PHYLOGENETIC RELATIONSHIPS BETWEEN MAN AND THE APES: ELECTROPHORETIC EVIDENCE!

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1 Evolution, 33(4), 1979, pp PHYLOGENETIC RELATIONSHIPS BETWEEN MAN AND THE APES: ELECTROPHORETIC EVIDENCE! ELIZABETH J. BRUCE AND FRANCISCO J. AYALA Department of Genetics, University of California, Davis, California Received June 22, Phylogeny was traditionally the subject matter of paleontology and comparative anatomy. However, in recent years it has become apparent that proteins and nucleic acids (the so-called "informational" macromolecules) store a wealth of information concerning evolutionary history. Protein sequencing, immunology, electrophoresis, and DNA hybridization are among the molecular techniques used in phylogenetic studies. These techniques have generally confirmed previously established evolutionary relationships, and in many cases have provided information where little or none was available from the fossil record. There are instances, nevertheless, when molecular studies lead to conclusions contrary to those reached by paleontologists or comparative anatomists. One case of disagreement concerns the evolutionary history of the hominoidsi.e., man, the great apes, and the Hylobatidae (gibbons and siamang). Paleontologists and comparative morphologists place the Asiatic orangutan much closer to the African apes (the gorilla and the chimpanzee) and to man than to the two other genera of Asiatic hominoids, the siamang and the gibbon. Molecular evolutionists, on the contrary, have proposed that the orangutan is much less closely related to the African apes and man, and may be as removed from these as the siamang and gibbon. Disagreement also exists with respect to the phylogenetic propinquity of the chimpanzee, the gorilla, and man. Paleontologists and comparative morphologists 1 This paper is dedicated to Professor Bernhard Rensch on the occasion of his 80th birthday, January 21,10. Revised March 7, 1979 generally propose that the gorilla and the chimpanzee are more closely related to each other than to man, while some molecular studies suggest that the chimpanzee is at least as closely related to man as to the gorilla. We have undertaken an electrophoretic study of 23 genetically controlled proteins in the six extant genera of hominoids in an attempt to provide light on the phylogeny of the hominoids. Inferences derived from protein sequencing and immunology are often based on the study of only one or very few proteins. Electrophoretic techniques provide less information for each protein than protein sequencing or even immunology, but this disadvantage might be more than compensated by the study of a fairly large number of proteins. Six polypeptide chains (the alpha and beta hemoglobin chains, myoglobin, carbonic anhydrase I, fibrinopeptides A and B), each encoded by a different gene locus, have now been sequenced in man, the chimpanzee, and the orangutan (several of these sequences are also known in the gorilla); moreover, immunological data exist in the hominoids for seven purified proteins. The electrophoretic data reported in this paper must be weighed together with this wealth of amino acid sequence and immunological information. MATERIALS AND METHODS We have studied a total of 69 individuals belonging to nine species and subspecies: Homo sapiens (man), Pan troglodytes (chimpanzee), Pan paniscus (pygmy chimpanzee), Gorilla gorilla, Pongo pygmaeus pygmaeus (Borneo orangutan), Pongo pygmaeus abelii (Sumatra orangutan), Hylobates lar (lar gibbon), Hylobates 1040

2 RELATIONSHIPS BETWEEN MAN AND APES 1041 TABLE 1. Species studied, with the number of individuals and of loci sampled in each. Family Hominidae: Num- Num- ber of ber of loci indi- sam- Species Common name viduals pled Source of sam piest 1. Homo sapiens man Graduate students, Davis, California Family Pongidae: 2. Pan troglodytes chimpanzee Yerkes RPC, Georgia 3. Pan paniscus pygmy chimpanzee 4 23 Yerkes RPC, Georgia 4. Gorilla gorilla gorilla Yerkes RPC, Georgia S. Pongo pygmaeus abelii orangutan (Sumatra) 8* 23 Yerkes RPC, Georgia 6. Pongo pygmaeus pygmaeus orangutan (Borneo) 3* 21 Yerkes RPC, Georgia Family Hylobatidae: 7. Hylobates lar lar gibbon 4 21 LEMSIP, New York 8. Hylobates concolor concolor gibbon 2 21 San Francisco Zoo, California 9. Symphalangus syndactylus siamang 1 21 San Francisco Zoo, California * A total of four additional organutans were studied for which the subspecies is unknown. t Yerkes RPC = Yerkes Regional Primate Center; LEMSIP = Laboratory of Experimental Medicine and Surgery in Primates. concolor (concolor gibbon), and Symphalangus syndactylus (siamang) (Table 1). Pongo, Hylobates, and Symphalangus are the only three genera of Asiatic apes; Gorilla and Pan are the only two genera of African apes. The ten gorillas studied are wild-born animals; all other animals are either wild born or unrelated to each other. The electrophoretic procedures followed are basically those of Ayala et al. (1972); additional details can be found in Bruce (1977). Table 2 lists the 23 proteins studied; 16 of them are red-cell proteins, the other seven are plasma proteins. The 23 proteins were selected for study because of their clear resolution in electrophoretic gels in a total of 36 primate species studied in our laboratory. All 16 red-cell proteins were electrophoresed in starch gels only; haptoglobin and leucine amino peptidase in polyacrylamide gels only; albumin, alkaline phosphatase, ceruloplasmin, and the esterases were electrophoresed both in starch and in polyacrylamide gels. Gel electrophoresis manifests banding patterns ("electromorphs") that are usually inherited as Mendelian traits; for a given protein, electromorphs with different mobility are, as a rule, determined by different alleles at a given locus. For simplicity, we shall in this paper speak of different alleles when referring to the alleles themselves or to the electromorphs they encode. The genetic control of each of the 16 red-cell proteins is known in man; we presume it to be the same in the other hominoids. Fifteen of the proteins are determined each by a single gene locus; hemoglobin a{3 consists of two a and two {3 polypeptide chains, and thus is determined by two gene loci. The seven plasma proteins (including two esterases) are assumed to be each encoded by a single gene locus, except haptoglobin which consists of two subunits, a and {3 (however, we did not dissociate haptoglobin subunits, so haptoglobin is here scored as a single locus). Allelic frequencies were determined for each locus in each species. Genetic identity (l) and genetic distance (D) were calculated using the method of Nei (1972). I may range in value from 0 (no alleles in common at any locus in the two species compared) to 1 (the same alleles in identical frequencies at every locus). D = -log,], may range in value from 0 to infinity, and may be interpreted as the num-

3 1042 E. J. BRUCE AND F. J. AYALA TABLE 2. Proteins studied with their tissue specificity and subunit structure. The chromosome assignments are for humans. Symbol Subunit Chromosome Protein for locus structure assignment Red-cell proteins: Adenosine deaminase Ada 1 20 Adenylate kinase Ak 1 9 Catalase Cat 4 autosomal Fumarase Fum 4 1 Glucose-6-phosphate dehydrogenase G6pd 2 X Glutamate oxaloacetatetransaminase Got-s 2 10 Hemoglobin a{3 Hb-a{3 4 a, 16 {3, 11 Isocitrate dehydrogenase Icd-s 2 2 Lactate dehydrogenase Ldh-A 4 11 Ldh-B 12 Malate dehydrogenase Mdh-s 2 2 NADH-Diaphorase Dia 1 Phosphoglucoisomerase Pgi 2 19 Phosphoglucomutase Pgm-l 1 1 Pgm Phosphogluconate dehydrogenase 6-Pgd 2 1 Plasma proteins: Albumin Alb 1 Alkaline phosphatase APh-l 1 Ceruloplasmin Cer 1 Esterase Est-A 1 Est-B 1 Haptoglobin Hpt-a{3 2 Leucine amino peptidase Lap 1 ber of electrophoretically detectable allelic substitutions per locus that have accumulated in the two species being compared since they last shared a common ancestor. The construction of a dendrogram based on genetic distances was made following the method of Sarich and Cronin (1976) (see also Fitch and Margoliash, 17). Essentially the procedure is as follows. The two most similar taxa are first clustered, then all other taxa are successively clustered by adding each time the taxon genetically most similar to the extant cluster. Genetic distances are partitioned between branches by calculating average distances to a reference group consisting of 16 species of Old World monkeys; the proportion of genetic distance between any two taxa under consideration is allocated to each branch on the basis of the genetic distance between each taxon and the reference group. RESULTS Electrophoretic Variation The alleles observed in the nine hominoid taxa are given in Table 3. At each locus, the most common allele found in man is called, all other alleles are named by reference to that one, by adding or subtracting the number of millimeters by which in our gels the protein encoded by each allele differs in migration from that encoded by allele. Thus, the allele found in Pan troglodytes codes for a protein which migrates 3 mm more than that of man at the Ada locus, but 4 mm less at the Ak locus. When only one allele has been found in our samples of a given species, we simply list the allele; when more than one allele is observed, the frequency of each allele is given in parentheses after the name of the allele. In the case of man we only give the most common allele; the detailed frequencies at most of

4 RELATIONSHIPS BETWEEN MAN AND APES 1043 TABLE 3. Alleles observed at each of 23 loci in nine hominoid taxa. When more than one allele has been observed in one taxon, the allelic frequencies are given in parentheses. Locus Homo Pan t. Pan p, Gorilla Pongo p. a. Pongo p. p. Red-cell proteins: Ada Ak Cat Fum G6pd Got-s Hb-af3 lcd-s Ldh-A Ldh-B Mdh-s Dia Pgi Pgm-l Pgm-2 6-Pgd Plasma proteins: Alb Aph-l Cer Est-A Est-B Hpt-af3 Lap (.33) (.67) (.12) (.88) (.20) (.80) 85 (.67) 95 (.33) 97 (.15) 105 (.85) (.71) (.29) (.86) (.14) 97 (.25) (.75) (.33) (.67) H. Sympha- H. lar concolor langus (.62) (.38) (.67) 108 (.33) the loci in our study and at many others can be found in the excellent studies of Harris and his collaborators (e.g., Harris and Hopkinson, 1972). In the following paragraphs, we compare locus by locus our results with the information available from other investigators. Ada. Previous studies have shown that some pongids carry an allele whose product migrates further anodally than that of man (Schmitt et al., 1970). Our results confirm this report and establish that Pongo pygmaeus abelii carries the human allele, while P. p. pygmaeus has the pongid allele. Schmitt et al. (1970) do not give the subspecies of the ten P. pygmaeus individuals surveyed in their study. Because of the identity of their Ada electrophoretic pattern with that of other pongids, and because of the scarcity of P. p. abelii, the Sumatran subspecies, we infer that their orangutans were probably P. p. pygmaeus, the Bornean subspecies. We could not clearly resolve the Hylobatidae pattern at the Ada locus. Ak. Previous studies have indicated that man and all apes (excluding gibbons which were not tested) carry the same allele, (Fildes and Harris, 16; Schmitt et al., 1971a). Barnicott and Cohen (1970) observed that one of their three orangutans, and one of their three gorillas exhibited banding patterns different from those

5 1044 E. J. BRUCE AND F. J. AYALA of the others. We have used a different buffer system (that of Ayala et ai., 1972), and find a series of differences. Pan troglodytes is fixed for allele, and G. gorilla is polymorphic for alleles and. Moreover, the two gibbon species are fixed for allele 92, and the siamang for allele. Cat. No previous data are available for nonhuman primates. We could not resolve the Hylobatidae electrophoretic pattern. Man and all great apes carry allele, but the pygmy chimpanzee is polymorphic for alleles and. Fum. No data have been previously reported for nonhuman primates. All hominoids carry allele, with the exception of Pan paniscus which is fixed for allele. Pan troglodytes and Pongo pygmaeus pygmaeus are not recorded because the gels were poorly resolved. Gopd. It has been previously reported that the great apes carry the same allele as humans (Barnicot and Cohen, 1970; Schmitt et ai., 1970). Kampf et al. (1971a) also showed that Symphalangus carries a different allele from the other apes (the gibbon was not tested). These results are confirmed by our study, which also shows that gibbons and siamang are fixed for the same allele,. Got-s. King and Wilson (1975) have found that humans and the chimpanzee have different alleles. Kampf et al. (1971b), using a buffer system different from that of King and Wilson, compared Pongo and man and found them identical. We have used still a third buffer system, and find no differences between humans, chimpanzees, and gorillas, but observe both species of Pongo and the Hylobatidae fixed for a different allele,. Hb ail. Our electrophoretic method does not separate the a and {3 hemoglobin chains, and thus both loci are treated together as if they were one single locus. Hoffman and Gottlieb (17) have shown that Pan troglodytes and Hylobates lar have banding patterns identical to Homo. According to Buettner-Janusch (16) two different banding patterns have been found in gibbons and orangutans. Our results are consistent with these previous results, but provide additional information. All great apes and H. lar share "allele" with man, but the two Pongo subspecies are polymorphic for "alleles" and ; Hylobates concolor and Symphalangus carry only "allele". Icd-s. No primate comparisons have been previously reported. We find two alleles. Allele is shared in common by man, the gorilla, the two gibbons, and the siamang. The two chimpanzees and the two orangutans share allele. Ldh-A and Ldh-B. No data are available from other investigators except for man and Pan troglodytes. Lactate dehydrogenase is a very conservative protein. We have observed no variants at the B locus. At the A locus all hominoids share allele, a different one from the common human allele. Mdh-s. Tariverdian et al. (1971a) and Shotake and Nozawa (1974) have shown that the pygmy chimpanzee carries the common human allele. Moreover, Shotake and Nozawa (1974) have shown that H. lar also carries the same allele as well as one coding for a more cathodal polypeptide. Our results confirm these findings-h. lar is polymorphic for alleles 93 and. The Sumatran orangutan is also polymorphic, but for alleles and. All other pongids and H. concolor are fixed for allele, while the siamang carries allele. Dia. The chimpanzee and the gorilla have been reported as carrying alleles different from man and from each other (Schmitt et ai., 1971b; King and Wilson, 1975). In our buffer system, humans and chimpanzees are indistinguishable; the gorilla is polymorphic for alleles 85 and 95. The lar gibbon is polymorphic for alleles and 108, but all other hominoids are fixed for allele. Pgi. According to Tariverdian et al. (1971b), Gorilla and Pongo are identical, butdifferentfrom Homo, while Pan troglodytes is different from all three. We confirm the differences between the three apes and man, but cannot distinguish Pan from the other two apes. The other hom-

6 RELATIONSHIPS BETWEEN MAN AND APES 1045 inoids share the same allele as the great apes. Pgm-l. Both the chimpanzee and the gorilla have previously been shown to be polymorphic for the common human allele and a second one, different in gorillas and in chimpanzees (Schmitt et al., 1970; Goodman et al., 1971; King and Wilson, 1975). Barnicot and Cohen (1970) have shown that H. lar possesses the common human allele. Our results are consistent with such previous data, except that no polymorphism was detected in the ten gorillas we examined. Moreover, we show that Pan paniscus, both subspecies of Pongo, and the two Hylobates species all carry allele, while the siamang has allele 103. Pgm-2. Schmitt et al. (1970) have reported that P. troglodytes and P. paniscus carry the common human allele, while Gorilla and Pongo have alleles different from each other and from Homo. The identity between man and P. troglodytes was confirmed by King and Wilson (1975). We confirm this identity as well as the difference between the gorilla and humans. We find the Sumatran Pongo to be polymorphic for alleles 97 and, and all other hominoids (with the noted exception of Gorilla) to be fixed for allele. 6Pgd. According to Barnicot and Cohen (1970), man, orangutan, and gibbon all have alleles different from each other, while gorilla exhibits a polymorphism, with one allele being the same as man's. We confirm the gorilla polymorphism, although both alleles are different from the common human allele. We also confirm the difference between man and Pan troglodytes, but not between man and the orangutan. Moreover, we find the two chimpanzee species to be different from each other, the two Pongo subspecies to be identical to each other (and to man), the siamang to carry the Pan troglodytes allele, and the two gibbons to be identical to each other but different from all other species. Alb. King and Wilson (1975) have reported that Pan troglodytes is fixed for an allele different from the human one, a difference we can not detect with our buffer system. We find all the great apes and the lar gibbon identical, while the concolor gibbon and the siamang are identical to each other but different from the other species. Aph-L. No data have been previously reported for nonhuman hominoids. We find all species studied to have the common human allele at this locus. Cer. According to King and Wilson (1975), Pan troglodytes is fixed for an allele which has a frequency of one percent in humans, while with our methods the P. troglodytes allele appears to be the same as the common human allele. We find that the gorilla and the Hylobatidae all share the same allele, which is different from that of all other hominoid species. Est-A. King and Wilson (1975) report that Pan troglodytes is fixed from an allele different from the human one, a difference that we cannot detect. We find Gorilla, Pongo, and the Hylobatidae to be fixed for alleles different from each other and from the human-chimpanzee allele. Est-B. No differences were detected by King and Wilson (1975) between man and P. troglodytes. We obtain the same result and find that all pongids share the human allele, while the three Hylobatidae species are fixed for allele. Hpt-OI.{3. Shim and Bearn (14) dissociated haptoglobins from various primates into subunits, and examined their electrophoretic patterns. The three hominoids tested-chimpanzee, orangutan, and gibbon-were found to differ from man at the alpha, but not at the beta locus. We have not separated the subunits, but our results are consistent with those of Shim and Bearn: all the nonhuman hominoids are different from man. Two electrophoretic patterns are found besides that of man: one ("allele" 105) is shared by the two chimpanzees and the orangutan, while the other (107) is shared by the gorilla and the Hylobatidae. King and Wilson also found Pan troglodytes to be different from humans. Lap. No data have been reported for nonhuman primates. We find that all

7 1046 E. J. BRUCE AND F. J. AYALA TABLE 4. Summary of genetic variation in nine hominoid taxa. Average number Frequency of Average heterozygosity Number of of individuals polymorphic Species loci sampled sampled per locus loci" obs. exp.t Homo sapiensi: Pan troglodytes Pan paniscus Gorilla gorilla Pongo pygmaeus abelii Pongo pygmaeus pygmaeus Hylobates lar H ylobates concolor Symphalangus syndactylus Nonhuman average * A locus is considered polymorphic whenever more than one allele has been observed in our samples. t Calculated under the assumption of Hardy-Weinberg equilibrium as if all animals were from the same random mating population. *Data from Harris and Hopkinson (1972). hominoids have the common human allele at this locus. A summary of the amount of variation found in the nonhuman hominoids studied is given in Table 4. The number of loci examined is fairly large, comparable to that used in typical studies of genetic variation in natural populations of various organisms. However, the number of animals studied is small in our case, which makes unreliable the values of polymorphism and heterozygosity given in the table. Some variation has been found in every species except H. concolor and S. syndactylus, of which only two and one individuals, respectively, were studied. The degrees of polymorphism and of heterozygosity are in every case lower in the nonhuman species than in humans (for which we use in the table the extensive data of Harris and Hopkinson, 1972). We conjecture that this difference is real, namely that at least the pongids have less genetic variation than humans at gene loci coding for structural proteins. In the case of Pan troglodytes our conjecture is reinforced by the results of King and Wilson (1975) who observed only one polymorphism (at the Pgm-l locus) among 44 loci studied, although they sampled a fairly large number of individuals at each locus (113 on the average). Genetic Distances and Phylogeny Table 5 gives the genetic identity, I, and genetic distance, D, for all pairwise comparisons between the nine taxa studied. Only one other study is available where pairs of hominoid species have been compared on the basis of an electropho- TABLE 5. Genetic identity, I (above diagonal) and genetic distance, D (below diagonal) between pairs of nine taxa representing all six extant genera of hominoids. I and D calculated according to the method ofnei (1972). Pengo Pongo H. Sympha- Homo Pan t. Panp. Gorilla p. a. p. p. H.lar concolor langus Homo sapiens Pan troglodytes Pan paniscus Gorilla gorilla Pongo pygmaeus abeiii Pongo pygmaeus pygmaeus Hylobates lar Hylobates concolor Symphalangus syndactylus

8 RELATIONSHIPS BETWEEN MAN AND APES 1047 TABLE 6. Genetic identity, I (above diagonal) and genetic distance, D (below diagonal) for pairwise comparisons between the six extant genera of hominoids. Homo Pan Gorilla Pongo Hylobates Symphalangus Homo Pan Gorilla Pongo Hylobates Symphalangus retic survey of many gene loci, namely King and Wilson (1975), who compared man and Pan troglodytes. They studied 44 loci and obtained a value of D = 0.620; our value is D = Possible reasons for this discrepancy will be discussed below. The genetic distances in Table 5 range from (between the two chimpanzee species) to (between the siamang and either man or the pygmy chimpanzee). The genetic distance between the two Pengo subspecies is 0.130, somewhat greater than that between the two chimpanzee species, and the same as between the two gibbon species. In general, the genetic distances between pongid species, or between these and man, or between the Hylobatidae species are comparable to those observed between sibling or closely related species of a great variety of organisms, although some of the hominoid comparisons are between different genera (Pan-Gorilla-Pongo) or between different families (the great apes vs. man). The genetic distances between the Hylobatidae and the other two families (Hominidae and Pongidae) range from to 1.099, and thus are comparable to those observed in other groups of organisms between species not closely related or between genera (see Ayala, 1975, for a summary). The average distances for comparisons between different hominoid genera are summarized in Table 6, where the data from Table 5 have been averaged for the two chimpanzee species, the two Hylobates species, and the two orangutan subspecies. Figure 1 presents a dendrogram based on the genetic distances given in Table 4, and constructed according to the method indicated in the Materials and Methods. Dendrograms constructed following the weighted and unweighted pair group methods of Sneath and Sokal (1973), using centroid averages, are given in Bruce (1977); these dendrograms are similar to the one given in Figure 1, except for small value differences. Dendrograms obtained using arithmetic averages, or the Wagner tree method of Farris (1972), also give similar results except for the position of the Hylobatidae which appear further removed from the other hominoids. We have superimposed shading on the region of Figure 1 where the branching of man, the chimpanzee, the orangutan, and the gorilla are represented. This is done to indicate that the differences in genetic distance between these genera are too small to allow determination of their precise branching order. The amounts of genetic change between the branching nodes in the shaded region are smaller than the probable experimental error of the methods used. The phylogenetic inferences to be drawn from the figure are (1) that the hylobatid lineage diverged first from a lineage leading to humans and pongids, and (2) that the human and the three pongid lineages diverged from one another at about the same time, but without being able to specify the precise branching order of these four lineages. DISCUSSION Phylogeny of the H ominoids Informational macromolecules-i.e., nucleic acids and proteins-document evolutionary history. Degrees of similarity

9 1048 E. J. BRUCE AND F. J. AYALA.15.se -"':':":"'--Homo --""---- Gorillo Pan poniscus Pan troglodytes Pongo pygmoeus pygmaeus Pongo pygmoeus abettt Hylobates lor Hytobates concotor Symphalangus FIG. 1. A dendrogram based on the genetic distances among nine species and subspecies of hominoids, constructed using 16 species of Old World monkeys as a reference group. The numbers along the branches are the estimated average number of electrophoretically detectable allelic substitutions per locus that have taken place. The dendrogram may be interpreted as a phylogenetic tree if it is assumed that greater amounts of genetic differentiation indicate increasing degrees of phylogenetic separation. The shading over the area where man and the great apes branch off from each other indicates that the differences in genetic distance between the four genera are too small to be reliable. Thus, the electrophoretic data suggest that man, the chimpanzee, the gorilla, and the orangutan are about equally related to each other; but do not allow determination of their branching order. in such macromolecules reflect, on the whole, degrees of phylogenetic propinquity. Protein sequencing, immunology, electrophoresis, and nucleic acid annealing have in recent years provided phylogenetic information complementary to that obtained by such "classical" methods as comparative anatomy and the study of the fossil record. The word "complementary" in the previous sentence is used advisedly. Both macromolecular evolution and morphological evolution reflect the same evolutionary history. The information gained from the study of macromolecules must be weighed together with that derived from comparative anatomy and paleontology in order to ascertain phylogenetic relationships among organisms. Among the methods employed in the study of a single gene or protein, the most phylogenetic information is gained from the nucleotide sequencing of genes, a technique that has only very recently become available. The amino acid sequence provides the second highest amount of information per gene locus concerning phylogeny, followed successively by immunological methods that evaluate the number of amino acid differences between proteins, and by electrophoresis. Protein sequencing is a rather laborious technique, and thus only a limited number of proteins, if any at all, have usually been sequenced in any given organism or group of organisms. Such techniques as microcomplement fixation involve considerably less work, but require that antibodies specific for a given protein be developed. The advantage of electrophoresis is that a fairly large number of proteins can be studied with a moderate amount of cost and work. DNA hybridization of single-copy sequences is a method by which the overall amount of genetic divergence between living species can be estimated, although it requires obtaining radioactively labeled DNA from one or more reference species, a far from simple procedure. Electrophoretic estimates of genetic differentiation are useful when the organisms compared are evolutionarily closely related. The reason is that when a protein is found to be different in two organisms, it is not known how many amino acid differences exist between the organisms. Electrophoretic estimates of genetic differentiation are, in fact, based on the number of proteins that are identical in the organisms being compared. If it is assumed that amino acid substitutions in different proteins are randomly distributed, the number of proteins with none, one, two, etc. amino acid differences will have a Poisson distribution. The zero class (i.e., the proportion of identical proteins) of such distribution allows estimation of the mean number of substitutions per protein. Whenever two organisms are phylogenetically far removed, the proportion of identical proteins may be zero and then there is no way of estimating the mean number of amino acid differences per protein; if the proportion of identical proteins is very small, estimates can be obtained but they are potentially subject to large errors.

10 RELATIONSHIPS BETWEEN MAN AND APES 1049 Another difficulty is that not all amino acid substitutions result in differential electrophoretic mobility of the proteins. Thus electrophoretic methods underestimate the amount of genetic differentiation between organisms. (For discussions of this problem see, e.g., Lewontin, 1974; King and Wilson, 1975.) In the study of closely related organisms, the limitations pointed out may, however, be compensated by the study of many different proteins. The amino acid sequence of a protein contains much more information than its electrophoretic mobility, but the study of a single or very few protein sequences may give a distorted view of phylogenetic propinquity, because it is not unlikely that an occasional protein may be no more different in two closely related than in two more distantly related organisms. Needless to say, as stated above, all available information should be used in the reconstruction of evolutionary history. Figure 2 gives a simplified view of the phylogeny of the hominoids, according to different sources of evidence. Although not all scholars in any given discipline agree, the phylogenies given may be taken as representative of the prevailing views. The phylogenies given are based on the following works: A. Fossil record, Walker (1976); comparative anatomy, Tuttle (1975) and Simpson (1945). B. Immunology, Sarich and Cronin (1976); protein sequencing, a reconstruction based jointly in Romero-Herrera et al. (1973, 1976a, b) and Wooding and Doolittle (1972); DNA hybridization, Hoyer et al. (1972), Kohne et al. (1972). C. Immunology, Dene et al. (1976), Goodman (1976), Sarich and Wilson (17); DNA hybridization, Benveniste and Todaro (1976); chromosome banding, Miller (1977). D. Electrophoresis, present paper. One major discrepancy apparent in Figure 2 concerns the phylogenetic position of the orangutan (Pongo) relative to the other hominoids. The fossil record (Walker, 1976) and comparative anatomy (Simpson, 1945; Tuttle, 1975)-A in Figure 2-support an early divergence be- A. FOSSIL RECORD MORPHOLOGY B. IMMUNOLOGY PROTEIN SEQUENCING DNA HYBRIDIZATION C. IMMUNOLOGY DNA HYBRIDIZATION CHROMOSOME BANDING D. ELECTROPHORESIS & Homo Pon Gorilla Pango Hylab. ~ ~ Hom o Pan Gorilla Pongo Hylob. Homo Pan Gorilla _Pongo Hylob. c Homo Pan Gorilla _pongo ----Hylob. FIG. 2. Phylogeny of the hominoids according to various sources of evidence. The electrophoretic phylogeny (D) is a simplified version of the dendrogram in Fig. 1. The sources for the other phylogenies are given in the text. The dark zones covering the precise position of branching points reflect degrees of uncertainty concerning phylogenetic events. tween the lineage leading to the Hylobatidae (gibbons and siamang) and the great apes; a divergence which, according to Walker (1976) and Simons (1976), occurred about 27 million years ago or more. The lineage leading to Pongo would have split from the human, chimpanzee, and gorilla lineages much later (about 17 million years ago, according to the authors quoted). Immunological studies through microcomplement fixation (Sarich and Cronin, 1976), DNA annealing (Hoyer et al., 1972), and the amino acid sequences of globins (Romero-Herrera et al., 1973, 1976a, b) and fibrinopeptides (Wooding and Doolittle, 1972)-B in Figure 2-are, however, consistent with a phylogenetic split of the Hylobatidae, and Pongo lineages both at about the same time from the Gorilla-Pan-Homo lineage and from each other. Immunodiffusion with antiserum against whole plasma (Dene et al., 1976; Goodman, 1976) supports an intermediate phylogeny-the Hylobatidae lineage would have separated first from the pongid-human lineage, but the orangutan lineage would branch off from the lineage leading to the other great apes and man

11 1050 E. J. BRUCE AND F. J. AYALA._---- Homo ~""----Pan "'----- Gorilla ~-----Pongo Hytobates Symphalangus FIG. 3. A phylogeny of the hominoids proposed on the basis of all presently available evidence, molecular as well as morphological and paleontological. before these separate from each other-c in Figure 2. The recent DNA hybridization data of Beneviste and Todaro (1976) also support the phylogeny shown in Figure 2C. The banding patterns of the chromosomes (Miller, 1977) support a phylogeny similar, but not identical, to that shown in Figure 2C-the orangutan would branch from the man-chimpanzeegorilla lineage before these branch from each other, but much after the branching of the hylobatids; thus, the split of the Pongo lineage should be placed somewhat closer to the human-african apes lineage than appears in the figure. Our electrophoretic data, obtained from the study of 23 different proteins in nine hominoid taxa-figure 1 and D in Figure 2-support the phylogenetic placement of Pongo close to the African apes proposed by paleontologists and comparative anatomists, and disagree with the molecular phylogenies which place Pongo as distant from man and the African apes as the hylobatids. The average genetic distance between Pongo on the one side, and Homo, Pan, and Gorilla on the other is D = 0.343, which is not significantly different from the average distance between pairs of the latter three species (D = 0.367). However, in our opinion, the evidence cited together with very recent data on the amino acid sequence of orangutan hemoglobin (Maita et al., 1978), supports on the whole a phylogeny with the same topology as that shown in Figure 2C. We moreover believe that additional evidence will be required before deciding the approximate point at which the Pongo lineage splits from the human-african apes lineage. In our view, the overall available evidence favors a phylogeny with the topology shown in Figure 3, similar to the topology of Figure 2C, but with Pongo somewhat closer to the African apes and man. The phylogenies shown in Figure 2 also disagree concerning the phylogenetic propinquity of Gorilla, Pan, and Homo. Paleontologists and comparative anatomists propose a closer relationship between the gorilla and the chimpanzee than between these and man; the immunological and protein-sequence phylogenies data support an equal distance among the three taxa (Wilson et al., 1977); while the chromosome banding and (to a lesser extent) electrophoresis suggest a somewhat closer relationship between man and the gorilla than between these and the chimpanzee. However, it should be noted with respect to the electrophoretic data that the dendrogram in Figure 1 shows the Gorilla set apart from the lineage leading to Homo, Pan, and Pongo, by an increment of t:j) = 0.01 (or less than 3% of the total difference between any of these genera); and similarly the greater similarity between Pan and Pongo than between these and Homo amounts only to t:j) = These increments of genetic change are not significantly different from zero, and hence the four genera are effectively equidistant according to the electrophoretic data. With respect to paleontological data, the proposed greater similarity between the gorilla and the chimpanzee is hardly based on any substantial evidence: the last fossil ancestral remains of the Pan and Gorilla lineages are the Dryopithecines, between about 13 and 22 million years ago; but Dryopithecus is also the presumed last common ancestral stock to Homo, Pan, Gorilla and Pongo. It seems that the most reasonable conclusion is to state that, at present, the molecular and paleontological evidence is on the whole consistent with the notion that Gorilla, Pan, and Homo are approximately equally distant phylogenetically from each other. The chromosome banding data indicating a closer relationship between Gorilla and Homo

12 RELATIONSHIPS BETWEEN MAN AND APES 1051 arr not sufficiently weighty to depart from the phylogeny suggested by the molecular data. Genetic Divergence Man and the apes are presently classified into a single superfamily, Hominoidea, consisting of six extant genera grouped into three families: Hominidae (genus Homo), Pongidae (genera Pan, Gorilla, and Pongo), and Hylobatidae (genera Hylobates and Symphalangus). The basic morphological similarities between humans and apes were already recognized by Linnaeus in' his classification of living beings (first published in 1735) by placing them together in the order of primates (Anthropomorpha), although in separate genera. (No taxonomic category between "genus" and "order" was recognized by Linnaeus; the category "family" was introduced later.) Linnaeus would later muse (1778) that he perhaps should have classified men and apes in a single genus: "I demand of you, and of the whole world that you show me a generic character... by which to distinguish between man and ape. I myself most assuredly know of none. I wish somebody would indicate one to me. But, if I had called man an ape, or vice versa, I would have fallen under the ban of the ecclesiastics. It may be that as a naturalist I ought to have done so." The general view of today's taxonomists is, however, that Linnaeus was correct in his original classification, and hence they place humans and apes in different genera (and indeed in different families after the introduction of the "family" category). According to Simpson (13): "Homo is both anatomically and adaptively the most radically distinctive of all hominoids, divergent to a degree considered familial by all primatologists." Molecular studies lend some support to Linnaeus' perception that man and the apes may be biologically no more different than some congeneric species are. Figure 4 shows average genetic distances among taxa in different categories for various kinds of organisms. The non primate data SuBSPECIES SPECIES GENERA FAMILIES D'osoph,'. Fish 1 Liz cr ds Mammols* Orangutan Drosophila: siblings Drosoph no: non- sibl ings Starfish Fish+ Salamanders Lizards Mommals Gibbol'ls Chimpanzees Starfish Fish Salamanders Mammals* Pongids Hylobolids Hominid-Pongid Hominid-Hylobotid { Ponqrd- Hytobutf d a o > < >--+--< o o KH o 00 o GENETIC DISTANCE FIG. 4. Average genetic distance (circles) for various groups of organisms at various levels of taxonomic separation. The open circles are based on data given in the present paper. The solid circles are calculated from the data in Ayala (1975). The bars given indicate the range observed in the genetic distance values. *The values for "mammals" do not include primate data. tsome very closely related genera are included among the fish species. are taken from the review by Ayala (1975); the ranges and mean values (calculated as un weighted averages between mean values usually obtained by different investigators) are given for comparison with the hominoid data reported in this paper. The primate data involve an intersubspecific comparison, namely between Pongo pygmaeus pygmaeus and P. p. abe Iii, the Bornean and Sumatran orangutans. On the basis of 21 proteins, the genetic distance between these two subspecies is D = (/ = 0.878, Table 5), a value comparable to those found between subspecies in other organisms (see Fig. 4). One single locus, Ada, is fully diagnostic between the two orangutan subspecies; subspecies, even morphologically indistinguishable ones, are often but not always unambiguously distinguished by electrophoretically detectable genetic differences (Ayala, 1973; Ayala and Dobzhansky, 1974). Two comparisons between congeneric species are made in our study. The genetic

13 1052 E. J. BRUCE AND F. J. AYALA distance is D = between the chimpanzees, Pan troglodytes and P. paniscus, and D = between the gibbons, Hylobates lar and H. concolor (Table 5). These distances are at the lower end of the range of values found between congeneric speciesin other organisms, and are similar to typical distances found between subspecies (Fig. 4). Even within the hominoids, the distance between the two orangutan subspecies is equal to that between the two gibbons, and larger than the distance between the two chimpanzees. Two loci (Ak and 6-Pgd) out of 22 are fully diagnostic between the two chimpanzee species, and two (Hb-af3 and Alb) out of 21 "loci" studied are fully diagnostic between the two gibbons. The average genetic distance between the three pongid genera (Pan, Gorilla, Pongo) is jj = ± 0.071, and between the two hylobatid genera (Hylobates and Symphalangus) is even smaller, jj = ± These intergeneric differences are typical of the values observed for comparisons between congeneric species, rather than those typically found between confamilial genera (see Fig. 4 and Ayala, 1975). The same is true of the genetic distance between Homo and the three pongid genera, jj = ± 0.008; although this involves interfamilial comparisons, the genetic distance observed is within the typical range observed for comparisons between congeneric species in most types of organisms studied. The average genetic distances for the other two interfamilial comparisons (jj = for Homo versus the Hylobatidae and jj = for the Pongidae versus the Hylobatidae) are somewhat higher than most average values observed between congeneric species, but somewhat lower than the genetic distances observed between confamilial genera in other groups of organisms. If differentiation at structural gene loci as observed in electrophoretic studies would be used as the criterion for taxonomic classification, it would seem appropriate to classify men, chimpanzees, gorillas, and orangutans all in a single genus, and the hylobatids in a separate genus but in the same family as the pongids and humans. Dene et al. (1976), based on their immunodiffusion studies, have proposed a genealogical classification that places orangutans, gorillas, chimpanzees, and humans all in a single family, Hominidae, consisting of two subfamilies: Ponginae including only the genus Pengo, and Homininae consisting of the three genera Gorilla, Pan, and Homo. Our results would favor the classification of all four genera in a single family, rather than in two as they presently are classified, but they would not support the classification of Pongo is a separate subfamily. Indeed the electrophoretic genetic distance between Pongo and the other three genera is about the same as that observed between the latter. King and Wilson (1975) and others have argued that the evolution biologically most significant-i.e., affecting morphology, reproductive incompatibility, and behavior-may not be accurately reflected by changes in structural gene loci, because it involves primarily changes in genetic regulation. This is an intriguing, albeit as yet far from substantiated, hypothesis. The notable differences in morphology and behavior between humans and pongids are apparent, and this can hardly be exclusively attributed to the fact that humans are more likely to notice differences involving them. The question still remains whether the relatively small degree of genetic differentiation observed among hominoids warrants their reclassification into categories at lower level than those commonly used at present. We believe that taxonomists should take into account recent molecular information concerning differences between taxa. But the taxonomist must use all available evidence and not only the molecular data alone. The considerable morphological and behavioral differences between humans and pongids justify their classification into different families. The present study reports for the first time genetic differences at many loci among all extant hominoid genera. King

14 RELATIONSHIPS BETWEEN MAN AND APES 1053 and Wilson (1975) have previously compared men and chimpanzees (Pan troglodytes) at many loci in a study using electrophoretic methods, although protein sequence and immunological information were taken into account. They estimated the genetic distance between man and the chimpanzee, based on a sampleof 44 gene loci, as D = Based on a sample of 22 gene loci, we have observed a genetic distance of D = between Homo and Pan troglodytes. They called attention to the astonishing degree of genetic similarity between men and chimpanzees. This notable similarity is now confirmed; indeed our results at face value indicate even less genetic differentiation than theirs. Why are the two estimates of genetic distance between man and the chimpanzee so different? One obvious possibility is that different sets of loci are sampled in both studies. This however, is not the main reason. Of the 22 loci studied by us in both man and Pan troglodytes, 18 were also studied by King and Wilson (these correspond to 20 of their loci, since King and Wilson examined separately the a and f3 hemoglobin chains, and the a and f3 haptoglobin chains). We have calculated the average genetic similarity, S (Rogers, 1972; 5 is a different statistic from the genetic identity, I, used in our study, and one usually giving lower numerical values than the latter; their measure of genetic distance, D, is however the same as ours) for the 24 loci surveyed by King and Wilson but not included in our study. The value obtained is S = 0.57, a larger value than the average, S = 0.52, observed for all 44 loci. That is, if these 24 loci were removed, the average similarity for the remaining 20 loci (those also included in our study) would be slightly lower than the overall average, and consequently the genetic distance would be even greater than it is. (Indeed, we have calculated the average similarity for the common 20 loci using their data and obtained S = 0.46.) It might also be the case that the four loci (five if we include Fum, which was studied in Homo and P. paniscus but not in P. troglodytes) included in our study but not in King and Wilson's, are less differentiated than the rest. But such is not the case. The average similarity for these four loci (Cat, Icd-s, Aph-L, and Lap) is I = 0.50, i.e., lower than the average for all 22 loci in our study. If these four loci were removed from our calculations, the genetic identity would be slightly higher, and the genetic distance even lower, in our study than they presently are. The main reason why King and Wilson obtained a higher estimate of D than ours, is that different electrophoretic methods were used in the two studies. More specifically, King and Wilson used buffers and other conditions that, on the average, lead to more differences between man and the chimpanzee than ours do. This can be evinced as follows. At ten of our 18 loci also surveyed by King and Wilson, the results of both studies are effectively identical. Of the other eight loci, there are five (Got-s, Dia, Alb, Cer, and Est-A) at which our techniques showed the chimpanzee electrophoretic band to be the same as the common one in man, while their techniques manifested different bands; at one additional locus (Pgm-1), they observed a greater degree of differentiation than we did; at the remaining two loci (Ak and Hpt-af3) we scored greater differentiation than they did. At both the Ak and Hptaf3 "loci" we found different bands in Homo and Pan troglodytes; King and Wilson did not detect the difference at the Ak locus, and found the haptoglobin f3 chain to be identical but the a chain different. On balance, then, it turns out that the electrophoretic methods employed by King and Wilson on the average allow detection of greater number of differences than our methods. King and Wilson pointed out that the degree of genetic differentiation observed in their study is similar to that observed between sibling and other closely related congeneric species. It deserves notice that most of the studies of other organisms used by King and Wilson for comparison generally employ electrophoretic methods much more similar to ours than to those

15 1054 E. J. BRUCE AND F. J. AYALA of King and Wilson (in fact many of the studies come from our own laboratory). Thus, King and Wilson's emphasis on the comparatively low degree of genetic differentiation between man and chimpanzee is further justified: if they would have used the techniques typically employed in other electrophoretic studies, they likely would have observed a degree of genetic differentiation even smaller than they did. One final point concerning rates of evolution. Our data suggest that, on the average, rates of genetic evolution may have been greater in the Hylobatidae than in the lineages leading to man and the great apes. This can be seen in Figure 1. The dendrogram has been constructed by calculating average genetic distances to a reference group consisting of 16 species of Old World monkeys. Yet the average amount of genetic change per locus along each lineage is 0.32 for the man-pongid phylad, but 0.49 (or 50% higher) for the hylobatid phylad. Our data also suggest (although the differences are too small to be given much weight) that within the man-pongid phylad, genetic evolution has proceeded at a faster rate in the human lineage than in the pongid lineages. Since the last common ancestor of man and the pongids, the average amount of genetic change per locus is estimated as 0.20 for Homo, 0.18 for Gorilla, 0.16 for Pongo, and 0.14 for Pan. Paleontological evidence indicates that chimpanzees may be the most similar, and humans the least similar, to the last common ancestors of the human-pongid lineages (see Simons, 1976; Walker, 1976, and references therein). SUMMARY We have studied allelic variation at gene loci coding for 23 proteins in nine species and subspecies of hominoids. The organisms studied comprise the three families and the six genera of extant hominoids as follows. Family Hominidae: man (Homo sapiens). Family Pongidae: chimpanzee (two species, Pan troglodytes and P. paniscus), gorilla (Gorilla gorilla), and orangutan (two subspecies, Pongo pygmaeus pygmaeus and P. p. abelii). Family Hylobatidae: gibbon (two species, Hylobates lar and H. concolor) and siamang (Symphalangus syndactylus). The degree of genetic variation observed in the nonhuman hominoids is low; the expected heterozygosity is 2.3%, compared with a 6.7% for humans. Although the number of individuals studied is very small (59 if humans are excluded), it seems safe to conclude that at least the large apes are genetically less polymorphic than humans. The phylogeny inferred from the electrophoretic data shows man, the chimpanzee, the gorilla, and the orangutan about equally divergent from each other, but considerably separated from the siamang and the gibbon. Thus, our data are not consistent with phylogenies proposed by some molecular biologists showing the orangutan as being as different from man and the African apes as the gibbon and the siamang are. On the other hand, our data are not incompatible with phylogenies showing the orangutan as slightly more different from man and the African apes than these are from each other-a phylogeny which in our view is favored by the available information when all sources of evidence are taken into account. Concerning the phylogenetic relationships between humans, chimpanzees, and gorillas, the electrophoretic data agree with other molecular data indicating that these three genera are about equally related to each other. This is inconsistent with phylogenies proposed by paleontologists and comparative anatomists showing the gorilla and the chimpanzee as more closely related to each other than either one is to man. The degree of genetic differentiation among man, chimpanzee, gorilla, and orangutan is similar to that observed among congeneric species in other groups of organisms, although they involve comparisons between genera (chimpanzee-gorilla-orangutan) or between families (man versus any of the great apes). The genetic distance between the Hylobatidae and the

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