Testing for Archaic Hominin Admixture on the X Chromosome

12 Pages·2008·0.28 MB·English

Copyright(cid:2)2008bytheGeneticsSocietyofAmerica
DOI:10.1534/genetics.107.080432
Testing for Archaic Hominin Admixture on the X Chromosome:
Model Likelihoods for the Modern Human RRM2P4
Region From Summaries of Genealogical Topology
Under the Structured Coalescent
Murray P. Cox,* Fernando L. Mendez,† Tatiana M. Karafet,* Maya Metni Pilkington,‡
Sarah B. Kingan,* Giovanni Destro-Bisol,§ Beverly I. Strassmann**
and Michael F. Hammer†,‡,**,1
*ARLDivision ofBiotechnology,†Departmentof Ecologyand EvolutionaryBiologyand ‡DepartmentofAnthropology,UniversityofArizona,
Tucson, Arizona85721, §Department ofAnimal andHuman Biology,University ofRome ‘‘LaSapienza,’’ and Istituto Italianodi
Antropologia,00185Rome, Italyand**Department ofAnthropology, UniversityofMichigan,AnnArbor,Michigan 48109
Manuscript receivedAugust 13, 2007
Acceptedforpublication November 11,2007
ABSTRACT
A2.4-kbstretchwithintheRRM2P4regionoftheXchromosome,previouslysequencedinasampleof
41globallydistributedhumans,displayedbothanancienttimetothemostrecentcommonancestor(e.g.,
aTMRCAof(cid:2)2millionyears)andabasalcladecomposedentirelyofAsiansequences.Thispatternwas
interpreted to reflect a history of introgressive hybridization from archaic hominins (most likely Asian
Homo erectus) into the anatomically modern human genome. Here, we address this hypothesis by
resequencingthe2.4-kbRRM2P4regionin131Africanand122non-Africanindividualsandbyextending
thelengthofsequenceinawindowof16.5kbencompassingtheRRM2P4pseudogeneinasubsetof90
individuals.WefindthatboththeancientTMRCAandtheskewinnon-Africanrepresentationinoneof
the basal clades are essentially limited to the central 2.4-kb region. We define a new summary statistic
called the minimum clade proportion (p ), which quantifies the proportion of individuals from a spe-
mc
cifiedgeographicregionineachofthetwobasalcladesofabinarygenetree,andthenemploycoalescent
simulationstoassessthelikelihoodoftheobservedcentralRRM2P4genealogyundertwoalternativeviews
of human evolutionary history: recent African replacement (RAR) and archaic admixture (AA). A
molecular-clock-basedTMRCAestimateof2.33millionyearsisastatisticaloutlierundertheRARmodel;
however,thelargevarianceassociatedwiththisestimatemakesitdifficulttodistinguishthepredictionsof
thehumanoriginsmodelstestedhere.Thep summarystatistic,whichhasimprovedpowerwithlarger
mc
samples of chromosomes, yields values that are significantly unlikely under the RAR model and fit
expectations betterundera range of archaicadmixture scenarios.
FOSSIL, archaeological, and genetic data all lend 2005a,b;PlagnolandWall2006;WallandHammer
support to the hypothesis that Homo sapiens orig- 2006). Early studies of nonrecombining regions such
inated in Africa (McBrearty and Brooks 2000; asmtDNAandtheYchromosomewereconsistentwith
McDougall et al. 2005; Garrigan and Hammer the hypothesis of a single origin followed by complete
2006). With the acceptance of the role of Africa in replacement,sometimesreferredtoastherecentAfrican
our species’ origin, there is now increasing interest in replacement (RAR) model. While many of the more re-
thequestionofhowtheancestralpopulationthatgave centlypublishedDNAsequencingstudiesofX-linkedand
risetoanatomicallymodernhumans(AMH)wasstruc- autosomallociarealsoconcordantwiththisRARmodel,
tured.DidAMHemergefromasingle,isolatedAfrican agrowingnumberarenot(Evansetal.2006;Garrigan
deme or from a subdivided ancestral population with and Hammer 2006).
geneflowamongsubpopulations?Arelatedquestionis Garrigan et al. (2005b) published one of the first
whether the expanding AMH population completely studiestopositrecentadmixturebetweenAMHandan
replaced or interbred with then contemporaneous ar- archaichumanpopulation.Aresequencingstudyof2.4
chaic populations such as Neanderthals and H. erectus kb of the ribonucleotide reductase M2 pseudogene 4
(Eswaran 2002; Templeton 2002; Garrigan et al. (RRM2P4) in a sample of 41 globally diverse humans
identified an unusual pattern of nucleotide polymor-
phismcomparedwithmostofthehumangenome.The
1Corresponding author: ARL Division of Biotechnology, Life Sciences
reconstructed gene tree revealed two clades of allelic
South,UniversityofArizona,Tucson,AZ85721.
E-mail:[email protected] sequences that were estimated to have diverged (cid:2)2
Genetics178:427–437(January2008)
428 M. P.Coxetal.
Figure 1.—Location of the RRM2P4 locus on
the X chromosome (top bar), positions of the
threesequencedregions(middlebar),andplace-
mentoftheRRM2P4pseudogenefragmentsrel-
ative to the RRM2P4 central region (bottom
bars).Verticalbarsinthecentralregionindicate
SNPs defining the two basal clades. All coordi-
nates correspond to the human genome UCSC
March 2006 build.
million years ago (MYA). One clade with very little se- highratesofrecombination($1.0cM/Mb).Theseloci,
quencevariationwasspecifictoAsians,whiletheother which represent selectively neutral X-chromosomal di-
more diverse clade of RRM2P4 sequences resembled a versity, will be published elsewhere. However, using
pattern typical of human variation and was globally thesedata,weshowherethatsimpleRARmodelsoften
distributed. By genotyping a diagnostic SNP in a large produceTMRCAvaluesthataresimilartothatofRRM2P4,
sample of humans, the divergent ‘‘Asian clade’’ was whilegenealogieswithskewingofbasalclademember-
shown to be frequent in Southeast Asians and nearly shiptowardnon-Africansremainstatisticaloutliers.
absent in sub-Saharan Africans. The greater genealog-
ical depth in Asia led to the hypothesis that RRM2P4
is a genomic remnant of introgressive hybridization
SUBJECTS AND METHODS
fromanAsianarchaicpopulation(H.erectus)intoAMH
groups expanding from Africa. However, despite the Regionssequenced:ResequencedatafortheRRM2P4
increasingavailabilityofgenomicdata,theuniqueness locus were generated within a ‘‘trio’’ design (Figure 1),
ofsuchdeeptimestothemostrecentcommonancestor which is an economical approach to jointly ascertain
(TMRCA) and of gene trees with non-African basal detailedpolymorphismpatternsandlarger-scalelinkage-
clades remains unclear. Importantly, we still do not disequilibrium profiles (Garrigan et al. 2005b). We se-
understand the likelihood of such genealogies under quencedthreegenomicsegmentsof1725bp½University
either the RAR model or models involving admixture of California, Santa Cruz (UCSC) March 2006 genome
witharchaichumans. coordinates143,212,138–143,213,863(cid:3),2341bp(coordi-
Garriganetal.’soriginalRRM2P4studywaslimitedby nates 143,219,154–143,221,495), and 1601 bp (coordi-
a paucity of sequence data and primarily qualitative nates 143,227,057–143,228,658), which were separated
analyses. Here, we extend earlier research by sequenc- by unsequenced regions of 5291 and 5562 bp, respec-
ing the 2.4-kb RRM2P4 region in 131 African and 122 tively.Ourcentralsubregionisthesameasthatdescribed
non-African individuals. We also determine the extent byGarriganetal.(2005b).
of sequence that exhibits the unusual pattern of poly- Sampling: The RRM2P4 central region was se-
morphism by selectively resequencing three DNA frag- quencedin131Africanand122non-Africanindividuals
mentstotaling5.6kbwithina16.5-kbwindowflanking (panel A). These samples were chosen without prior
RRM2P4. We infer the likelihood of the observed information about RRM2P4 lineage status. DNA sam-
RRM2P4 genealogy using a suite of summary statistics ples representing Mandenka from Senegal (n ¼ 16),
andMonteCarlocoalescentsimulationsundertheRAR BiakaPygmies fromtheCentralAfricanRepublic(n¼
model and a range of archaic admixture (AA) models 16), Khoisan from Namibia (n ¼ 9), French Basque
(Nordborg2000;PlagnolandWall2006).TheRAR (n¼16),HanChinese(n¼16),andNasioifromBougain-
models are parameterized by approximate Bayesian ville(n¼16)werepurchasedfromtheCentred’Etude
computation (ABC) conditioned on resequence data duPolymorphismeHumain(Cannetal.2002).Samples
from an additional data set of 19 neutral, unlinked X- of Baka Pygmies from Cameroon (n ¼ 23) were pro-
chromosomalloci.Thisresequencedatasetrepresents vided by Giovanni Destro-Bisol and the Dogon from
12Mbfrom19regionsoftheXchromosome,whichare Mali (n ¼ 32) were provided by Beverly Strassmann.
unlinked from genes (and each other) by medium to Samples from the Dinka of southern Sudan (n ¼ 21)
ArchaicAdmixture ontheX Chromosome 429
were collected in Tucson, Arizona, with informed con-
sent. Samples from three Siberian populations, the
Selkups (n ¼ 32), Forest Nentsi (n ¼ 28), and Tundra
Nentsi(n¼3),weredescribedpreviously(Karafetetal.
2002). Non-population-based samples (n ¼ 25) from
the Y chromosome consortium cell lines (Y Chromo-
some Consortium 2002) were also included in this
panel.Resequencedataforthefulltrioweregenerated
inasecondpanelof42Africanand48non-Africanin-
dividuals(panelB)fromthreeAfricangroups(Khoisan,
Mandenka, and Biaka) and three non-African groups
(French Basque, Han, and Nasioi) (samples as de-
scribed above). All sampling protocols were approved
bytheHumanSubjectsCommitteeattheUniversityof
Arizonaandbytheinstitutionsofallcollaboratorswho
providedDNAsamples.
Recombinationanalysisandgenetreedating:Ratesof
linkage disequilibrium across the sequence were de-
termined using LDhat (McVean and Spencer 2006).
Figure 2.—Schematics of two demographic models. The
TheRRM2P4centralregionshowsonlylimitedsignsof
recent African replacement (RAR) model grows exponen-
recombination, and a most parsimonious tree was tially from a 104 effective population size at 80 KYA, and
reconstructed by breaking low-frequency reticulations. 500 individuals found the non-African population at 60
The TMRCA of the tree and the age of its polymor- KYA, experience a constant-sized bottleneck for 5 KYA, and
phismswereestimatedwithGenetree(Griffiths2007). grow exponentially from 55 KYA. The ancient admixture
(AA) model includes introgression from an ancestral homi-
Genetree employs a full maximum-likelihood method
nin source with constant population size of 103; the popula-
that is based on the standard coalescent (Kingman tion leading to that of anatomically modern humans and
1982) and assumes an infinite-sites mutational model. ancestral hominins diverged 2 MYA. Modern effective sizes
Likelihoodsurfacesforthepopulationmutationrate,u, andtheintercontinentalmigrationratewereinferredbyap-
andthepopulationgrowthrate,b,weregeneratedun- proximate Bayesiancomputation.
derapanmicticsingle-dememodelandanislandmodel
of population structure. TMRCA values were inferred
frommaximum-likelihoodparameterizationsunderboth the intergeneration interval (e.g., a 25-year mean in-
models. tergeneration interval) does not alter our conclusions
Demographicmodels:SummariesoftheRRM2P4cen- (ourunpublisheddata).
tral region were compared with values obtained from TheRARmodeldepictstwopanmictic,exponentially
simulationsunderastructuredcoalescent (Nordborg growingWright–FisherdemesrepresentingAfricanand
1997;Hudson2002)todeterminethelikelihoodofthe non-Africanpopulations.GrowthbeginsintheAfrican
observed genealogy. We employed a framework for deme 80,000 years ago from a single ancestral popula-
humandemographysimilartothatdevelopedbyPlagnol tion(N ¼104)andcontinuesuntilitreachesitscurrent
e
and Wall (2006), but modified (see below) to yield effectivesize.Asmallgroup(N ¼500)splitsfromthe
e
the number of segregating sites, S, consistent with an Africandeme60,000yearsagotoformthenon-African
independent data set of 19 X chromosome loci se- deme.Thissubgroupexperiencesabottleneckfor5000
quencedinthesameindividuals(panelB).Wepresent years before expanding exponentially to its current
resultsfromtwodemographicmodels:atwo-demeRAR effective size. Modern effective sizes and the intercon-
model and an AA scenario similar to Nordborg’s tinental migration rate were parameterized by approx-
(2000)isolationandadmixturemodel(Figure2).Alter- imateBayesiancomputation(seebelow).
nativeRARmodelswithvaryinglevelsofrecentpopula- TheAAmodelincorporatesinstantaneousadmixture
tion subdivision (e.g., one-deme and six-deme models) fromanancestralhomininsourceintotheRARmodel
did not differ significantly from the two-deme RAR describedabove.Anancestralhomininpopulationwith
model reported here (our unpublished data). These constanteffectivepopulationsizeof103splitsfromthe
models are not meant to represent the true history of ancestorsofmodernhumansat2MYA.Weconsidereda
human populations, but they do let us explore the ef- range of admixture rates (0–5%) from ancestral hom-
fects of expansion and replacement vs. archaic admix- ininstomodernhumansinAsia,coupledwithaseriesof
tureonpatternsofgenomicvariation.Coalescentdates admixture times (10–55 KY before present). We could
(scaledby3N generations)weretranslatedtochrono- notinferanoptimizedAAmodelbecausethepaucityof
e
logical time using a 28-year mean intergeneration candidatelociisnotsufficientbothformodeltraining
interval(Fenner2005).Theuseofalowerestimateof andforsubsequentstatisticaltesting.
430 M. P.Coxetal.
Model fitting via approximate Bayesian computa-
tion:WeparameterizedtheRARmodeltoreflectwhatis
known about deep human demography and, conse-
quently,toproducesimulateddatasetsthatmimicreal
genomicdatasets.Parameterinferencerapidlybecomes
computationally intractable at high-dimensional state
spaces, such as those associated with complex demo-
graphicmodels.Therefore,wefixedsomedemographic
parameters that have been inferred elsewhere, e.g.,
thetimeofonsetofpopulationgrowthandnon-African
bottleneck size (Plagnol and Wall 2006, Figure 2).
However, a lower-dimensional state space of modern
populationsize,N ,andtheintercontinentalmigration
0
ratepergeneration,m,wereinferredexplicitlybyABC
(Beaumontetal.2002).
Essentially,wegenerated104coalescentsimulationsfor
each of 105 different sets of demographic parameters,
Q¼{N ,m},thatweredrawnrandomlyfromtwouniform
0
distributions,N 2U½104; 105(cid:3) andm 2U½10(cid:4)11; 10(cid:4)8(cid:3):
0
Each set of coalescent simulations was compared with
resequence data from 19 noncoding regions on the X
chromosome (i.e., panel B, 42 African and 48 non- Figure 3.—Schematic of the minimum clade-proportion
Africanindividuals;datatobepublishedelsewhere).We statistic, pmc.See textfordetails.
estimatedu(¼3N m)foreachdemographicparameter
0
set using the average mutation rate of these 19 loci,
the minimum quantity of individuals from a specified
8.33 10(cid:4)10/bp/year(range4.83 10(cid:4)10–1.63 10(cid:4)9),in-
geographicregionorethnicgroup(suchasAfricans)in
ferred from sequence divergence (assuming a human/
eachofthetwobasalcladesofabinarygenetree(Figure
chimpanzeedivergencetimeof6MYA).Furthermore,we
3). (This statistic is described fully in the appendix.)
chose to condition our ABC inference on the mean
The p statistic has an intuitive interpretation. Con-
number of segregating sites, S, that was observed across mc
theadditionaldatasetof19Xchromosomeloci(S(cid:2)¼26). siderabinarytreeofAfricanandnon-Africansequences
(Figure 3); clades (C , C ) can be defined as the two
Thisstatisticwaschosenbecauseitvariesprimarilywith 1 2
basal branches that diverge from the coalescent of all
themutationrate(aknownparameter)andtreedepth
sampledindividuals.Here,thenumberofAfricanchro-
(aparameterwewishedtoinfer).Weselectedthe0.01%
mosomecopiesis(k ¼1,k ¼6)andthetotalnumber
ofrandomdemographicparametersets,Q,whosecoa- 1 2
ofchromosomecopiesis(n ¼4,n ¼10).FromEqua-
lescent data sets produced an average number of seg- 1 2
regating sites closest to the observed value (S(cid:2)¼26). tionA1,theminimumcladeproportionisthelesserof
(k =n ¼1; k =n ¼ 6); here, p ¼1: The p statistic
MeanvaluesforN0andmweredrawnfromthissubsetof 1 1 4 2 2 10 mc 4 mc
was calculated directly from coalescent genealogies
demographic parameter sets. These values represent
usingcustomsoftware(codeavailableonrequest).
best-fit estimatesfor themodern effectivesize, N ,and
0
migration rate, m, of the real-world demography un-
derlyingourobserved19Xchromosomeneutral-locus
RESULTS
genomicdataset.
Test statistics: We calculated the approximate like- Patterns of DNA sequence variation within and
lihoods of two summary statistics: the TMRCA and the around RRM2P4: We sequenced 2.4 kb of the central
minimumproportionofAfricansinoneofthetwobasal RRM2P4 segment, which encompasses the processed
clades. Values were inferred from 105 replicates under pseudogene (Figure 1), in 131 African and 122 non-
all demographic models. We note that these tests are African individuals. A total of 22 segregating sites were
conservative, because both summaries are determined identified,producing23uniquehaplotypes(supplemen-
directlyfromcoalescentgenealogies.Resolutionofthe tal Table 1 at http://www.genetics.org/supplemental/).
underlying genealogy is constrained by S for real data SimilartothepreviousstudyofGarriganetal.(2005b),
sets(cf.Nordborg 2000).Thedistribution ofTMRCA levels of nucleotide diversity are statistically higher in
values was extracted from the output of Hudson’s non-Africans(u ¼0.136)thaninAfricans(u ¼0.0768;
p p
(2002) ms using custom software (code available on P > 0.001; Table 1). Also, the central subregion still
request). shows minimal evidence of recombination despite a
Here, we also define a new summary statistic, the sixfoldincreaseinthesizeofthedataset(supplemental
minimum clade proportion (p ), which characterizes Figure 1 at http://www.genetics.org/supplemental/).
mc
ArchaicAdmixture ontheX Chromosome 431
TABLE 1
Estimates ofsummarystatistics for the RRM2P4central and flankingregions
Lower5% 95% confidence
Region Deme n Length S u /bp(%) quantile u /bp(%) interval
w p
59 Global 83 1727 5 0.058 — 0.068 (0.052, 0.081)
African 37 1727 5 0.069 — 0.084 (0.059, 0.098)
Non-African 46 1727 3 0.040 — 0.056 (0.034, 0.073)
Central Global 253 2320 22 0.155 0.120 0.110 (0.093, 0.126)
African 131 2320 16 0.126 0.071 0.077 (0.066, 0.087)
Non-African 122 2320 15 0.120 0.104 0.136 (0.105, 0.163)
39 Global 83 1612 7 0.088 — 0.061 (0.037, 0.081)
African 37 1612 4 0.060 — 0.081 (0.044, 0.108)
Non-African 46 1612 6 0.085 — 0.041 (0.014, 0.067)
Statisticalconfidencewasdeterminedfrom104bootstrapreplicates.SNPdiversity,u ,issignificantlylowerintheflankingre-
W
gionsthaninthecentralregion(a,0.05).Africanu -valuesdonotdiffersignificantlyamongregions(a.0.05),butnon-African
p
u -andu -valuesaresignificantlyhigherinthecentralregion.Withinsequencedregions,non-Africanshavesignificantlyhigher
p W
u relative to Africans in thecentral region only (P >0.001,underlined). Thiseffect is not observed in the 59 (P ¼0.966) or
p
39regions(P¼ 0.957).
As before, this low level of recombination permitted To determine the length of sequence within and
partial reconstruction of a single nonreticulating gene around the RRM2P4 locus that shows the unusual
tree for the central region (Figure 4). This genealogy genealogical features, we sequenced two additional
has the same two unusual characteristics described by fragments of 1725 and 1601 bp that flank the central
Garrigan et al. (2005b): a deep TMRCA and a basal region(Figure1)inasubsetofAfricanandnon-African
cladecomposedalmostentirelyofAsiansequences.For individuals (panel B). Levels of nucleotide diversity in
example, of the 253 individuals resequenced for the African vs. non-African populations were more similar
centralregion,21areintheleftmostdivergentcladein toaveragepatternsforthegenome,withAfricanvalues
Figure4(‘‘cladeA’’),and20ofthesearefromEastAsia (u ¼0.0836and0.0805)greaterthannon-Africanvalues
p
and Oceania. A single Dogon from Mali was the only (u ¼ 0.0556 and 0.0412) in the 59- and 39-flanking
p
African member of this clade. This haplotype carries a regions, respectively (Table 1). Recombination rates
SNP(1639)thatoccurs inbothbasalclades.Anorigin are low (0.43 cM/Mb) for the 59 and central RRM2P4
of this haplotype through recombination rather than subregions, but are substantially elevated (16 cM/Mb)
homoplasyismorelikelygiventhelowmutationrateof betweenthecentraland39subregions(supplementalFig-
the RRM2P4 locus together with its moderate rate of ure1athttp://www.genetics.org/supplemental/).Thisre-
recombination.BecausetheparentalcladeAformwas combinationhotspoteffectivelyunlinksthecentraland
found only in Asia and this haplotype is shared with a 39 subregions, which therefore have largely indepen-
Melanesian, we suggest that this recombinant lineage dent evolutionary histories. A network representing
may have originated in Asia and migrated recently to sequences of the entire 16.4-kb region illustrates the
Africa. decouplingofthesetwogenomicregions(supplemental
We used both molecular-clock and coalescent ap- Figure 2 at http://www.genetics.org/supplemental/).
proaches to estimate the TMCRA of the RRM2P4 cen- The unusual genealogy is less apparent in the 59 frag-
tralsubregiongenetree.Outgroupcomparisonsreveal mentdespitesomelinkagedisequilibriumwiththecen-
an average of 22 nucleotide substitutions between all tral region. (Recombination rates in these regions are
human and chimpanzee central RRM2P4 region se- about one-third the X chromosome average; supple-
quences.Givenanaverageof8.53nucleotidedifferences mental Figure 1.) However, only five segregating sites
observedbetweenthetwohumanRRM2P4lineages(i.e., were identified in the 59-flanking region, a reduction
the average number of mutations between sequences of63% overthecentral region diversity(Table1), and
across the base of the human gene tree), we estimate noderivedpolymorphismswereidentifiedinthe59re-
thatthetwodeepesthumancladesdiverged(cid:2)2.33MYA gion on chromosomes carrying central region clade A
(assuming a 6-MYA human–chimpanzee divergence haplotypes(supplementalTable2athttp://www.genetics.
time). We also inferred the TMRCA of the RRM2P4 org/supplemental/). Because the unusual pattern of
central subregion using a full maximum-likelihood polymorphism is most apparent in the central region,
method under both a panmictic and an island model. we focus further analyses solely on this portion of the
These models yielded TMRCA values of 1.24 and 2.88 sequencedregion.
MYA,whichbracketthemolecular-clockdate(datanot RAR model parameters: TheRARmodelwasparame-
shown). terizedbyABC.Becausethemodelcannotbeparameterized
432 M. P.Coxetal.
Figure 4.—Time-scaled gene tree of the RRM2P4 central region. Polymorphisms are labeled with the nomenclature of
Garriganetal.(2005b)andareequivalenttothatpublication’sFigure1.Italicizedandunderlinedpolymorphismsindicateprob-
ablereticulationpoints.TheproportionsofAfricanandnon-Africanindividualscarryingeachlineageareindicatedbeneaththe
tree.Notetheoverrepresentationofnon-Africansintheleftmostbasalclade(‘‘cladeA’’).Molecular-clockestimatesoftheTMRCA
andmutation agesareshown in millionsof years onthevertical axis.
on the test locus (here, RRM2P4), we conditioned the Both the distribution of TMRCA values and the pro-
modelonaseparatetrainingdatasetof19independent portion of African individuals in one of the two basal
X chromosome noncoding regions. These resequence clades were inferred under both models. The mean
dataaretooextensivefordetaileddescriptionhereand TMRCAunderthetwo-dememodelwas1.15MYA,with
arethesubjectofaseparatepublication.However,basic datesexceeding2.12MYAbeingstatisticaloutliers(a¼
summaries of these 19 loci relevant to the current 0.05). While the maximum-likelihood method assum-
analysisarepresentedinsupplementalTable3athttp:// ingpanmixiareturnedameanTMRCAvaluesimilarto
www.genetics.org/supplemental/. The two-deme RAR thatproducedundertheRARmodel½i.e.,P(TMRCA.
modelwasparameterizedbygenerating105randomN 1.24 3 106 j RAR) ¼ 0.350(cid:3), both the molecular clock
0
and m values (see full description in subjects and ½P(TMRCA . 2.33 3 106 j RAR) ¼ 0.030(cid:3) and the
methods) and accepting the 0.01% that best matched maximum-likelihoodmethodassuminganislandmodel
the number of segregating sites observed in 19 inde- ½P(TMRCA.2.883106jRAR)¼0.008(cid:3)yieldedTMRCA
pendentXchromosomenoncodingloci(supplemental valuesthatareunusuallyold.UndertheAAmodel,the
Figure 3 at http://www.genetics.org/supplemental/). TMRCAdistributionisshifteddeeperintimerelativeto
The optimal parameters were inferred as a modern RAR models, and genealogies exceeding 3 MYA would
effective size, N , of 12,300 (range 12,000–12,500) and be expected under our archaic admixture scenario
0
anintercontinentalmigrationratepergeneration,m,of (Figure5,aandb).Themolecular-clock-basedestimate
3.62 3 10(cid:4)9 (range 6.75 3 10(cid:4)10–8.24 3 10(cid:4)9). Using ofthetrueTMRCAisnotanoutlierundertheAAmodel
parametervaluesattheextremesoftheserangeshadlittle asspecifiedhere½P(TMRCA.2.333106jAA)¼0.140(cid:3).
effect on the following statistical analyses (our unpub- The geographical distribution of lineages on the
lisheddata).Importantly,theparameterizedRARmodel central RRM2P4 genealogy is also skewed: one of the
producessimulateddatasetsthatyieldsummaries(such two basal clades (Figure 4, clade A) is found infre-
as growth rates and effective sizes) that are consistent quently in Africans (0.048) but commonly in non-
with other demographic inferences (e.g., from Gene- Africans (0.952). This pattern can also be compared
tree).Althoughthereisstrictlynowaytoassessthetrue with the empirical distribution of p , as determined
mc
history of our samples, this best-fit RAR model is a fromtheadditionaldatasetof19Xchromosomeloci,
reasonablefirstapproximationforhumandemographic among which the smallest minimum African clade
history,asreconstructedfromtheXchromosome. proportion is 0.214. The likelihood of observing the
Assessing the uniqueness of the RRM2P4 genealogy: same, or a more extreme, proportion of Africans (i.e.,
The uniqueness of the RRM2P4 central region was 1)inabasalcladewasstatisticallysignificantunderthe
21
estimatedusingasimulation-basedsummary-likelihood RARmodel½P(p #0.048jRAR)¼0.031(cid:3),butnotso
mc
approach under the RAR and AA models (Figure 2). under the AA model ½P(p # 0.048 j AA) ¼ 0.24(cid:3). To
mc
ArchaicAdmixture ontheX Chromosome 433
Figure5.—Distributionandcumulativeprobabilityof(aandb)TMRCAsand(candd)minimumcladeproportionsunderthe
optimaltwo-demeRARmodel(solidcurve)andthecorrespondingmodelwith5%introgressionfromancestralhomininsat50KYA
(dashedcurve).Themolecular-clockdateisshownbyasolidverticallineinaandb;thepanmicticandislandmodeldatesareshownby
dottedverticallines(totheleftandright,respectively).Theadmixturepeakwouldincreaseasadmixtureoccurredmorefrequently
andshiftrightwithdeeperstructurebetweenadmixingdemes.Theobservedp isillustratedbyasolidverticallineincandd.
mc
furtherexploretheeffectsofarchaicadmixtureonthe 2001; Satta and Takahata 2004). Garrigan et al.
minimumcladeproportion,wetooktheoptimizedRAR (2005b)also genotyped asinglediagnosticSNP to test
model and incorporated variable rates of admixture for the presence of clade A in a larger number of
(0.5–5%)occurringatvariabletimes(10–55KYbefore samples (n ¼ 570 from 17 globally distributed popula-
present)andran105simulationsateachpointona103 tions).Theydiscoveredadecreasingfrequencygradient
10 grid of parameter space (Figure 6). Basal clades centered on southern China (where the clade A is
dominatedbynon-Africansareobservedmoreoftenon present .50%) and extremely low frequencies of the
averageasarchaicadmixturebecomesmorerecentand ‘‘Asian’’ divergent lineage in Europe, the Middle East,
morefrequent.Higherratesofadmixturemakeitmore andAfrica.(SeeFigure1inGarriganetal.2005b.)To
likely that one of the two basal clades derives entirely explaintheprevalenceofbasalRRM2P4lineagesinEast
from the descendants of Asian ancestral hominins Asiatheyfavoredamodelofrecentadmixturebetween
(p / 0), and more recent admixture makes it less divergent AMH and H. erectus populations; although
mc
likely that migrants will carry admixed lineages into they could not rule out founder effects leading to the
Africanpopulations(also,p /0). lossofoneofthetwodivergentlineagesinAfrica.Here,
mc
we resequence this locus in a much larger sample of
humans, extend the length of the sequenced region,
DISCUSSION
and test unusual aspects of the genealogy statistically,
Garriganetal.(2005b)describeda2.4-kbregionon usingamodel-basedcoalescentsimulationframework.
theXchromosomewithunusualgenealogicalstructure We generated resequencing data 59 and 39 of the
in a sample of 41 humans: a deep TMRCA and a basal central2.4-kbregioninapanelof90individualstosee
clade composed entirely of Asian (n ¼ 3) sequences. whether the pattern originally described by Garrigan
This differs from most genealogies observed to date, et al. (2005b) extended farther along the X chromo-
where African individuals dominate at least one of the some. We found that a strong recombination hotspot
two basal clades (Labuda et al. 2000; Takahataet al. almostcompletelydecouplesthecentraland39regions,
434 M. P.Coxetal.
linkage with a third selected locus located farther 59
unlikely. In sum, it appears that polymorphisms that
clearlydefinetheunusualRRM2P4genealogyarefound
only within the stretch of noncoding DNA associated
withthepseudogene(Figure1)andthatthesesitesare
unlikelytobeaffectedbyrecentpositiveselection.
Oursixfoldlargerdatabaseofcentralregionsequences
doesnotresultinasubstantialchangeinthetopologyof
thegenetreedescribedbyGarriganetal.(2005b)orin
the partitioning of its two deepest branches. However,
wedidobserveseveralnewlineagesatlowfrequencyand
discovered some novel evidence of recombination. Of
the21individualsidentifiedwiththelessfrequentbasal
lineage, only one was from Africa. This Dogon in-
dividual from Mali was the same African individual
identified as carrying a clade A lineage on the basis of
the SNP-based genotyping assay in Garrigan et al.
(2005b). Our extended sequence database identified
Figure 6.—Effect of admixture parameters on the mini- onlyasinglenewpolymorphismincladeAand11new
mumcladeproportion.FewerAfricansarefoundinonebasal
polymorphismsinthemorediversegloballydistributed
cladeasarchaicAsianadmixtureoccursmorefrequentlyand
cladeB(compareFigure1ofGarriganetal.2005bwith
morerecently.Thelikelihoodsurfaceisgeneratedbyinterpo-
lationfrommeanp valuesof105simulationsateachpoint ourFigure4).Thisbringsthetotalnumberofhaplotypes
mc
ina10310grid(dots)coveringtheparameterspaceofad- inthedivergentAsiancladeAto3,comparedwithatotal
mixture timeandadmixture proportion. of20haplotypesintheotherbasalclade.Interestingly,
the new clade A haplotype was identified only in two
individuals:theaforementionedDogonindividualand
and despite linkage disequilibrium with the 59 end of anindividualfromBougainvilleIsland,Melanesia.This
the sequence, the pattern of higher Asian diversity for is consistent with the possibility that recent migration
the central region was not found for the other two carriedthisrarelineagebetweencontinents.Although
regions. While it is possible that this reduced diversity we cannot exclude the possibility that unsampled
may simply reflect the stochastic nature of the muta- Africanpopulationscarrythiscladeathigherfrequency
tional process given the relatively short length of se- thanweobservehere,ourgeographicalcoverageofthe
quenceexamined((cid:2)2kb),wesuggestthatthelineage Africancontinentisstillquiteextensive.
history of the central region has been at least partly Our simulations show that the 2.33-million-year
decoupledfromthoseofthe59and39regionsthrough molecular-clock-based TMRCA estimate is a statistical
recombination. BLAST results show that the RRM2P4 outlier under the RAR model (Figure 5, a and b), but
processed pseudogene has sequence conservation to notunderanAAmodelwithancestralstructuredating
the rhesus macaque (Macaca mulatta). Therefore, the from 2 MYA. However, maximum-likelihood estimates
highdiversityofthecentralregiondoesnotresultfrom of the TMRCA inferred under different models of
increasedmutationatthetimeofpseudogeneinsertion humanpopulationstructurespanawiderangeoftimes.
becausehumanvariationtracesbackonlytotheHomo ThelargevarianceinTMRCAestimatesisalsoexpected
lineage(i.e.,theTMRCAofhumanhaplotypesismuch because the short length of the central subregion se-
more recent than the insertion time). Central region quencelimitsthenumberofsitessegregating between
diversity is also unlikely to reflect paralogous gene thetwobasalclades.Unfortunately,thevarianceofTMRCA
conversionbecausetheRRM2P4sequencehasnoclose isalwayslargerelativetothemeananddoesnotdecrease
matchestootherregionsinthehumangenome.Finally, appreciablywithincreasedsamplesize(Griffithsand
increased central region diversity is unlikely to reflect Tavare´ 1994; Tang et al. 2002; Basu and Majumdar
linkagetoageneunderlong-termbalancingselection. 2003).Indeed,asweincreasedthesamplesizefrom41
The nearest 59 gene is SPANX-N2, which encodes a (Garrigan et al. 2005b) to 253, we found no new
protein of uncharacterized function. SPANX-N2 is 580 polymorphic sites on the basal branches of the gene
kb,or(cid:2)1.8cM,distantfromtheRRM2P4pseudogene, tree,whichsuggeststhatwehavesampledsufficientlyto
and the intervening region contains at least three hot- observe the basal node of the genealogy for the entire
spotsof(cid:2)16cM/Mb(eachsimilartothehotspot39of global population. There is only a remote probability
theRRM2P4locus).Whilewecannotexcludethepossi- that we have not observed the deepest split of the
bility that an unknown 59 functional variant may be RRM2P4 tree in our data set of 253 individuals (P (cid:5)
linked to the RRM2P4 locus, the disparity in diversity 0.0079)(Saundersetal.1984;KlimanandHey1993).
between the RRM2P4 59 and central regions makes Our TMRCA estimates, while consistent with the AA
ArchaicAdmixture ontheX Chromosome 435
model specified here, are unlikely to be improved by gions depends largely on the amount of admixture
samplingadditionalindividuals. between the two archaic populations (Wall 2000).
WealsoconsideredtheobservationofGarriganetal. Moreover, unless admixture was recent and involved
(2005b)thatAsiansamplesareoverrepresentedinone highly divergent populations, the power to detect ar-
ofthetwobasalcladesoftheRRM2P4tree.Thispattern chaicadmixtureislow(Nordborg2000).Inthecaseof
continuedtoholdevenafterincreasingthesizeofour RRM2P4, divergence may have started at the time of
DNA sequence data set. To assess how unusual this separationofH.ergaster/H.erectuspopulationsinAfrica
aspect of the RRM2P4 genealogy is under alternative (cid:2)2MYA(AntonandSwisher2004).Yet,thelengthof
modelsofhumanevolutionaryhistory,wedefinedanew the divergent sequence is short, possibly as a result of
summarystatistic,p ,whichquantifiestheskewinthe thenearbyrecombinationhotspotorbecauseadmixture
mc
proportionofindividualsfromtwopopulationsamong occurred in the more distant past and recombination
the two basal clades of a gene tree. This summary has subsequently broken down the admixed chromo-
statistic is applicable to RRM2P4 because the central some. In any case, identifying longer sequences with
region is essentially tree-like. We observed p ¼ 0.048 greater divergence would allow for more sophisticated
mc
forthecentralregionofRRM2P4,whichissignificantly tests of archaic admixture. For example, Wall (2000)
unlikely under the RAR model (P ¼ 0.031, Figure 5, c suggestedanumberofsummarystatisticsthatarebased
and d). To examine the sensitivity of the p statistic onboththelevelofdivergencebetweentwocladesand
mc
under a range of archaic admixture parameters, we theamountofrecombinationbetweenthem.
simulated coalescent genealogies and varied both the Recent population structure is another factor that
admixture proportion and the timing of introgression may affect the probability of sampling a locus with
(Figure6).Althoughgenealogieswithsmallp values a genealogy showing signs of archaic admixture. As
mc
are more common as the admixture proportion in- pointedoutbyNordborg(2000),populationstructure
creases(i.e.,upto(cid:2)5%)andintrogressionbeginsmore mayactuallyincreasethepowertodetectarchaicadmix-
recently (i.e., as recently as (cid:2)10 KYA), the RRM2P4 tureifwesamplesufficientlyamongdemes,becausewe
genealogyisnotasignificantoutlierunderanyofthese wouldexpecttheintrogressedallelestostillbepresent
AAmodelparameterizations.Thisfurthersupportsour in the area of the world where admixture took place.
conclusionthattheRRM2P4genealogyfitsexpectations In the case of RRM2P4, individuals carrying the less
betterunderascenarioofarchaicadmixture. frequent divergent lineage are concentrated in East
WhiletherearelimitationswithbothTMRCAandp Asia, suggesting that admixture may have occurred at
mc
for distinguishing predictions of the RAR and AA the Asian end of the global distribution of human
models, they do represent independent summaries of populations.Ontheotherhand,itisimportanttopoint
thedataand,thus,complementoneanother.Asalready out that current population structure is unlikely to re-
mentioned, the power of these two test statistics de- flect ancient patterns directly. Following a demic ex-
pendsondifferentaspectsofsampling.Varianceinthe pansion,whatwasoncesubdivisionbetweentwoAfrican
estimateoftheTMRCAisimprovedbylongersequences populationsmaynowappearasstructurebetweenAfri-
of the region with tree-like ancestry (in the case of can and non-African populations. For loci with more
RRM2P4thisislimitedbythesmallcentralregionand ancientTMRCA,thereisanincreaseinpowertodetect
flankingrecombination),butonlyslightlybyincreasing archaic admixture even if it occurred at more ancient
thesamplesize.Ontheotherhand,estimatesofp can times(Nordborg2000).ThismeansthatforRRM2P4,
mc
beimprovedbyincreasingthesamplesize,becausethe whichhasanancientTMRCA,wecannotbeconfident
variance of p decreases approximately as the inverse about where archaic admixture may have occurred
mc
of the sample size (analyses not shown). If the p is geographically. In this regard, it is interesting to note
mc
genuinelyanoutlier undertheRAR model,increasing thatagrowingnumberoflocihavebeendiscoveredwith
thenumberofindividualssampledincreasesthepower two deeply divergent lineages where both the major
to reject RAR. Indeed, when we use the SNP data of and the minor types are present only in African pop-
Garrigan et al. (2005b), which included 177 Africans ulations (Barreiro et al. 2005; Garrigan et al. 2005a;
and 393 non-Africans, we reject the RAR model with Hayakawa et al. 2006). This supports modelsinwhich
greaterconfidence(p ¼0.0189,P¼0.014). anatomicallymodernhumansdescendfromastructured
mc
Furtherevidenceinsupportofanarchaicadmixture ancestral African population (Garrigan and Hammer
modelawaitsanalysisofadditionallociexhibitinggene- 2006). We find some support that elevated admixture
alogical properties similar to the central RRM2P4 re- among highly divergent African subpopulations just
gion. Several candidates have already been identified prior to the recent African expansion could explain
(Hardingetal.1997;Zie˛tkiewiczetal.2003;Stefansson thepatternofpolymorphismatRRM2P4(supplemental
etal.2005;Shimadaetal.2007),butmostlackrigorous Figure 4 at http://www.genetics.org/supplemental/),
statistical analyses under a range of demographic but note that this model has little power to explain
models, including ancient admixture alternatives. The why RRM2P4 clade A lineages are geographically re-
frequency at which we expect to find introgressed re- strictedtoEastAsiatoday.Fornow,thislocusrepresents
436 M. P.Coxetal.
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APPENDIX
Here,wedefineanew summarystatistic,theminimumcladeproportion (p ).Initssimplest form,thisstatistic
mc
characterizestheproportionofindividualsfromaspecifiedgroup(e.g.,Africans)ineachofthetwobasalcladesofa
binarygenetree.Theminimumcladeproportioncanthusbedefinedas
(cid:2) (cid:3)
k k
p ¼min 1; 2 ; ðA1Þ
mc
n n
1 2