation The spatially explicit model takes into account evolutionary processes such as population structure, drift, and natural selection (Itan et al., 2009), imple￾mented here for a semidominant allele. The model also considers various demographic processes, including population growth, sporadic long-range migration, cultural diffusion of farming technology, gene flow between demes and between cultural groups, and the effects of the spread of farming on carrying capacities (see Modeling the Origins and Spread of 370A in an Approximate Bayesian Computation Framework and Figure S2 for details). Approximate Bayesian Computation We applied an ABC inference framework to estimate parameters of interest (Bertorelle et al., 2010; Csille´ry et al., 2010). We compared summary statistics (370A allele frequency in 29 populations) recorded after each simulation to observed frequencies (Table S1) and accepted only those simulations in which the differences were sufficiently small. We calculated the Euclidean distance (d) between the simulated and observed statistics for each simulated data set and retained those with the smallest values. Parameter sets were obtained according to the associated d. See Modeling the Origins and Spread of 370A in an Approximate Bayesian Computation Framework for details on posterior density and choice of simulation cut-off. Mouse Strains To construct mice carrying the 370A allele, a targeting vector containing the T1326C point mutation was injected into J1 ES cells (Brigham and Women’s Hospital Transgenic Mouse Facility, Boston USA). Chimeric mice that trans￾mitted the knockin allele to the germline were recovered from a correctly targeted clone after injection into C57BL/6 blastocysts. The chimera was bred to a ubiquitously expressing b-Actin Cre line (gift from Susan Dymecki, Harvard Medical School, Boston USA). Mice were subsequently bred onto an FVB (Charles River Laboratories) background for five generations, by which the Cre transgene was also removed (Generation and Statistical Analysis of the 370A Knockin Mouse for details of knockin construction). We obtained dlj mice on a C3HeB/FeJ background from Jackson Laboratories and crossed them onto an FVB background for one generation. Mouse work was performed in accordance with protocols approved by the Harvard Medical Area Standing Committee on Animals. Mouse Hair Size Hair from the back of P19–P21 pups was mounted on slides in Gelvatol and analyzed on a Nikon eclipse E1000 microscope to score medulla cells. A minimum of 700 hairs were scored from each mouse. 370V (n = 12), 370V/ 370A (n = 11), and 370A (n = 13) animals were analyzed. Cell 152, 691–702, February 14, 2013 ª2013 Elsevier Inc. 699