class II oligonucleotide typing (DQA and DQB) to analyze families of Yanomami indians settling in villages in Southern Venezuela. There exist complex networks ...
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Microsatellite and HLA class II oligonucleotide typing in a population of Yanomami Indians L. Roewer, M. Nagy, P. Schmide, J. T. Epplena and
G. Herzog-Schroderb Institut fiir Gerichtfiche Medizin, Humboldt-Universitiit, Hannoversche Str. 6, 10115 Berlin, Germany; aMolekulare Humangenetik, MA, Ruhr-Universitiit, Universitiitsstr. 150, 44780 Bochum, Germany; bForschungsstelle fiir Humanethologie, Max-Planck-Gesellschaft, Von-der Tann-Strasse 3-5, 82346 Andechs, Germany Summary We have used three different microsatellites (on chromosome 12 and Y) together with HLA class II oligonucleotide typing (DQA and DQB) to analyze families of Yanomami indians settling in villages in Southern Venezuela. There exist complex networks of biological relationship between villages as a result of wife exchange, village fissioning and changing patterns of alliances associated with inter-village warfare. Social status in this society is largely determined by the kinship system. Polygyny is common, especially among headmen, with additional wives, frequently being chosen among the sisters of the first wife. Our preliminary results mainly obtained from inhabitants of the village HAP show the expected allele distribution in populations with a high degree of consanguinity: (i) deficiency of observed heterozygotes at the autosomal loci and (ii) almost all men carry the same Y chromosomal allele. Nevertheless in the Yanomami village two thirds of the described autosomal microsatellite alleles were identified. Several paternities were clarified.
Introduction
One of the few peoples of the Amazon lowland who stayed in relative isolation until recent times are the Yanomami indians. Traditionally the Yanomami settled around small rivulets in the headwaters of the Orinoco, Rio Negro and Rio Branco. The Yanomami live in self-contained communities of 40 to 200 inhabitants and subsist by simple agriculture, hunting and gathering. The number of Yanomami living in Venezuela is estimated at 10,000 (Herzog-Schroder, in press). The kinship system of the Yanomami is a classificatory one according to the Iroqouis type (i.e. siblings and parallel cousins are terminologically equivalent but distinguished from cross-cousins) and the bilateral cross-cousin is the prescriptive marriage-partner. Husbands or wives are found within or without the local group. Due to an imbalanced sex-ratio and polygyny, women are in short supply, so that many men are forced to obtain women from other villages. Within a village 95% of the inhabitants are cognative relatives. 50% of a village population stands in an in-law relationship to an ego. The other 50% are "culturally relatives", i.e. the group among which a spouse can not be chosen.
222
I
VENEZUELA
,/ • 0&1.,
Otr Vanomaml
•
k.th. .. 1.. 1on
iJ
sb
BRASILI
is
N
l00~m
Figure I. Territory inhabited by the Yanomami in Southern Venezuela and Northern Brazil.
223
These proportions warrant that each individual can marry his or her ideal partner according to the cross-cousin marriage rule (Chagnon, 1980). The band-society of Yanomami indians has been studied with respect to their blood groups, serum proteins and erythrocyte enzyme systems (Gershowitz et ai. , 1972; Weitkamp et ai., 1972; Weitkamp and Neel, 1972; Tanis et ai., 1973; Ward et ai., 1975). A few studies have been carried out involving DNA analysis in Amerindian populations (Wallace et ai., 1985; Schurr et ai., 1990; Kidd et ai., 1991; Guerreiro et ai. , 1992).
B
4815LR
e
d
c
4804 Figure 2. DNA samples of a large German family typed at 3 microsatellite loci. For methodological details see the materials and methods section. The autosomal loci 4804LR and 4815LR were PeR-amplified simultaneously, the V-chromosomal 27H39LR locus was amplified separately. The lengths of the alleles c, d, e (4804LR) are 159 bp, 163 bp and 167 bp, respectively; the allele lengths of D, E, F, G are 246 bp, 250 bp, 254 bp and 258 bp, respectively; the allele fJ (27H39LR) is 190 bp long.
224 Table I. Characteristics of autosomal and Y-chromosomal polymorphic loci in Germans and Yanomami indians from the village of HAP
Locus ( chromosomal localization)
4804 LR (12)
4815 LR ( 12)
Allele frequencies [Heterozygosity rates: observed/expected] Allele
a b c d e f
A
B C D E
F G H
27H39 LR (Y)
Germans
Yanomami
no. of chromosomes 208
44"
12 12 121 37 22 4 [0.612/0.611]
0 3 35 4 2 0 [0.364/0.353]
no. of chromosomes 188
no. of chromosomes 32a
6 6 18 47 47 35 21 8 [0.846/0.815]
0 0 I 4 13 13 2 0 [0.687/0.670]
no. of chromosomes 48
no. of chromosomes
ex
8 21
Y 0
II
P
[gene diversity:
8 0.706
no. of chromosomes
lla
10
1 0 0 0.165]
no. of chromosomes 350 HLA-DQA
03 04
54 9 no. of chromosomes 622
HLA-DQB
0402
16
aFor the calculations of allele frequencies and heterozygosity rates in the Yanomami sample only largely unrelated individuals were included.
225 The micro satellite DNA markers used in this study are of the GATA type located on chromosome 12 (4804 LR and 4815 LR) and Y (27H39 LR) (Roewer et aI., 1992). 6 alleles are described for 4804LR, 8 for 4815LR and 4 for the 27H39LR locus in a number of caucasoids (Roewer et aI., 1992; and Tab. 1). Because of the shortness of the PCR amplified length variable alleles informative typing of old and degraded DNA is possible. The sensitivity of PCR allows the collection of hairs instead of blood for discriminative DNA analysis. HLA class II genes are analyzed by the PCR-based oligotyping method.
Materials and methods
Genomic DNA was extracted from 3-10 hairs by an "in-one-tube extraction" method (Kawasaki, 1990). Repeat flanking oligonucleotide primers for the (GATA)n micro satellite loci were used as described previously (Roewer et aI., 1992). Approximately 100-250 ng DNA was used for a 25-J.LI PCR sample with 2 U Taq polymerase in the buffer recommended by the suppliers (Promega). The MgCl 2 concentration was 2.5 mM. Amplifications were performed for 30 cycles with 30 sec denaturation at 94°C, 30 sec primer annealing at 51 °c and 90 sec extension at 72°C in a PCR thermocycler (Biomed). Simultaneous multilocus PCR using the 3 primer pairs was carried out under identical conditions. Gel purification of the repeat-containing fragments, radioactive labelling of the fragments, PAGE and X-ray exposure were performed as previously described (Roewer et aI., 1991). A standard ladder of the known alleles was used for sizing the bands. HLA class II oligotyping (DQA 1 and DQB 1 loci) was done following the protocols of the 11th International Histocompatibility Workshop in Yokohama (1991). Results
89 individuals were analyzed at the (GATA)n micro satellite loci 4804LR, 4815LR (both linked on chromosome 12) and 27H39LR (chromosome V). 75 individuals originate from the village HAP, the remaining ones stem from the surrounding villages WA W, SHI, ARI, MAR and MAA (Tab. 2). In HAP 33 males and 42 females live in 3 generations. We found several polygyneous families and 2 polyandreous families. 23 individuals of the ancestral generation, who are possibly also more or less related, were chosen for the calculation of observed allele frequencies, heterozygosity rates and gene diversities (Nei, 1973) (see Tab. 1). The
226 Table 2. Microsatellite typing of 89 Yanomami indians from 6 villages (F father; M mother; C child; m male; f female; 04 locus 4804LR; 15 locus 4815LR; Y locus 27H39LR); closely related individuals are put to groups if possible HAP
Sex
04/15 /Y
HAP
1 2 3 4 5 6
F M CI C2 C3 C4
m f m m f f
ee/EF /a cd/DF /cd/DE/a cd/DE/a cd/DF /cd/DE/-
44 45 46 47 48 49
F M CI C2 C3
7 8 9 10
F M CI C2
m f m m
cc/DF /a be/EF /ee/DF /a be/ED/a
50 51 52
M CI C2
II 12 13 14 15 16 17 18 19
F M CI C2 C3/M F Cl/F M C
m f f m f m m f f
ee/EE /a ee/EF /ee/EF /ee/EE /a ee/EG /ee/FF /a ee/FG/a ee/EG/ee/GG/-
20 21 22 23 24 25 26 27 28
M Cl C2 C3 C4 C5/M Cl C2 C3
f f f f m f m f m
cd/EE //ee/ ee/EF /cd/ /-
f
cd/DF /-
m f m f f m m f m f f m
eel D/a
40
F M Cl C2 C3/M F Cl C2 C3 C4 C5 C6
41 42 43
F Cl C2
m f m
56 29 30 31 32 33 34 35 36 37 38 39
/
/a
cd/EF /ee/ /a ee/EF /cd/EF IP
// ee/ 1ee/EF 1-
ce/
ee/
ee/ /a ee/EF /a ee/ /ee/ /a /ee/ /ee/ ee/EF /
be/CE /a be/EG/ce/CG/a
04/15 /Y
m f f f m
ee/EF /a be/ F/be/DF /ee/FF /ee/DF /a ee/FF /-
f f m
ee/EE /ee/EE /ee/ /a
m m m
ee/FF /a ee/FG /a be/EF fa.
f m f f m f m f m f f f f m f m f f f
ee/FF /ee/ /a /be/ /ee/ cd/DF fa. be/ /ee/ /a /ee/
C4
53 54 55 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75
Sex
M Cl C2
F M C
/
/a
ee/
/
/ /-
ee/ ee/
/-
ee/EG/cd/ //ee/ ee/EF /-
eel /
/a //-
WAW 1 4 7 8 9 10 13
F M Cl C2
m m m f f f m
/
/a
/P
ee/EF /a ee/DE/ee/DF /ee/DE/-
/
fa.
MAH I 4 II 19
m m m m
/P
m
/P
m
/a
m
fa.
/a /a /a
MAA
ARI
SHI
227 alleles found in the Yanomami are not different from those observed in a German control sample. Both populations were tested for different allele frequencies using Fisher's exact test (SAS Institute Inc., 1991). For each locus allele-wise comparisons were evaluated allowing for multiple testing. Appropriate thresholds for the error probability P, calculated by division of 0.050 by the number of alleles, were 0.008 (4804LR), 0.006 (4815LR), and 0.012 (27H39LR), respectively. Significant differences were found for 4804LR, with allele c being significantly more frequent in the Yanomami sample (P = 0.005), and for 27H39LR, with allele !Y. being dramatically overrepresented among Yanomami males (P = 6.52 X 1O~6). For locus 48l5LR, the deficiency of allele G (2/33 vs 21/188) respresents a borderline result (P = 0.0098). According to the test the allele distribution at the autosomal loci appears to be not substantially different from that in caucasoids. Rare alleles are lacking in the investigated Yanomami most probably because of the small sample size. As expected the observed heterozygosity rates differ markedly between the populations. In contrast to the diversity at the chromosome 12 loci we see in HAP at the Y chromosomal locus 27H39LR always the !Y. allele with only one exception. In this case the allele fJ was typed, a man (HAP 28) whose father is dead and whose descent is unknown due to a strong taboo of mentioning deceased relatives. Also WA W 4, MAA 1 and MAH 1 carry the allele fJ (MAA 1 is the father of MAH 1, and WAW 4 is the son-in-law of MAA 1). Historically the villages ARI and WAW are relatively isolated, whereas the village MAA is a split of MAA and SHI. All the investigated villages are located within walking distance. Other Y chromosomal alleles are lacking in the investigated population. Sequence analysis confirmed identity between !Y. alleles in Germans and Yanomami (data not shown). Several families were additionally typed at the MHC loci DQA 1 and DQBl. Due to the limited amount of DNA extracted from hairs the sample size was too small to calculate H LA -DQ allele frequencies for Yanomami indians. Despite the comparatively homogeneous Yanomami population two exclusions from paternity were found: a) HAP 49 is not the son of HAP 3 but probably of HAP 44: b) Hap 41 is not the son of HAP 57. Further details concerning family relationships are shown in figures. In the pedigree depicted in Fig. 3 the "strange" woman HAP 18, brought by HAP 17 from another village, is probably his true cross-cousin. Neither of the parents of HAP 17 has direct siblings so he probably chose to look back at least two generations to keep the idea of the cross-cousin marriage rule. Figure 4 shows the situation of two possible fathers for the deceased woman HAP X. The typing at the locus 4815LR increases the probability that HAP 54 is her father rather than HAP 1.
228 r-----------
EE
I
~,,
EF
• Figure 3. Pedigree of a HAP Yanomami family. Capitals symbolize the typed 4815LR alleles. Hatched symbols mark putative relatives of the mother of HAP 15 who transmit the rare allele G. The family of HAP 18 lives in another village. Symbolized is the hypothetic inheritance of the allele G typed in the "stranger", HAP 18. HAP 17 and HAP 18 are probably marriage partners corresponding to the preferential cross-cousin marriage rule. Additionally typed HLA class II alleles: DQA: HAP 15: 03, 04; HAP 16, 17: 03. DQB: HAP 15. 16. 17: 0402.
J
OF
® 6
DE
DE
OF DE
CG
EG
Figure 4. Pedigree of a Yanomami family of HAP. Capitals symbolize the typed 4815LR alleles. HAP I and HAP 54 represent two putative fathers of the deceased woman HAP X. The rare allele G identified in the children of HAP X increases the paternity probability of HAP 54.
Discussion
Anthropological field observations, especially the ethological research, is generally dependent upon more or less reliable information by the observed people themselves. DNA micro satellite typing via peR is a promising method for more objectivity in anthropology. One can individualize minimal amounts of DNA e.g. in hairs which are easier to
229 collect from people and are easy to preserve for months than blood. Microsatellites based on tetranucleotide repeats yield short PCR-amplifiable alleles with a simpler profile (less slippage artifacts) than seen at the frequently used CA repeat loci (Roewer et aI., 1992; Edwards et aI., 1992). Microsatellite polymorphisms are in the informative range of some serological markers, but the information increases by typing two or more loci consecutively or even better simultaneously. In a multiplex PCR one hair root is enough to type the 3 above-mentioned markers. The MHC class II oligotyping requires more DNA but delivers more higher information. The Y chromosomal micro satellite is essential to yield information about male descendence lineages in the investigated populations. The village HAP seems to be founded by close male relatives after fissioning of a larger community. Probably females contribute mainly to the considerable variability checked at the autosomal loci. These women were either captured or came from other villages to which exist alliances. The preferential cross-cousin marriage rule (ccm) was additionally confirmed in some cases with the help of micro satellite typing. In all cases one has to have the ethnological field research data in mind contributing to the probability of paternity. The similar allele distribution in unrelated Germans and Yanomami reflects the origin of the polymorphisms by slippage mutation events without influence of selection pressure. Acknowledgements We are indebted to the friendly cooperating people of the Yanomami. We would like to thank Dr. Michael Krawczak for valuable comments. This work was supported by the VW-Stiftung.
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230
°
Roewer L, RieB 0, Prokop (1991) Hybridization and polymerase chain reaction amplification of simple repeated DNA sequences for the analysis of forensic stains. Electrophoresis 12: 181-186 Roewer L. Arnemann J, Spurr NK, Grzeschik K-H, Epplen JT (1992) Simple repeat sequences on the human Y chromosome are equally polymorphic as their autosomal counterparts. Hum Genet 89: 389-394 SAS Institute Inc. (1991) SAS/ST AT user's guide, release 6.03 edition. SAS Institute Inc., Cary NC, pp 283-357 Schurr TG, Ballinger SW, Gan Y-Y, Hodge JA, Merriwether DA, Lawrence DN, Knowler WC, Weiss KM, Wallace DC (1990) Amerindian mitochondrial DNAs have rare Asian mutations at high frequencies, suggesting they derived from four primary maternal lineages. Am J Hum Genet 46: 613-623 Tanis RJ, Neel JV, Dovey H, Morrow M (1973) The genetic structure of a tribal population, the Yanomama Indians. IX. Gene frequencies for 18 serum protein and erythrocyte enzyme systems in the Yanomama and five neighboring tribes: nine new variants. Am J Hum Genet 25: 655-676 Wallace DC, Garrison F, Knowler WC (1985) Dramatic founder effects in Amerindian mitochondrial DNAs. Am J Phys Anthropol 68: 149-155 Ward RH, Gershowitz H, Layrisse M, Nee! JV (1975) The genetic structure of a tribal population, the Yanomama Indians. IX. Gene frequencies for 10 blood groups and the ABH-Le secretor traits in the Yanomama and their neighbors: the uniqueness of the tribe. Am J Hum Genet 27: 1-30 Weitkamp LR, Neel JV (1972) The genetic structure of a tribal population, the Yanomama Indians. IV. Eleven erythrocyte enzymes and a summary of protein variants. Ann Hum Genet 35: 443-444 Weitkamp LR, Arends T, Gallango ML, Neel JV, Schultz J, Shreffler DC (1972) The genetic structure of a tribal population, the Yanomama Indians. III. Seven serum protein systems. Ann Hum Genet 35: 271-279
