Blood Groups of Baboons. Population Genetics of Feral Animals W. W. SOCHA,' A. S. WIENER,' J . MOOR-JANKOWSKI AND C. J . JOLLY 'Primate Blood Group Reference Laboratory and World Health Organization Collaborating Centre for Primate Haematology, at the Laboratory for Experimental Medicine and Surgery in Primates (LEMSIP), of the New York University Medical Center, New York,New York;3Department of Anthropology of the New York University, New York,New York I0016 KEY WORDS Baboons Olive baboons Hamadryas baboons. Human-type blood groups . Simian-type blood groups ABSTRACT Blood and saliva from 495 Ethiopan baboons were collected in the field and tested for their human-type A-B-0 groups while 493 blood samples were tested for their simian-type blood factors AP, Bp, Cp, GP, NP, ca and hu. Four series of feral animals were tested: 194 olive baboons, a troop of 82 and another of 90 hamadryas baboons, and a series of 129 baboons classified as olive/ hamadryas hybrids. In addition, 126 baboons from other sources were tested for their human-type A-B-0 groups and 131 for their simian-type blood groups. Human-type groups A, B and AB but not group 0 were found in combined series of 621 animals. Gene frequency analysis also indicated the absence of group 0. Population analysis of the data obtained for the 493 Ethiopian baboons has shown that the simian-type blood groups AP and BP are independent of one another. In contrast Bp and GP appear to be determined by corresponding allelic genes; if confirmed by population data on additional series of animals, this would define the first baboon blood group system found. There is a close association between the blood group specificities ca and hu, the exact nature of which still remains to be clarified. Blood group ca, originally believed to be species specific, is found to be polymorphic in both olive baboons and hamadryas as well as in the hybrids; hu, on the other hand, present in all hamadryas tested, is polymorphic only in olive baboons. Baboons, like other Old World monkeys and apes, have homologues of the human-type AB - 0 blood groups (Moor-Jankowski e t al., '64; Wiener and Moor-Jankowski, '69). In addition, baboons have their own, numerous, simian-type blood groups (Moor-Jankowskiet al., '67; Wiener et al., '701, analogous to the multiplicity of blood groups in man and in lower animals. Reagents for demonstrating the simian-type blood groups of baboons have mostly been produced by isoimmunization, and also by cross-immunization (Moor-Jankowski et al., '73;Moor-Jankowski et al., '74). Former attempts to classify the various simian-type agglutinogens of baboons into systems have been unsuccessful. This was due in part to the unavailability of families for genetic studies and in part to the lack of large AM. J. PHYS. ANTHROP., 47: 435-442 enough samples from normal feral populations to be used for methods of population genetic studies. Recently, we had the opportunity to test blood and saliva samples of large series of baboons collected by F. L. Brett in Ethiopia. Details of that field study are reported separately (Brett et al., '761, including a troop by troop detailed analysis of the findings for the human-type A-B-0 group. The purpose of this article is to analyze the blood group findings further, applying the methods of population genetics with special reference to the simian-type blood groups. MATERIAL AND METHODS Baboons caught in the wild were subjected ' Supported by USPHS, NIH Grant GM 12074 and Contract HR 4-2184. 435 436 W. W. SOCHA, A. S. WIENER, J. MOOR-JANKOWSKI A N D C. J. JOLLY TABLE I Human-type A - B - 0 bloodgroups Phenotypes A Population EthiOpiQ Hamadryas troop A troop K combined Olive baboons Hybrids Subtotal AB B Estimated gene frequencies Tntal NO. No 'b 0 15 15 0.00 16.67 8.72 76 92.68 31 34.44 107 62.21 6 7.32 44 48.89 50 29.07 82 90 172 5 2.58 133 68.56 56 28.87 5 3.88 81 62.79 43 25 5.05 321 64.85 28 22.22 39 30.95 No. % No % Allelism 0 A B xz,t, p 0.037 0.411 0.233 0.198 0.170 0.963 0.589 0.747 0.733 0.830 0.12 0.008 5.93 1.26 0.10 >0.70 >0.90 c0.02 >0.20 194 0.0 0.0 (a) 0.0 (b) 0.069 0.0 33.33 129 0.0 0.206 0.794 0.06 >0.80 149 30.10 495 >0.70 Other sources Total 53 360 59 46.82 208 to physical examination and taxonomic classification k f . Brett e t al., '76).Their blood specimens were collected both with and without anticoagulant and their saliva samples were immediately inactivated in a boiling water bath, as described in a previous publication (Moor-Jankowski et al., '64).The inactivated saliva samples as well as the blood specimens were stored in liquid nitrogen in the field and then brought to New York City and stored there in liquid nitrogen, Batches of blood were thawed and reconstituted (Rowe e t al., '72) immediately before testing in our laboratory. The human-type A-B-0 groups were determined in 495 Ethiopian baboons by quantitative inhibition tests for A-B-H in the saliva, and by testing the baboon sera for their content of anti-A and anti-B agglutinins after removal of non-specific heteroagglutinins by absorption with human group 0 red cells. Since the introduction of this technique (Wiener and Moor-Jankowski, '63) thousands of primate animals have been tested without difficulty and with readily reproducible results confirmed by population genetics, family studies (Moor-Jankowski and Wiener, '721, transplantation of A-B-0 blood group compatible simian organs (Murphy e t al., '69) to man and cross-circulation of human patients with baboons (Fortner e t al., "71). The simian-type baboon blood groups were determined in all but two Ethiopian baboons by testing the red cells with a battery of antisera, anti-Ap, anti-Bp, anti-CP, anti-GP, anti- 126 621 NP, anti-ca and anti-hu, described in detail in earlier papers (Wiener et al., '70; Moor-Jankowski e t al., '73; Moor-Jankowski e t al., '74). In general, these reagents produce their most definitive reactions by the antiglobulin technique. The techniques of grouping simian blood was reviewed by Socha e t al. ('72). For the purpose of population analysis, the Ethiopian baboons were divided into four series on the basis of their physical characteristics and geographic location, namely, two geographically widely separated groups of hamadryas of 82 (Troop A) and 90 (Troop K) animals respectively, a series of 194 olive baboons (Papio anubisj and a series of 129 animals deemed to be olive/hamadryas hybrids, giving a total of 495 animals. In addition, there were 131 baboons (of which only 126 could be tested for their human-type A-B-0 blood groups) derived from a variety of other sources; subspecies of those baboons had not been determined. RESULTS In tests for human-type A-B-0 blood groups, with only few exceptions, Landsteiner's rule held, namely, there was a reciprocal relationship between the blood group substances present in saliva and the agglutinins present in the serum, confirming the accuracy of the AB-0 group determination. As can be seen in table 1, among the total of 621 animals tested for the A-B-0 blood groups only groups A, B and AB, but not group 0 were found. The fre- 437 BLOOD GROUPS OF BABOONS quencies of the three blood groups in different baboon troops were used for estimating gene frequencies and for testing the hypothesis of inheritance. Table 2 summarizes results of tests performed on 493 blood samples of Ethiopian baboons and 131 blood samples of baboons from other sources, using baboon isoimmune sera of seven different specificities. As can be seen, t h e simian-type polymorphism was found in all baboon troops for all specificities tested except one, namely, hu, which was present in all the 172 hamadryas baboons. Because of the previously reported ambiguous observations on the relationship between simian-type specificities AP and EP, the present findings on the relatively large sample of animals are of special interest. Table 3, shows distributions of the four types defined by antiAP and anti-Bp reagents, as well as the results of Ap-Bp gene frequency analysis in different series of feral baboons. The results obtained are discussed in detail later in the article. Table 4 shows the distribution of four blood types defined by another pair of reagents, anti-BP and anti-GP. Original observations on simian-type specificities Bp and Gp suggested the possibility of an antithetical relationship between those two factors, and the present series of baboons provided the opportunity to test the possibility that Ep and GP might be transmitted by triple allelic genes. The results of the gene frequency analysis are discussed later. Finally, the table 5 presents the results of tests with still another pair of reagents, namely, anti-ca and anti-hu, which detect specificities supposed to be constrasting "species" characteristics of the red cells of two groups of baboons: specificity ca of olive and yellow baboons (cynocephalusanubis) and specificity hu of hamadryas and chacmas (hamadryas-ursinus). Discussion of the results of gene frequency analysis is deferred to the latter part of the article. Two-by-two contingency tests have been carried out for associations between all pairs of simian-type specificities other than Ap-Bp, Gp-Ep and ca-hu. With only few exceptions the tests indicated the absence of associations between any of the pairs of antigens analyzed. In the few cases where the P values were less than 0.5, the chi-square value is only just beyond the level of statistical significance. Bearing in mind that when numerous 2 X 2 N O " mml-2 m $: m ooom 9999 m m x 2 ooot- m m oooc M * i moYzz N O N - m wm t -m ( occn ~ m m 2 m Q, 3 N m w"2R ri *z 0 N 438 W. W. SOCHA, A. S. WIENER, J . MOOR-JANKOWSKI AND C. J. JOLLY TABLE 3 Distribution o f A P - B p t y p e s B l d types op Population No. % Ethiopia Hamadryas troop A troop K combined Olive baboons Hybrids 13 3 16 33 15.85 3.33 9.30 17.19 40 31.01 Subtotal 89 Other sources Total 49 138 np AP No. %, 0 0 0 62 0.00 0.00 0.00 32.29 30 23.26 92 37.40 34 126 No. 9: 63 76.83 83 92.22 146 84.88 37 19.27 29 22.48 212 25.95 34 Gene frequencies APBP Total Ap Bp 6 4 10 60 7.32 4.44 5.81 31.25 82 90 172 192 0.037 0.022 0.030 0.400 0.602 0.817 0.695 0.302 1.22 0.14 1.01 0.20 20.20 >0.50 >0.20 >0.50 30 23.26 129 0.269 0.263 0.82 >0.30 100 25.95 246 tests are carried out, even in the absence of association one of twenty of the tests could be expected to give results a t the borderline or statistical significance, we do not consider any of the seeming associations to be meaningful. DISCUSSION Significant differences in the distribution of the human-type A - B - 0 blood groups have been found for different series of baboons in the present as well as in previous studies, beginning in 1964 (Moor-Jankowski et al., '64). These differences in distribution appear to result from genetic drift caused by geographical separation and isolation among the various troops of animals. Thus, the A - B - 0 blood group distribution in 192 olive baboons (P. anubis) in the present study was different from that observed in the previous series of 174 P. anubis but of different geographical origin. Paradoxically, the distribution of the A - B - 0blood groups in one troop of hamadryas baboons in the present study does not differ markedly from that of a group of olive baboons from the previous study, but differs markedly from that of the second troop of hamadryas described here. These findings demonstrate that for a single category of inherited markers, genetic drift can produce wider differences among populations within a species than those existing between species. Not one group 0 animal was found among the 621 baboons which represent the largest number investigated by us in a single study; Independence rZili X, No 14 114 P 493 10.69 131 624 therefore, one might conclude that in this series there were no carriers of the amorph gene 0, so that only two allelesA and B had t o be postulated. Therefore, these gene frequencies were calculated by direct count, and the values obtained used to compute the expected numbers of animals in each of three groups, A, B and AB. The results of x 2 tests (cf. table 1) indicate good fit for the 2-allele theory for the population of olive baboons, for hybrids, as well as for each of two troops of hamadryas, when analyzed separately. Interestingly enough, when the test for goodness of fit was applied for the combined series of 172 hamadryas, the value of chi-square obtained (xt = 5.93) seemed to reject the theory of inheritance based on the presence of only two alleles. When, on the other hand, a different method of estimating the gene frequencies was used, this time based on assumption that allele 0 is present but infrequent (Wiener and Moor-Jankowski, '691, chi-square proved to be only 1.26, showing a much better fit. Thus, pooling of the two series with widely different distributions of blood groups had resulted in a misleading artifact. Thus, contrary to our previous assumption (Moor-Jankowski et al., '641, the presence of gene 0 in yellow, olive and hamadryas baboons remains unproven. On the other hand, however, two group 0 animals were actually found in a series of 188 P. papio from Senegal (Wiener et al., '69);a third group 0 baboon found in the same study was of unknown origin and was not evaluated for his morphological characteristics. It thus ap- 0 0.00 3 0 0.00 3 Oiher sources Total 0.00 1.56 0.00 0.00 Subtotal 0 0 0 3 X No. 65.85 60.00 62.79 19.27 X 191 7 5.34 39 30.23 184 54 54 108 37 hu No. +- 0 ca -+ 10 % 7.75 157 107 81.68 50 TABLE 5 21 16.03 82 131 624 129 493 82 90 172 192 Total ++ 34.14 40.00 37.21 58.33 Ib 273 17 12.98 256 80 62.01 28 36 64 112 No. hulca 624 131 129 493 82 90 172 192 Total 0.098 0.156 0.126 0.084 0.577 0.263 0.571 0.748 0.641 0.291 0.722 1.0 1.0 1.0 0.527 0.450 0.566 0.640 0.600 0.5444 Ca Gene frequencies Hu @ 6.12 0.125 0.831 0.30 3.12 0.05 0.02 6.5 4.7 - P - Pill XZili 12.1 8.93 27.8 XZIII 1.02 0.75 0.18 0.60 0.06 p <0.001 <0.01 <0.001 P Allelism 0.003 3.4 3.4 9.8 0.4 XtCH -0.31 -0.88 r 0.95 0.07 0.07 <.01 0.55 p Allelism Chi-square for Independence Independence 0.160 0.331 0.096 0.233 0.625 @ Gene frequencies Bp Distribution of the specificities huand ca 24 18.32 76 61 11 8.53 X 27 20.93 52 No. 13 15.85 26 28.89 39 22.67 12 6.25 M BPGP 3.66 2 2.22 5 2.91 20 10.42 3 No. GP 0.00 0 0.00 0 0.00 40 20.83 0 No. huica types 33 25.19 283 __ 53 40.46 183 48 37.21 250 68.29 67.78 68.02 44.27 X 43 33.33 130 No. Blwd types 56 61 117 85 % BP 10 12.19 1 1.11 11 6.39 75 39.06 No. OP Ethiopia Hamadryus troop A troop K combined Olive baboon8 Hybrids Population Total Other sources Subtotal Ethiopia Hamadryas troop A troop K combined Olive baboons Hybrids Population Distribution o f B P - G P t y p e s TABLE 4 440 W. W. SOCHA, A. S. WIENER, J. MOOR-JANKOWSKI AND C. J. JOLLY pears that while the distribution of the A-B-0 blood groups may vary greatly among as well as within hamadryas, yellow and olive and Guinea baboons, the presence of group 0, though rare, has been demonstrated conclusively for Guinea baboons alone. Anti-Ap and anti-Bp, the first two reagents for typing red cells of baboons produced by isoimmunization (Moor-Jankowski e t al., '671, have been studied most intensively. The fact that both specificities are nonreactive or only poorly reactive by the ficin method, as compared with the antiglobulin method, suggests that the agglutinogens and antibodies involved are comparable to those of the human M-N systems; this was one of the reasons why we originally assumed that AP and BP belonged to one and the same blood group system. However, while no families were available for investigation, previous studies by the method of population genetics on two series, one of yellow baboons and the other of olive baboons, indicated that AP and BP are independent of one another (Wiener et al., '70). On the other hand, a different series of 184 Guinea baboons showed all four types, Op = 20, AP = 56, Bp = 12 and A p B p = 96. In that case, it was evident that AP and BP exhibited a rather strong positive association incompatible both with allelism and independence, unless one invoked a fourth allele which produced an antigen having both specificities AP and Bp, Thus, our previous results did not provide enough data to determine whether the Ap-Bp baboon blood groups are transmitted by allelic genes or are independent from one another. The population genetics analysis performed in the present study leaves little doubt that AP and BP are actually independent of one another. As shown in table 3, the distribution of the Ap-Bp blood types in each of the four series of baboons from Ethiopia demonstrates that AP and BP are independent from each other, and, therefore, presumably belong to different blood group systems. I t is instructive to observe the effect of pooling the four series. When the statistical test for independence was applied to the combined series of 493 baboons, the very high chi-square value of 17.31 was obtained, making it appear as if there were a strong negative association between the two agglutinogens AP and BP (r = -0.19). Of course, this was an artificial association created by pooling several series of baboons which produced a stratification effect. A relationship between agglutinogens Bp and GP was suggested by the previous observation that reagents of specificities anti-BP and anti-Gp could both be produced in baboons by cross-immunization with red cells of geladas fTheropithecus geladd (Moor-Jankowski e t al., '71), even though anti-BP and anti-Gp sera have been produced also by isoimmunization. Though the possibility was not mentioned in the original paper, we did notice that Bp and GP appeared to be antithetically related, and the present new series of baboons provided the opportunity to test the possibility that BP and GP might be transmitted by triple allelic genes. In combination, the two antisera, anti-BP and anti-Gp,detect four blood groups (cf. table 4). The most striking results were obtained with the series of 129 hybrids with the high chi-square value of 6.12 for independence as against only 0.003 for allelism. Thus, the distribution for the 129 hybrid animals conformed closely with the prediction under the hypothesis of triple allelic genes for the BP and GP blood groups, and argued against independence (P < 0.02). Also in the case of the series of 192 olive baboons the findings favor the hypothesis of triple allelic genes (x2(11= 0.4) rather than independence ( x * ( ~ = , 3.12). Paradoxically, however, for the series of 172 hamadryas baboons, the opposite results were obtained, since chi-square for allelism was 9.8, and for independence only 0.30. Still, it will be recalled that the 172 hamadryas baboons consisted of two troops of animals, and when the two troops were analyzed separately, the paradox was again resolved since the chisquare for allelism was reduced from 9.8 to only 3.4 for each troop. These data provide still another example of the paradoxes which arise when statistical analyses are applied to series composed of a mixture of several different groups of animals. In conclusion, therefore, the analysis of the distribution of the Bp-Gp blood groups in three series of feral Ethiopian baboons supports the hypothesis that the resulting four blood types are transmitted by triple allelic genes and not by independent pairs of genes. This conclusion is reinforced by the fact that these two factors, but not others, are shared by baboons with geladas (Moor-Jankowski e t al., '74). 44 1 BLOOD GROUPS OF BABOONS Therefore, if confirmed by further studies, the resulting B p - G p system would represent the first simian-type blood group system found in baboons represented by more than a single blood factor. As mentioned before, the anti-ca and antihu reagents appear a t first to be “species specific.” If t h a t were so, the tests with these two reagents would have shown only two contrasting types for ca and hu specificities, namely + - and - +. However, the tests carried out on large numbers of baboons show that all four combinations of these two specificities can occur, namely + -, - and - (cf. table 5). Of the four possible types, the rarest was the one lacking both antigens hu and ca; among the total of 624 baboons tested, only three were found of that type (confirmed by repeated tests). The existence of only three major types, -, - and suggested that the antigens hu and ca might be determined by contrasting allelic genes, but the findings shown in table 5 do not support that simple hypothesis. Thus, in two series, one of olive and the other of hybrids, the frequencies of the heterozygous hu-ca type were 58.3%and 62.0% respectively, whereas theoretically, the frequency of that type could not have exceeded 50%if hu and ca were alleles in population a t Hardy-Weinberg equilibrium. The distribution of the blood groups hu and ca among the hybrid animals was intermediate between the distribution for the hamadryas and olive baboons, as to be expected. On the other hand, the hybrid animals showed the highest frequencies of the heterozygous type hulca. All the 172 hamadryas had the hu antigen, for which the olive baboons and hybrids were polymorphic. In contrast, baboons of all three series were polymorphic for ca. In conclusion, the findings indicate a negative correlation between the hu and ca antigens, but the exact nature and cause of this is not yet apparent. Moreover, the results provide further evidence of the relationship between serological specificities determining t h e species differences and those determining individual differences. For example, hu, shared by all animals of the two troops of hamadryas, proved to be polymorphic in olive baboons and their hybrids. On the other hand, specificity ca which in our original study appeared to be “species specific” for olive and yellow + + +, + + ++ baboons, in the present study was shown t o be polymorphic not only in hamadryas baboons but also in olive baboons and their hybrids. This result underlines the importance of testing large series of animals before concluding whether a newly found serological specificity is type specific or species specific. We are aware that in the present study we have applied to monkeys methodology originally developed for such studies in human populations in which panmixia is assumed (though it does not always occur) (Rosin, ’56; Moor-Jankowski and Huser, ’56-57). The breeding patterns of primate species vary. Those of chimpanzee appear also to be different from those of man, yet the results obtained by applying human population genetics methodology to our blood group findings in chimpanzees could later by fully confirmed when families became available for testing (Wiener et al., ’75). Similarly, we anticipate that the conclusions presented in this paper for baboons will be confirmed when baboon families become available for blood grouping. LITERATURE CITED Brett, F. L., C. J. Jolly, J. Moor-Jankowski, W. W. Socha and A. S.Wiener 1976 Human-type A-B blood groups in wild Ethiopian baboons. Yearbook of Phys. Anthrop., in press. Fortner, J. G., E. J. Beattie, Jr., M. H. Shiu, J. S. Howland, P. Sherlock, J. Moor-Jankowski A. S. Wiener 1971 The treatment of hepatic coma in man by cross-circulation with baboon. In: Medical Primatology 1970.Goldsmith and Moor-Jankowski, eds. S. Karger, Baselmew York, pp. 62-68. Moor-Jankowski, J. K., and H. J. Huser 1956-57 Seroanthropological inveatigation in t h e Walser a nd Romanch isolates in the Swiss Alps and their methodological aspects. Acta. Genet., 6: 527-531. Moor-Jankowski, J., and A. S. Wiener 1972 Red cell antigens in primates. In: Pathology of Simian Primates. R.N. T-W-Fiennes, ed. S. Karger, Baseli’New York, pp. 270-317. Moor-Jankowski, A. S. Wiener and E. B. Gordon 1964 Blood groups of apes and monkeys. I. The A-B-0 blood groups in baboons. Transfusion, 4: 92-100. Moor-Jankowski, J., A. S. Wiener, E. B. Gordon and J. H. Davis 1967 Blood groups of baboons demonstrated with isoimmune sera. Nature, 214: 181. Moor-Jankowski, J., A. S. Wiener and W. W. Socha 1974 A new taxonomic tool. 11. Serological differences between baboons and geladas demonstrated by cross-immunization. Fol. Primatol., 22: 59-71. Moor-Jankowski, J., A. S . Wiener, W. W. Socha, E. B. Gordon and J. H. Davis 1973 A new taxonomic tool: Serological reactions in cross-immunized baboons. J. Med. Prim., 2; 71-84. Murphy, G.P., A. W. Weber, H. D. Brede, F. P. Retief, C. P. Retief, J. A. van Zyl, H. Groenewald and J. J. W. van Zyl 1969 The significance of human ABO blood groups in the 442 W. W. SOCHA, A. S. WIENER, J. MOOR-JANKOWSKI AND C. J. .JOLLY survival of untreated baboon renal allotransplants. Amer. Surg., 35: 292-300. Rosin, S. 1956 Die Verteilung der ABO Blutgruppen in der Schweiz bearbeitet under Einfuhrung neuer Methoden der Auswertung und Darstellung. Arch. J. Klaus-Stiftung, Vererb. Forsch., 31: 17-127. Rowe, A. W., J. H. Davis and J. Moor-Jankowski 1972Preservation of red cells from the nonhuman primates. Primates in Medicine, 7: 117-130. Socha, W. W., A. S. Wiener and J. Moor-Jankowski 1972 Methodology of primate blood grouping. Transplant. Proc., 4: 106-110. Wiener, A. S., and J. Moor-Jankowski 1963 Blood groups in anthropoid apes and baboons. Science, 142: 67-69. 1969 The A-B-0 blood groups of baboons. Am. J. Phys. Anthrop., 30: 117-122. Wiener, A. S., W. W. Socha and J. Moor-Jankowski 1974 Homologues of the human A-B-0 blood groups in apes and monkeys. Haematol. (Budapest), 8: 195-216. Wiener, A. S., W. W. Socha, J. Moor-Jankowski and E. B. Gordon 1970 Studies on t h e AP-BP blood groups of baboons. Am. J. Phys. Anthrop., 31: 433-438. 1975 Family studies on simian-type blood groups of chimpanzees. J. Med. Primatol., 4: 45-50.