Am. J. Hum. Genet. 50:1301-1307, 1992

The Y-associated XY275 Low Allele Is Not Restricted toIndigenous African Peoples

Amanda Spurdle, Michele Ramsay, and Trefor Jenkins

Medical Research Council Ecogenetics Research Unit, Department of Human Genetics, School of Pathology, South African Institute for MedicalResearch and University of the Witwatersrand, Johannesburg

Summary

The level of linkage disequilibrium between the XY275 MspI polymorphism and the X and Y boundaries wasinvestigated in 21 different southern African populations. A full range of frequencies of the high allele wasobserved on the 1,013 X chromosomes studied, in keeping with published data. In previous studies fixationof the high allele on the Y chromosome was observed in all but two groups -a Pygmy and a Tsumkwe Sanpopulation. However, in the present study of 673 Y chromosomes, the low allele was found to be associatedwith the Y chromosome in several different Bantu-speaking negroid groups, the Khoisan-speaking negroidDama, the Khoisan, two groups of mixed ancestry, and the South African Asiatic-Indian population. Thediscovery of the low allele on Y chromosomes of caucasoid individuals suggests that more than one classof Y chromosome gave rise to the present-day non-African population. The data also fail to provide support

for the theory that Africa is the site of origin of Homo sapiens, but they equally do not exclude it.

Introduction

The pseudoautosomal region of the human sex chro-mosomes is defined as the region of strict homologyon the distal tip of the short arms that pair at meiosisand undergo homologous recombination (Weissen-bach et al. 1987). Recombination in the pseudoau-tosomal boundary region is limited at its proximal endby the pseudoautosomal boundary, i.e., the interfacebetween pseudoautosomal and sex chromosome-spe-cific regions. The pseudoautosomal boundary of thehuman Y chromosome is defined by the insertion of a303-bp Alu repeat sequence (Ellis et al. 1989), anevent thought to have created the boundary. How-ever, recent studies on the pseudoautosomal bound-aries of great apes and Old World monkeys indicatethat the Alu element did not create the present-dayboundary but was only inserted at the preexistingboundary after the great ape and Old World monkeylineages diverged (Ellis et al. 1990c).

ReceivedJuly 19, 1991; final revision receivedJanuary 22, 1992.Address for correspondence and reprints: A. B. Spurdle, Depart-

ment of Human Genetics, South African Institute for Medical Re-search, P.O. Box 1038, Johannesburg 2000, South Africa.© 1992 by The American Society of Human Genetics. All rights reserved.0002-9297/92/5006-0019$02.00

Polymorphisms of the pseudoautosomal boundaryregion have been studied in an attempt to formallydefine the boundary in terms of recombination (Elliset al. 1990b). The X chromosomes were found to bepolymorphic at five positions in a 300-bp region, whileall Y chromosomes were identical except for one distalpolymorphism shared with the X chromosome (Elliset al. 1 990b). This MspI polymorphism was originallytermed "XY274" (Ellis et al. 1990a) and was erron-eously believed to result from a C-to-T transition 274bp distal to the Alu insertion site. The polymorphismhas since been shown to be a G-to-T transversion lo-cated 275 bp from the boundary and has been renamed"XY275" (Ellis 1991). This correction does not sig-nificantly affect the interpretation of the primary data(Ellis 1991), although it does exclude prediction of theancestral form of the polymorphism. The two-allelepolymorphism may be detected either by hybridiza-tion of pseudoautosomal probe HfO.2 to Mspl-digested DNA or by PCR amplification and MspI di-gestion analysis of the boundary region (Ellis et al.1990a). The alleles are termed "high," for absence ofthe MspI site, or "low," for presence of the site (Elliset al. 1990a).The polymorphism was tested on a number of

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different populations, and a full range of high allelefrequencies was observed on the X chromosome (Elliset al. 1990a). However, XY275 was shown to be fixedas the high allele on the Y chromosome in all but twopopulations- a Pygmy population and a Kalahari Sanpopulation (Ellis et al. 1990a). The discovery of (low,PABY) chromosomes in two African populationsonly, in conjunction with the strong linkage disequi-librium observed between XY275 and the Y bound-ary, led Ellis et al. (1990a) to suggest that an ancestralHomo sapiens population possessing both high andlow alleles was African in origin and that a single classof Y chromosomes with the high allele migrated outof Africa at the time of the African/non-African splitabout 90,000 years ago.

The present study was undertaken (a) to determinethe level of linkage disequilibrium between XY275and the pseudoautosomal boundary in a number ofdifferent southern African populations, and (b) to re-

late the results to theories put forward by Ellis et al.

Material and Methods

Subjects

The subjects were unrelated male individuals be-longing to various southern African populations as

indicated in table 1. Their appropriate geographicalareas of origin are shown on the map in figure 1.The Bantu-speaking negroid group includes differ-

ent chiefdoms, each speaking a different Bantu lan-guage. The Zulu, Xhosa, Ndebele, and Swazi chief-doms are classified linguistically as the Nguni, whereasthe southern Sotho, Pedi (northern Sotho), and Tswanaare grouped as the Sotho/Tswana. Likewise the Herero-speaking group includes the Herero and Himba chief-doms. The Tsonga population is represented bypooled samples including individuals from the Tsongaand Shangaan chiefdoms. The migration of the Bantu-speaking negroids from their postulated area of originin west-central Africa is believed to have followed at

least two routes (Huffman 1982)-a general south-

Table I

XY275 High Allele Frequency in Southern African Populations

Frequency of Frequency ofNo. of X-associated No. of Y-associated

Population;' Chromosomes High Allele (SE) Chromosomes High Allele (SE)

Nguni ............. 182 .10 (.02) 76 .99 (.01)Zulu ............. 70 .04 (.02) 39 .97 (.03)Xhosa ............. 32 .16 (.06) 10 1.00 (.00)Ndebele ............. 9 .11 (.10) 9 1.00 (.00)Swazi ............. 71 .13 (.04) 18 1.00 (.00)

Sotho/Tswana ....... 103 .13 (.03) 86 .92 (.03)Southern Sotho ... 45 .18 (.06) 27 .85 (.07)Pedi ............. 40 .10 (.05) 34 .97 (.03)Tswana ............. 18 .06 (.06) 25 .92 (.05)

Venda ............. 21 .62 (.11) 21 1.00 (.00)Tsonga ............. 60 .15 (.05) 23 1.00 (.00)Herero ............. 41 .17 (.06) 37 1.00 (.00)Himba ............. 33 .03 (.03) 35 1.00 (.00)Ambo ............. 65 .14 (.04) 35 .95 (.04)Lemba ............. 36 .11 (.05) 40 1.00 (.00)Dama ............. 58 .22 (.05) 26 .92 (.05)Nama ............. 73 .10 (.04) 22 .77 (.09)Omega San ........... 52 .25 (.06) 47 .81 (.06)SA European ......... 53 .58 (.07) 51 .98 (.02)SA Indian ............. 67 .22 (.05) 61 .79 (.05)SAJewish ............. 69 .72 (.05) 33 1.00 (.00)"Colored" ............. 56 .13 (.04) 56 .98 (.02)Richtersveld .......... 44 .20 (.06) 22 .68 (.10)

NOTE. -The XY275 polymorphism is represented by only two alleles; thus the frequency of the lowallele is reciprocal to that of the high allele.

a Data for different chiefdoms were pooled according to the major linguistic classification (in boldfacetype) after a x2 test indicated no significant difference between the frequencies in the chiefdoms.

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Y-associated XY275 Low Allele

Figure I Geographical location of the southern African populations included in the present study

bound course (the eastern Bantu) and a southwesterlyroute across central Africa toward the western partsof the continent (the western Bantu). The Herero andthe Ambo are the only representatives of the westernBantu group.The Lemba are Venda-speakers considered by eth-

nographers to be of alien origin (Van Warmelo 1974).Many factors distinguish them from the other Bantu-speakers (Van Warmelo 1974): physically, manyLemba have a distinctive appearance including angu-lar features with a prominent hooked nose; the menused to wear a long cotton upper garment (khanzu),as found along the east coast of Africa; among them-selves they spoke a language not understood by theirhosts in southern Africa; marriage was strictly endoga-mous; certain foods are forbidden, e.g., pork, certainother animals, and the flesh of cattle not kosher-killed

according to their law; circumcision is practiced; andunintelligible prayers are recited (and responded to) atcertain ritual ceremonies. Although the Lemba them-selves claim to belong to one of the lost tribes of Israel,certain facts about them suggest that they are descen-dants of Semitic traders, presumably Arabs, from theeast coast. One such fact was the observation that theirritual prayers may represent mangled suras from theKoran (Van Warmelo 1974).The Dama are a Khoisan-speaking negroid group.

Although these people have typical negroid features,culturally and linguistically they do not reveal any sim-ilarities to the Bantu group (Malan 1980). They speakthe same Khoi language as the Nama, in agreementwith their historical enslavement by Khoi pastoralists(Jenkins 1982).The Khoisan group is composed of the Khoi (for-

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merly referred to as "hottentots") and the San (for-merly "bushmen"). These medium-statured peoplehave a yellow skin color, flat triangular face, and ste-atopygia (Malan 1980); and they speak languagescomposed of clicks and other guttural sounds (Nurseand Jenkins 1977). The !Kung San group collected atthe Omega military camp in northern Namibia origi-nate from southern Angola.The South African (SA) caucasoid group includes

peoples of western European and Asiatic-Indian ori-gin, as well as Ashkenazim Jews from eastern Europe.The Johannesburg "colored" group is a population ofmixed ancestry, resulting from admixture of Euro-pean caucasoid, Khoisan, Malay, and Bantu-speakingnegroid people. The Richtersveld "colored" group hasresulted from admixture between European caucasoidtrekboers (farmers), who moved into the northernCape area in the 18th century, and indigenous Namawomen. They have remained relatively isolated overthe ensuing years.

Hybridization Studies

Probe HfO.2 was extracted according to standardprocedures (Maniatis et al. 1982). Human genomicDNA was extracted from packed cells or buffy coats,using the method of Sykes (1983). MspI restriction-enzyme digests of human genomic DNA (5-10 ig)were separated by electrophoresis on 1.2% agarosegels in 1 x TBE. DNA was transferred by the methodof Southern (1975) to Hybond-N nylon membranes,and filters were baked at 801C for 1-2 h. Baked filterswere prehybridized and hybridized according to man-ufacturer's specifications. The 32P-dCTP oligolabeledprobe (Feinberg and Vogelstein 1983) was hybridizedto prehybridized blots for 20-48 h. Filters werewashed twice in 2 x SSPE, 0.1 % SDS at room temper-ature (for 15 min each), once in 1 x SSPE, 0.1% xSDS at 420C (for 30 min), and once in 0.1 x SSPE,0.1% SDS at 65°C (for 20 min). Fragments were visu-alized by autoradiography after 1-6 d exposure withKodak XAR film backed with DuPont Cronex intensi-fying screens.

PCR Studies

Sequence data supplied by Nathan Ellis were usedto design three primers for the amplification ofXY275and of the X and Y boundary regions. The primersequences from 5' to 3' were as follows: XY or pseudo-autosomal l9mer, CTG AGA GTG GAA GTG TCGC; Y-specific 22mer, AGA AAA CTA GTA TTT TCC

CCT C; and X-specific 20mer, AAC AAG CTC ATCAGC GTG AC.PCR reactions were carried out in a Perkin Elmer

Cetus DNA thermal cycler. The final reaction volumewas 25 gl, and each reaction used 6.25 pm of theappropriate primers (XY and X, or XY and Y), 6.25nm of each dNTP, 5-10 gg of acetylated BSA, 2 unitsof Promega Taq polymerase, 2.5 gl of 10 x PromegaTaq polymerase buffer, and approximately 0.5,g ge-nomic DNA. The reaction was overlaid with 1 dropof mineral oil (Sigma) and incubated for 30 cycles(94°C for 48 s, 57°C for 48 s, and 72°C for 90 s),with a 10-min extension at 72°C. Amplification ofsamples was verified by electrophoresis of a 5-pl ali-quot on 0.8% agarose. The remaining 20 il of ampli-fied product was digested with 6 units of Promega orAmersham MspI at 370C for 2 h and was analyzed byelectrophoresis through 2% composite FMC agarosegels (3:1 Nusieve GTG; Seakem HGT). PCR amplifi-cation and digestion products are shown in figure 2.

Results and Discussion

The frequencies of the XY275 high allele in thedifferent populations studied are shown in table 1.Data were generated or confirmed by PCR analysis,with the exception of those for high/high homozy-gotes detected by hybridization studies. A wide rangeof P, values occurs in these southern African popula-tions, a phenomenon observed by Ellis et al. (1990a)in their study of Caucasians, Oceanic populations,Amerindians, and Africans. The P, value of .58( ± .07) in the SA European population correlates wellwith the .59 observed by Ellis et al. (1990a) in north-ern Europeans. The latter sample includes a few Ash-kenazi Jewish individuals (N. A. Ellis, personal com-munication). The SA Ashkenazi Jewish populationhas a much higher P. value, .72 ( ±.05), possibly be-cause of genetic drift; when the approximately 40,000Jewish immigrants came to South Africa in 1880-1910, they nearly all came from Lithuania, mostly infamily groups and from only a few towns or villages(shtetls). The SA Asiatic-Indian population is distinctfrom the other two caucasoid groups and has a P. ofonly .22 ( + .05). Likewise, the negroid, Khoisan, andhybrid groups have much lower P. values, .03-0.25,with the exception of the Venda, who have a P, of .62(± .11). The Venda are relatively recent immigrantsto southern Africa, and their unusually high P, valuemay reflect frequencies found in central African

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Y-associated XY275 Low Allele

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Bantu-speaking populations, none of which has beenscreened to date. The Venda sample also representsindividuals from a geographically localized area, andsampling error cannot be excluded as a possible expla-

Figure 2 Top, Schematic representation of the XY275 PCRamplification and MspI digestion products expected from usingprimers described in Material and Methods. An asterisk marks theXY275 polymorphism. Numbers above each schematic chromo-some represent fragment sizes (in bp). Left, Separation ofundigestedand Mspl-digestedXandYboundary amplificationproductson2%composite gels (see Material and Methods). Appropriate fragmentsizes are indicated (in bp) to the left. Lane 1, Undigested Y amplifi-cation product of 1,122 bp. Lane 2, Undigested X amplificationproduct of 9S0 bp. Lanes 3 and 4, Mspl-digested X amplificationproducts displaying low allele (181 bp and 156 bp). Lanes 5 and6, MspI-digested Y amplification products displaying low (181 bpand 156 bp) and high (337 bp) alleles, respectively. A spuriousamplification product of approximately 250 bp is observed in Xamplification products but does not interfere with interpretation ofdigestion results.

nation for the high P,, value observed in this rathersmall sample. It is interesting that the Px values ofcaucasoids in general are much higher than those inAfrican populations (table 1), a phenomenon also ob-

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Spurdle et al.

served in the populations screened by Ellis et al.(1990a). These findings may be seen as lending sup-

port to the theory of an African/non-African split, as

suggested by Wainscoat et al. (1986), on the basis ofnuclear polymorphisms, and by Cann et al. (1987),on the basis of mtDNA polymorphisms.The P, values differ greatly from the P. values (table

1). As described by Ellis et al. (1990a), strong disequi-librium between XY275 and the Y boundary is ob-served, with the high allele occurring almost exclu-sively in association with the Y chromosome. The lowallele is found in association with the Y chromosomein the Omega San population, at the relatively highfrequency of .19 ( ± .06). This is in agreement with theobservation, by Ellis et al. (1 990a), ofthe (low, PABY)haplotype in 6 / 14 San individuals from a distinct pop-ulation from Tsumkwe. (The Omega and Tsumkw(populations are both !Kung or northern Bush-lan-guage speakers.) The Khoi Nama also possess (low,PABY) chromosomes, at a frequency of .23 (+ .09).The frequency of .32 (+.10) in the hybrid Rich-tersveld population, which is thought to have Namaorigins (Nurse et al. 1985), indicates that Nama gene

flow into this population was not strictly maternal,although it may initially have been so. The (low,PABY) haplotype occurs sporadically in the differentBantu-speaking negroid groups (at frequencies of .00-.15), as well as in the Khoisan-speaking Dama (.08 +.05).

Ellis et al. (1990a) reported fixation of the highallele on the Y chromosome in caucasoids. However,(low, PABY) haplotypes have been observed in the SAEuropean and Asiatic-Indian populations sampled inthe present study (table 1). Although the occurrence ofa single (low, PABY) chromosome in the SA Europeanpopulation may be due to historical gene flow fromother southern African groups, the substantial fre-quency (.21 + .05) of the (low, PABY) haplotype inSA Asiatic Indians is believed to be significant. It ispossible that this rather high frequency is due to a

sampling effect and may not be representative of theAsiastic-Indian population as a whole, since a rela-tively small group of Indians from the provinces ofGujerat, Madras, and Bengal gave rise to the SAAsiatic-Indian population (Bernstein et al. 1986). The(low, PABY) haplotype occurs at similar frequenciesin the Hindu and Muslim religious groups (5/26 and3/14, respectively), and the differences are not sig-nificant (P = .65). This is not totally unexpected,since the spread of the Muslim religion to the Indian

subcontinent, with its ancestral Hindu population,only occurred about 1,000 years ago.The possibility that (low, PABY) chromosomes

were generated by crossover between the X and Y inthe Asiatic population cannot be excluded; the calcu-lated rate of recombination between XY275 and theboundary is 6 x 10-5/bivalent (N. A. Ellis, personalcommunication). Analysis of the XY275 polymor-phism in populations of the Middle East would proba-bly shed light on this question. However, one mighthave expected the notable presence of (low, PABY)chromosomes in other non-African populations,which has not been the case (Ellis et al. 1990a; presentstudy). It is especially surprising to note that the Ron-donia Surui and Karitiana populations studied by Elliset al. (1990a) possess only (low, PABX) and (high,PABY) chromosomes (as calculated by maximum-likelihood methods), and thus no crossover between Xand Y appears to have taken place in these Amerindiangroups.The discovery of Y-associated low alleles in the

Caucasoid SA Asiatic-Indian population suggests thatthe conclusions drawn by Ellis et al. (1 990a) may havebeen premature. Although mitochondrial data (Cannet al. 1987) and autosomal nuclear data (Wainscoatet al. 1986; Cavalli-Sforza et al. 1988) both indicatean African origin of modern man, the XY275 datacannot be said to support the hypothesis. Neither dothe data refute it. One must also acknowledge, how-ever, that the population relationships inferred fromY-specific markers-or from markers in linkage dis-equilibrium with the Y chromosome (as is the casewith XY275)-may not correspond to those obtainedfrom studies with autosomal or mtDNA markers. Ifmodern humans arose in Africa, then it is clear thatmore than one class of Y chromosome was alreadypresent, and the frequency differences in present-daypopulations are due to the operation of genetic driftafter the populations migrated out of Africa.

AcknowledgmentsOur sincere gratitude is extended to Dr. Nathan Ellis for

the gift of probe HfO.2, for primer sequence data used todetermine primer structure, and for his critical review of adraft of the manuscript; to the Nambia Blood TransfusionService, Windhoek State Laboratories, and Highveld BloodTransfusion Service for their assistance in the collection ofvaluable blood samples; and, last, to Himla Soodyall forMspI population blots used in the initial population screening.

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