Population Genetics of Y-Chromosome Short Tandem Repeats in Humans

Anna Perez-Lezaun,1 Francesc Calafell,1,* Mark Seielstad,2,3 Eva Mateu,1 David Comas,1 Elena Bosch,1

Jaume Bertranpetit1

1 Laboratori d’Antropologia, Facultat de Biologia, Universitat de Barcelona, Barcelona, Diagonal 645, 08028 Barcelona, Spain2 Department of Organismic and Evolutionary Biology, Harvard University, 26 Oxford St., Cambridge, MA 02138, USA3 Department of Genetics, Stanford University, 300 Pasteur Drive, Stanford, CA 94305, USA

Received: 3 January 1997 / Accepted: 25 April 1997

Abstract. Eight human short tandem repeat polymor-phisms (STRs) also known as microsatellites—DYS19,DYS388, DYS390, DYS391, DYS392, DYS393,DYS389I, and DYS389II, mapping in the Y chromo-some—were analyzed in two Iberian samples (Basquesand Catalans). Allele frequency distributions showed sig-nificant differences only for DYS392.Fst and gene di-versity index (D) were estimated for the Y STRs. Thevalues obtained are comparable to those of autosomalSTR if corrections for the smaller effective populationsize on the Y chromosome are taken into account. Thissuggests that Y-chromosome microsatellites might be asuseful as their autosomal counterparts to both humanpopulation genetics and forensics. Our results also rein-force the hypothesis that selective sweeps in the Y chro-mosome in recent times are unlikely. Haplotypes com-bining five of the loci were constructed for 71individuals, showing 29 different haplotypes. A haplo-type tree was constructed, from which an estimate of7,000 to 60,000 years for the age of the Y-chromosomevariation in Iberia was derived, in accordance with pre-vious estimates obtained with mtDNA sequences andnuclear markers.

Key words: Y chromosome — STR — Microsatellite— Basques — Catalans — Haplotype

Introduction

The value of polymorphisms in the Y chromosome forhuman evolutionary studies has been largely recognized(Jobling and Tyler-Smith 1995) and used in studies ofhuman evolution at the species level (Dorit et al. 1995;Hammer 1995; Whitfield et al. 1995) as well as in re-gional and populational studies (Hammer and Horai1995; Underhill et al. 1996; Roewer et al. 1996, amongothers). Y-linked loci are of special interest because theyare haploid and paternally inherited and, beyond thenearly telomeric pseudoautosomal region, the lack of re-combination implies that the Y chromosome is inheriteden bloc as a haplotype. However, since the first Y poly-morphisms were described more than 10 years ago(Casanova et al. 1985; Lucotte and Ngo 1985), the use-fulness of the Y chromosome for human population andevolutionary studies has been limited by the scarcity ofcommon polymorphisms found (Jakubiczka et al. 1989;Malaspina et al. 1990; Spurdle and Jenkins 1992). Sev-eral studies (Dorit et al. 1995; Hammer 1995) pointed toa low level of sequence variation in the Y chromosome.However, short tandem repeat (STR) population varia-tion in the Y chromosome has recently been described,and it appears to be highly relevant for molecular studiesof human evolution (Ciminelli et al. 1995; Roewer et al.1996; Cooper et al. 1996; Ruiz-Linares et al. 1996).

In the present study, eight Y-specific short tandemrepeats were typed in two populations from the IberianPeninsula (Catalans and Basques). The Basques are anoutlier in the genetic variation of Europe, as shown byclassical autosomal markers (blood groups, protein elec-

* Present address:Department of Genetics, Yale University School ofMedicine, 333 Cedar St., New Haven, CT 06520, USACorrespondence to:J. Bertranpetit; e-mail jaumeb@porthos.bio.ub.es

J Mol Evol (1997) 45:265–270

© Springer-Verlag New York Inc. 1997

tromorphs, and HLA; Calafell and Bertranpetit 1994a,b;Cavalli-Sforza et al. 1994), and they may retain a geneticdiversity that originated prior to the spread of the Neo-lithic in Europe (Calafell and Bertranpetit 1993). To thecontrary, the Catalans fit into the European geneticcon-tinuum (Calafell and Bertranpetit 1994b). Both sampleshave already been analyzed for 20 nonsyntenic autoso-mal STRs (Pe´rez-Lezaun et al. 1997), and those resultswill be used as a reference point against which Y-chromosome variation will be compared. To the best ofour knowledge, this is the first study in which both au-tosomal and Y-chromosome STRs are typed and con-trasted.

Short tandem repeats in a haplotype framework canpotentially answer some unresolved questions regardingthe populations bearing the variation and the productionand maintenance of the genetic variation (Tishkoff et al.1996). The aim of this article is to evaluate the degree ofgenetic variation within and between populations foundin Y-chromosome STRs as compared to autosomal STRsand to analyze the phylogeny and age of Y-chromosomehaplotype variation in these populations.

Materials and Methods

DNA was extracted from fresh blood of unrelated, autochthonous in-dividuals whose four grandparents were born in the same region(Gipuzkoa in the Basque Country and Girona in Catalonia). Five fe-males were also included as controls. Eight Y-linked STR loci weretyped: DYS19, DYS388, DYS389I, DYS389II, DYS390, DYS391,DYS392, and DYS393. All of them are tetranucleotide repeat poly-morphisms, except DYS388 and DYS392, which are trinucleotide re-peats.

A touchdownPCR thermal regime (Don et al. 1991) with a 10-mlfinal reaction volume was run in a Perkin Elmer 9600 thermal cycler.The same conditions described in Seielstad et al. (1994) were used forthe amplifications; PCR primers were fluorescently labeled. PCR prod-ucts for DYS19, DYS388, DYS390, DYS391, and DYS392 were run ina standard 6% denaturing sequencing gel using an ABI 373A auto-mated sequencer. PCR products for DYS389I, DYS389II, and DYS393were run in an ABI 377 sequencer. ABI GS500 was used as internalsize standard. GeneScan 672 was used to collect the data, track lanes,and measure fragment sizes. A minimum of 51 Basque and 25 Catalansamples were analyzed for each STR.

Gene diversity (D) was computed for each locus asD 4 1 − ∑pi2,

were pi are the allelic frequencies; this formulation is equivalent toheterozygosity in an autosomal marker (Weir 1990). In the equilibrium,and for an autosomal locus,D 4 4Nm/(4Nm + 1) (Hartl and Clark1989, p 124), whereN is number of individuals andm is the mutationrate. Assuming that an equal number of males and females contributegenes to the following generation, there would be an effective 1:4 ratioof Y to autosomal chromosomes. Using that ratio, it can easily beshown that, for the same population size and mutation rate,Dau 4

4Dy/(3Dy + 1), whereDau is gene diversity in autosomal markers andDy is gene diversity in Y-chromosome markers. Thus, intrapopulationvariability can be compared between autosomal and Y-chromosomeSTRs.

Interpopulation variability was estimated throughx2 contingencytests andFst (Wright 1951). In a stepwise mutation model, and for twocompletely separated populations,Fst 4 t/(t + 4N) (slightly modifiedfrom Slatkin 1995, eq. 17), wheret is the separation time between twopopulations. Then,Fst values in autosomes and the Y chromosomeshould be related as

Fstau

~1 − Fstau!=

Fsty4~1 − Fsty)

whereFstau is Fst for an autosomic locus andFsty is for an STR in theY chromosome.

Five different STRs (DYS19, DYS388, DYS390, DYS391, andDYS392) were used for the construction of haplotypes, because com-plete haplotypes with all eight markers were available for only a fewindividuals. A phylogenetic tree of STR haplotypes was constructedusing the methods described in Morral et al. (1994) and Bertranpetitand Calafell (1996). A stepwise mutation model was assumed, andgains or losses of more than one repeat were allowed, although with amuch smaller probability than gains or losses of one repeat (Di Rienzoet al. 1994). After a putative ancestral haplotype was identified, themean number of mutations accumulated per haplotype and locus wascounted and used to estimate the age of the variability. If we assumethat mutations from a known ancestor accumulate according to a Pois-son distribution (Hudson 1990), thenl 4 mt, wherel is the meannumber of mutations from the putative ancestor per individual andlocus, m is mutation rate, andt is time in generations. Cooper et al.(1996) obtained several estimates for the mean mutation rate for fivetetranucleotide Y-linked STRs (three of which overlap with the set usedin our haplotype analysis) that ranged from 1.2 × 10−4 to 1.03 × 10−3.These two extreme mutation rates were used to bracket the age of thehaplotype tree.

Results and Discussion

Intra- and Interpopulation Variability

All loci tested were found to be polymorphic and Yspecific (no amplification was obtained when usingDNA from females) and presented from three to six al-leles. Primers for DYS389 amplified two different Y-specific loci: DYS389I and DYS389II. Both amplifiedproducts differed more than 100 bp and thus they couldbe scored separately. The two PCR products obtained forboth loci have been shown not to be completely inde-pendent as DYS389I variation is also included inDYS389II. Primers and PCR conditions have to be re-designed to extract all the variability information in-cluded in both loci (de Knijff unpublished data). Theconsideration of both loci as independent may slightlyoverestimate the variability of DYS389II. As the numberof STRs described in the Y chromosome is still low, andtaking into account that possible errors in the calcula-tions of intra- and interpopulation variability may behigher if DYS389II is discarded, we have included it insome parts of the study.

Allele frequency distributions are shown in Table 1.There were no significant differences between Basquesand Catalans, except for DYS392 (x2 4 10.95,df, P <0.001). In this locus, allele 248 was the most frequent inCatalans, whereas allele 254 was the most common inBasques. The number of alleles and genetic diversities(D) are shown in Table 2. The meanD value for Y-linkedSTRs is 0.435. The same value for 20 unlinked autoso-mal STRs in the same population samples (Pe´rez-Lezaunet al. 1997a,b) was 0.730. Correcting for differences in

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effective size between autosomes and the Y chromosome(see Methods), a value ofD 4 0.755 was obtained forthe Y-chromosome markers, which is in close agreementwith that found in 20 autosomes in the same samples (P4 0.636, Mann-Whitney’s U test). Although the equa-tion on which this correction is based was derived underan infinite-allele mutation model, it has been shown to bea good approximation for a stepwise mutation model

(Hartl and Clark 1989, p 136), which seems to fit theevolutionary pattern of both autosomal (Di Rienzo et al.1994) and Y-linked (Cooper et al. 1996) STRs. Thus, thelevel of polymorphism in Y-chromosome STRs is re-markably similar to that in autosomal STRs. A recentselective sweep in the Y chromosome (Dorit et al. 1995)would have greatly reduced genetic polymorphism, andan inordinately fast mutation rate would have beenneeded to restore polymorphism to levels comparable tothose found in autosomes. Moreover, although severalmutation ranges are given by Cooper et al. (1996), theypointed out that the most likely range of estimated mu-tation rates for Y-linked tetranucleotide STRs is from 1.2× 10−4 to 4.8 × 104, lower than previous estimates for 12GATA repeats in chromosome 19 (2.1 × 10−3, Weberand Wong 1993). The most likely interpretation of thisresult is that STRs in the Y chromosome have evolvedwith mutation rates similar to, and one-quarter the effec-tive size of, autosomal STRs, and that no recent selectivesweep can be detected.

Figure 1 shows the comparison betweenFst valuesobtained from 20 autosomal STRs (Pe´rez-Lezaun et al.1997) and eight Y-linked STRs when Catalan andBasque samples are compared. MeanFst is 0.0387 in YSTRs and 0.0098 in autosomal STRs, with significantdifferences (P 4 0.005, Mann-Whitney’s U test). Cor-recting for the smaller effective population of Y chro-mosomes, theFst value obtained for the Y chromosomebecomes 0.0100, in close agreement with that observedin the same sample for 20 autosomal STRs (P 4 0.980,Mann-Whitney’s U). Again, the evolution of Y-chromosome STRs follows the pattern observed in auto-somes and confirms the extent of the Basque differen-tiation. For the whole European continent, autosomalFstis 0.0160 (Cavalli-Sforza et al. 1994); thus, the geneticdifference between Basques and Catalans, in the Y chro-mosome or in the autosomes, amounts to two-thirds ofthe overall autosomal European differentiation. We pre-viously suggested (Pe´rez-Lezaun et al. 1997) that thedifferentiation of STR polymorphism in human popula-tions is governed by drift rather than by mutation; theeffective population size of chromosome Y is around

Table 1. Allele frequencies for eight Y STRs—DYS19, DYS388,DYS390, DYS391, DYS392, DYS393, DYS389I, and DYS389II—inCatalan and Basque populations

Allele Catalans Basques

DYS19 186 0.040 0.077190 0.800 0.789194 0.160 0.096202 0 0.038

n 4 25 n 4 52

DYS388 126 0.034 0129 0.863 0.887132 0.034 0.075135 0.034 0138 0.034 0141 0 0144 0 0.038

n 4 29 n 4 53

DYS390 207 0.069 0211 0.172 0.170215 0.690 0.773219 0.069 0.057

n 4 29 n 4 53

DYS391 279 0.033 0.039283 0.400 0.392287 0.567 0.530291 0 0.039

n 4 30 n 4 51

DYS392 248 0.563 0.250251 0.031 0254 0.375 0.750257 0.031 0

n 4 32 n 4 52

DYS389I 245 0 0.018249 0.097 0.125253 0.742 0.534257 0.161 0.305261 0 0.018

n 4 31 n 4 56

DYS389II 361 0 0.018365 0.065 0.196369 0.580 0.393373 0.226 0.339377 0.129 0.018381 0 0.036

n 4 31 n 4 56

DYS393 120 0.063 0.071124 0.812 0.834128 0.125 0.095

n 4 32 n 4 42

Table 2. Number of alleles, allele sizes, and averageD values for theeight Y-linked STR analyzed

STR nAllelesfound

Size(bp) D

DYS19 77 4 186–202 0.354DYS388 82 6 126–138 0.224DYS390 82 4 207–219 0.413DYS391 81 4 279–291 0.547DYS392 84 4 248–257 0.495DYS389I 87 5 245–261 0.501DYS389II 87 6 361–381 0.641DYS393 74 3 120–128 0.306

Average — 4.5 — 0.435(±0.048)

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one-quarter of that in autosomes, and, therefore, drift hasa deeper impact on Y-chromosome variation. Our re-sults, as well as those obtained by Roewer et al. (1996)in two closely related populations (Germans and Dutch),confirm that STR polymorphisms are especially suitablefor detecting differences between populations that havediverged recently. The role of drift would, thus, haveproduced a wider divergence among populations for theY chromosome than when autosomic markers are con-sidered.

Haplotype Phylogeny and the Age ofY-Chromosome Variability

When the overall sample (Basques and Catalans) and asubset of five Y-linked STRs were considered, a sampleof 71 individuals presented 29 different haplotypes(Table 3). This level of polymorphism is higher than thatobtained with sequence variation (Dorit et al. 1995;Hammer 1995) or length variation in one single locus(Hammer and Horai 1995), but it is similar to that foundin East Anglians with five STR loci (Cooper et al. 1996).

For loci DYS19/388/390/391/392, haplotype 1 (190/129/215/287/254) was found in 15 out of 49 (31%)Basque individuals and in three out of 22 Catalans(14%); the most common haplotype in Catalans is num-ber 20 (190/129/215/287/248) found in six individuals(27%). Figure 2 shows a haplotype tree obtained bymaximum parsimony using haplotypes from both popu-lations. Branch lengths are proportional to the number ofmutations: in six cases (branches connecting 9 and 11; 11and 12; 7 and 26; 3 and 23; 3 and 14; and 24 and 29),mutations at more than one locus were needed to explainthe relation between haplotypes; this is not surprisinggiven the number of individuals analyzed and their smallgeographic distribution. Haplotypes found in either

Basques and Catalans or in both are randomly distributedin the tree, which can thus be analyzed as a whole.

Apparently, two haplotypes may be ancestral. Haplo-type 1 (Table 3) is the most common, and thus morelikely to be the ancestral (Watterson and Guess 1977); itshigher frequency among Basques, a known relict popu-lation in Europe (Calafell and Bertranpetit 1994a), addssupport to this hypothesis. On the other hand, haplotype2 seems to have generated more diversity, and longerbranches spring from it, which would imply that moretime was needed to generate such levels of diversity, andthus it would be the oldest. The mean number of muta-tions accumulated from the ancestor was slightly smallerif the ancestor was assumed to be haplotype 1; however,given the small differences, the results obtained would bevery similar if haplotype 2 had been considered the an-cestor.

The mean number of mutations accumulated (l) perlocus from haplotype 1 was 0.366. With mutation ratesbetween 1.03 × 10−3 and 1.2 × 10−4 (Cooper et al. 1996),the variation observed in the two Iberian populationscould have been generated in 355 to 3,051 generations,or, if a generation time of 20 years is assumed, of 7,100to 61,020 years. If haplotype 2 was considered the an-cestor, a very similar result was obtained:l would be0.388 and the time needed to generate all the diversity inthe tree would be 7,540 to 64,660 years. In any case,Cooper et al. (1996) pointed out a more likely and nar-rower range of mutation rates between 1.2 × 10−4 and 4.8× 10−4 which may bracket the diversity found between15,000 and 61,000 years. It is obvious that an empiricalestimation of the mutation rates for Y chromosomalSTRs is urgently needed.

By combining Basques and Catalans, we obtained asample that encompasses most of the genetic variabilityin the Iberian Peninsula. Basque and Catalan haplotypes

Fig. 1. Comparison ofFst values(×104) obtained for 20 autosomaland eight Y-linked STR markers.

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appear to be scattered randomly throughout the tree,which may imply, barring convergent evolution causedby selection, that the variability observed was generatedlong before the split between the ancestors of Basques

and Catalans and probably predates the differentiation ofEuropean populations. The age found is in agreementwith the previous hypothesis, as well as with the datesobtained with mtDNA D-loop sequences (Bertranpetit etal. 1995, Comas et al. 1997) that place the origin of theBasque differentiation before the Neolithic and mostlikely in the Upper Paleolithic. The concordance betweenY-chromosome, nuclear, and mtDNA ages could rule outa significant effect for sex-specific migration patterns inthe origin of the principal factors of the genetic differ-entiation of the Iberian populations.

The discovery of new polymorphisms in the Y chro-mosome adds new, exciting tools to the study of humanpopulation genetics, and their higher levels of resolutionmake them especially indicated for the study of closelyrelated populations.

Note Added at Proof

A recent publication (Heyer E, Puymirat J, Dieltjes P, Bakker E, deKnijff P. Estimating Y chromosome specific microsatellite mutationfrequencies using deep rooting pedigrees. Hum Mol Gen 6:799–803,1997) suggests that the mutation rate for 9 Y STRs is higher than thevalues usually accepted. If the rate estimated was an accurate measureof mutation rates for all STRs on the Y chromosome, the age of our tree

Fig. 2. Parsimony tree of haplotypes obtained after the analysis ofSTRs DYS19, DYS388, DYS390, DYS391, and DYS392 in two popu-lations of the Iberian Peninsula.

Table 3. Haplotypes found when joining Catalan and Basque genotypes for five different Y STR markers (1: DYS19, 2: DYS388, 3: DYS390,4: DYS391, 5: DYS392)

No.

HaplotypeMutations from

ancestral haplotypeFrequency(Cat/Bas)1 2 3 4 5 1 2 3 4 5 Total

1 190 129 215 287 254 0 0 0 0 0 0 3 152 190 129 215 283 254 0 0 0 1 0 1 2 93 190 129 215 283 248 0 0 0 1 1 2 2 04 190 129 211 283 254 0 0 1 1 0 2 0 25 190 129 211 283 248 0 0 1 1 1 3 0 16 190 135 211 283 248 0 1 1 1 1 4 1 07 194 129 211 283 248 1 0 1 1 1 4 0 18 194 129 207 283 248 1 0 2 1 1 5 1 09 194 132 207 283 248 1 1 2 1 1 6 1 0

10 194 132 219 283 248 1 1 3 1 1 7 0 111 202 132 211 283 248 2 1 3 1 1 8 0 112 202 132 215 279 248 2 1 4 2 1 10 0 113 190 129 219 283 254 0 0 1 1 0 2 0 114 190 126 219 283 248 0 1 1 1 1 4 1 015 186 129 215 283 254 1 0 0 1 0 2 0 116 186 129 219 283 254 1 0 1 1 0 3 0 117 194 129 215 283 254 1 0 0 1 0 2 1 018 190 144 215 283 248 0 1 0 1 1 3 0 119 190 129 215 287 251 0 0 0 0 1 1 1 020 190 129 215 287 248 0 0 0 0 2 2 6 421 190 129 211 287 254 0 0 1 0 0 1 1 422 190 129 211 287 257 0 0 1 0 1 2 1 023 186 129 215 279 248 1 0 0 2 1 4 0 124 190 129 215 291 254 0 0 0 1 0 1 0 125 190 144 215 287 254 0 1 0 0 0 1 0 126 194 138 211 279 248 1 2 1 2 1 7 1 027 194 129 215 287 254 1 0 0 0 0 1 0 128 194 129 211 287 254 1 0 1 0 0 2 0 129 186 132 215 291 248 1 1 0 1 1 4 0 1

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would be much younger. Nonetheless, the most conservative mutationrate obtained by Heyer et al. for the 4 STRs that overlap the ones wehave used to estimate the age of the variability, would be 1.2 × 10−3,which is very close to the higher value we have used.

Acknowledgments. This research was supported by the Direccio´nGeneral de Investigacio´n Cientıfico Tecnica (Spain) grants PB92-0722and PB95-0267-C02-01, by the Human Capital and Mobility contractsto J.B. (network ERCHRXCT92-0032 and ERB-CHRX-CT920090),by Direccio General de Recerca, Generalitat de Catalunya(1995SGR00205 and 1996SGR00041), and by Institut d’Estudis Cat-alans. The work was also possible thanks to fellowships to A.P.-L.(FP93-38110903, Spanish Ministry of Education and Science), and toD.C. (FI/93-1151), E.B. (FI/96-1153), and F.C. (postdoctoral fellow-ship) by the Comissionat per a Universitats i Recerca, Generalitat deCatalunya.

Short tandem repeats DYS19, DYS388, DYS390, DYS391, andDYS392 were genotyped by A.P.-L. in L.L. Cavalli-Sforza’s laboratoryat the Genetics Department of Stanford University (grant: NIHGM28428).

We especially thank G. Barbujani and L. Excoffier for their sug-gestions and comments on the manuscript and P. de Knijff for theinformation on DYS389I/II. We also thank Dr. Joan Profito´s and histeam from the Hospital de Girona (Programa de Donacio´ Sanguı´nia) forhelping us to collect the Catalan samples.

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