Molecular evidence on Primate phylogeny from DNA sequences

AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 94:3-24 (1994)

Molecular Evidence on Primate Phylogeny From DNA Sequences

MORRIS GOODMAN, WENDY J. BAILEY, KENJI HAYASAKA, MICHAEL J. STANHOPE. JERRY SLIGHTOM. AND JOHN CZELUSNIAK Denartments ofAnatomv and Cell Biolom (M.G., K.H., M.J.S., J.S.. J.C.) an2 Molecula; Biology and Genetics (WJ.B.1, Wayne State University School of Medicine, Detroit, Michigan 48201; Molecular Biology Unit 7242, The Upjohn Company, Kalamazoo, Michigan 49007 (J.S.)

KEY WORDS DNA sequence evidence, P-globin gene cluster, Hominoid classification, Maximum parsimony analysis

ABSTRACT Evidence from DNA sequences on the phylogenetic systematics of primates is congruent with the evidence from morphology in grouping Cercop- ithecoidea (Old World monkeys) and Hominoidea (apes and humans) into Catar- rhini, Catarrhini and Platyrrhini (ceboids or New World monkeys) into Anthro- poidea, Lemurifonnes and Lorisifonnes into Strepsirhini, and Anthropoidea, Tarsioidea, and Strepsirhini into Primates. With regard to the problematic relationships of Tarsioidea, DNA sequences group it with Anthropoidea into Hap- lorhini. In addition, the DNA evidence favors retaining Cheirogaleidae within Lemuriformes in contrast to some morphological studies that favor placing Cheirogaleids in Lorisifonnes. While parsimony analysis of the present DNA sequence data provides only modest support for Haplorhini as a monophyletic taxon, it provides very strong support €or Hominoidea, Catarrhini, Anthropoidea, and Strepsirhini as monophyletic taxa. The parsimony DNA evidence also rejects the hypothesis that megabats are the sister group of either Primates or Der- moptera (flying lemur) or a Primate-Dermoptera clade and instead strongly supports the monophyly of Chiroptera, with megabats grouping with microbats at considerable distance from Primates. In contrast to the confused morphological picture of sister group relationships within Hominoidea, orthologous noncoding DNA sequences (spanning alignments involving as many as 20,000 base positions) now provide by the parsimony criterion highly significant evidence for the sister group relationships defined by a cladistic classification that groups the lineages to all extant hominoids into family Hominidae, divides this ape family into subfamilies Hylobatinae (gibbons) and Homininae, divides Homininae into tribes Pongini (orangutans) and Hominini, and divides Hominini into subtribes Gorillina (gorillas) and Hominina (humans and chimpanzees). A likelihood analysis of the largest body of these noncoding orthologues and counts of putative synapomorphies using the full range of sequence data from mitochondrial and nuclear genomes also find that humans and chimpanzees share the longest common ancestry. 0 1994 Wiley-Liss, Inc.

It is estimated that there are l’-loo Received October 6,1992; accepted June 16,1993.

Wendy J. Bailey’s current address is Department of Biology,

Kenji Hayasaka’s current address is Department of Anatomy

lion living species inhabiting the earth (Ehr- lich and Wilson, 19911, of which about 223 are members of the order Primates (Nowak,

primates may seem insignificant, Homo sa-

thus, not surprisingly, the study of primate

0 1994 WILEY-LISS. INC

Yale University, New Haven, CT 06511.

1991). Although in numbers of species the

piens is the species Of the Order and

and Cell Biology, University of Michigan School ofMedicine, Ann Arbor, MI 48109.

Address reprint requests to Morris Goodman, Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI 48201.

4 M. GOODMAN ET AL.

phylogeny at the molecular level is a very active area of evolutionary research. The picture of primate phylogeny emerging from molecules is congruent in major features with the picture from morphology but has already provided sharper resolution of some of the blurred features in the morphological picture and has answered some of the phylogenetic questions that conflicting results from different morphological studies have failed to answer. The importance of these morphological studies with their long history should not be underestimated. They have provided our basic foundation of knowledge on primate phylogeny and have, therefore, defined the issues that could be profitably attacked at the molecular level of DNA sequences.

FROM GRADES TO CLADES IN PRIMATE SYSTEMATICS

Depending on the authors, the more than 200 primate species cluster into 60 or 65 genera (Fleagle, 1988; Nowak, 1991) and these genera into 13 or 15 families (Nowak, 1991; Fleagle, 1988). Both morphological and molecular studies agree that the clus- tering of these families to form superfami- lies yields six major monophyletic groups. They consist of Malagasy lemurs (Lemuroi- dea), Asian lorises and African galagos and pottos (Lorisoidea), Indonesian and Philip- pine tarsiers (Tarsioidea), New World monkeys and marmosets (Ceboidea), Old World monkeys (Cercopithecoidea), and apes and humans (Hominoidea) (Koop et al., 198913). Morphological and molecular studies also agree that cercopithecoids and hominoids are sister groups comprising infraorder Ca- tarrhini, which in turn groups with Platyr- rhini (the infraorder for ceboids) to form (either as a suborder or semisuborder) the more inclusive taxon Anthropoidea (Koop et al., 1989b). Similarly, both morphology and molecules depict lemuroids and lorisoids as sister groups comprising the suborder Strepsirhini. However the morphological picture is ambiguous with regards to whether the dwarf and mouse lemurs (family Cheirogaleidae) are cladistically closer to other Malagasy lemuroids or to the main- land lorisoids, with some recent morphological studies tending to favor the latter

(Schwartz, 1986; Fleagle, 1988). In contrast the molecular picture from nuclear DNA sequences (Bonner et al., 1980, 1981; Tagle et al., 1988; Koop et al., 1989b; Bailey et al., 1992b), and also from mitochondria1 (mt) DNA sequences (A. Yoder, this symposium), favor the former, i.e., cheirogaleids placed in Lemuroidea (or in Lemuriformes when the separation between lemuriforms and lorisiforms is drawn at the infraordinal level).

With regard to cladistic relationships among the major branches of Primates, the most uncertainty pertains to the placement of tarsioids. The results of morphological studies favor either a single trichotomous separation of strepsirhines, tarsioids, and anthropoids or two closely spaced dichotomous separations in which either tarsioids or strepsirhines are closest to anthropoids (Culotta, 1992). The view that tarsioids are cladistically closest to strepsirhines is also argued (Schwartz, 1986). In fact Schwartz groups tarsiers closest to lorisiforms. Thus Schwartz (1986) supports the commonly employed division of Primates into suborders Prosimii and Anthropoidea (Simpson, 1945; Fleagle, 1988; Martin, 1990), the former for the primates that appear to be more primi- tive in morphology, the lemuriforms, lorisiforms, and tarsiiforms, and the latter for the primates with enlarged brains and other derived or “advanced features of morphology, the ceboids, cercopithecoids, and hominoids. In contrast to Schwartz (1986), neither Simp- son (1945) nor Fleagle (1988) nor Martin (1990) claim that this division reflects cladistic relationships. Rather this division is thought to reflect grade relationships, i.e., relationships based on levels of morphological organization (Simpson, 1961; Fleagle, 1988; Martin, 1990). However, use of the grade concept in taxonomy can be criticized in that its definition of levels of organization is arbitrary and anthropocentric (e.g., in judgments of advanced) and retains para- phyletic assemblages even when current research shows how such assemblages can be replaced by monophyletic groupings, i.e., by a more consistent genealogical (cladistic) arrangement.

Ever since Darwin (1859) proposed that taxonomic classifications should group species according to their recency of common

DNA EVIDENCE ON PRIMATE PHYLOGENY 5

ancestry, i.e., their closeness genealogically, research in systematics has been increas- ingly concerned with identifying monophyletic groups or clades and with revising tra- ditional taxonomic classifications so that they reflect the cladistic hypotheses best supported by the data. Efforts to replace grades with clades have been accelerated by the growth of Willi Hennig‘s school of phylogenetic systematics (Hennig, 1966) or cladistics and by the proliferating studies in the rapidly developing field of molecular phylogenetics. One of the older proposed re- visions of the classification of major primate groups depicts the view, supported by cladistic evidence from a range of morphological characters, that tarsiers are the closest living relatives of anthropoids. This alternative classification divides Primates into Strepsirhini and Haplorhini, the former for lemuroids and lorisoids and the latter for tarsioids and anthropoids (Hill, 1953,1955). Amino acid sequence evidence from lens a A-crystallin and a and p hemoglobin chains supports the cladistic validity of dividing Primates into Strepsirhini and Haplorhini (deJong and Goodman, 1988; Czelusniak et al., 1990). As will be reviewed later in this paper, most of the molecular evidence from DNA sequences also supports the strepsirhine-haplorhine division of Primates.

Two other issues of primate systematics, with much older histories than the issue of the sister group of tarsiers, concern the boundaries of the order and the taxonomic placement of humans. The issue of the boundaries of Primates arose when Lin- naeus (1758) divided the mammals into eight orders and placed the genera Homo, Simia, Lemur, and Vespertilio in his first order, the one that he called Primates. As Linnaeus did not use intermediate taxonomic categories between the genus and the order, his genera tend to be equivalent in ranking to higher taxonomic groupings in modern classifications. Thus his genus Homo most resembles Simpson’s superfamily Hominoidea in that Linnaeus (1 758) grouped under Homo the species Homo sapiens for humans and the species Homo troglodytes apparently for man-like or anthropoid apes from Borneo and Africa. As the Simia of Linnaeus (1758) includes both ca-

tarrhine and platyrrhine monkeys, it resembles Simpson’s suborder Anthropoidea except that Simpson (1945) includes Homi- noidea in this suborder for simian or anthropoid primates. The genus Lemur of Lin- naeus (1758) is most like Simpson’s gradistic suborder Prosimii. Linnaeus placed three species in his genus Lemur, Le- mur catta from Madagascar, Lemur tardi- gradus from Ceylon, and Lemur volans from Asia. Clearly the first two species represent lemuriforms and lorisiforms, and the third species is the so-called flying lemur (actually a glider not a flier) that now has its own genus (Cynocephalus) in its own order (Der- moptera). However, the Lemur of Linnaeus, even with inclusion of flying lemur, still bears an uncanny resemblance to Simpson’s Prosimii. It does so because recent fossil evidence suggests that the extinct family Ple- siadapidae, which Simpson (1945) placed in Lemuriformes, has Cynocephalus (i.e., flying lemur) as its closest cladistic relative among extant mammals (Beard, 1990; Kay et al., 1990). Beard (1989) further argues, on the basis of his cladistic analysis, that the plesiadapiforms should be transferred from Primates to Dermoptera and that these two orders are sister taxa of a supraordinal clade, Primatamorpha.

All the species in Vespertilio, the fourth genus in the Primates of Linnaeus (1758), were bats. As early as the end of the 18th century, bats were given their own order Chiroptera. However the idea that bats are relatively close relatives of primates is still alive. One of the more persistent proposals for a supraordinal clade that contains Pri- mates places orders Primates, Scandentia (tree shrews), Dermoptera, and Chiroptera into a superorder called Archonta (Gregory, 1910; Szalay, 1977; Novacek, 1990). No- vacek (1990) depicts Dermoptera and Chi- roptera as closest relatives or sister taxa within Archonta, which conflicts with the view that the sister taxon of Dermoptera is Primates (Beard, 1989). The “flying primate” hypothesis of Pettigrew (1986, 1991) adds a further twist to such conflicting views from morphological studies concern- ing the phylogenetic relationships of primates, dermopterans, and bats. Pettigrew challenges the classical hypothesis of bat

M. GOODMAN ET AL. 6

monophyly which groups megabats (suborder Megachiroptera) with microbats (suborder Microchiroptera) within the order Chi- roptera. Instead Pettigrew’s hypothesis holds that true flight evolved twice in mammals, once in the early descent of microchi- ropterans and separately in the descent of megachiropterans after they diverged from a supposed common ancestor with primates and dermopterans.

Pettigrew (1986, 1991) found that the brains of primates, flying lemur, and megabats share certain characteristics of the vi- sual and motor neural pathways that are absent from microbats. Derived features of the glans penis are also found only in primates, flying lemur, and megabats (Smith and Madkour, 1980). Thus there is a set of morphological features that may be viewed as shared derived homologies or synapomorphies grouping megabats with primates and flying lemur, and there is another set of morphological features including wing structure, cranial vascular features, and fetal membranes (Baker et al., 1991) that are taken as synapomorphies grouping megabats with microbats in support of the classical hypothesis of bat monophyly. Clearly at least one of these two sets of morphological features is composed of false homologies or homoplasies, i.e., features that are similar in different species due to causes other than common ancestry such as selection for convergent adaptations. In contrast to these conflicting results from different sets of morphological features as to whether megabats share a more recent common ancestor with primates or with microbats, molecular studies are providing mounting evidence from DNA sequences that megabats are most closely related to microbats (Mindell et al., 1991; Adkins and Honeycutt, 1991; Bai- ley et al., 1992a; Stanhope et al., 1992; Am- merman and Hillis, 1992). Moreover, the molecular evidence does not place Chi- roptera in a clade close to Primates or Der- moptera, but while excluding Chiroptera from an archontan clade, does not reject the hypothesis of such a clade including Pri- mates, Dermoptera, and Scandentia.

Although the emerging molecular picture of primate phylogeny places bats at a far distance from primates, the molecular find-

ings show Linnaeus was exceptionally far sighted in grouping anthropoid apes (his Homo troglodytes) with humans. Other pre- evolutionary naturalists consigned a whole kingdom to humans and placed the human kingdom on a pedestal above the animals but not as high as the angels. Even as late as the 19th century, as reviewed by Darwin (1871) in his book The Descent ofMan, most naturalists gave humans an exclusive mammalian order, the Bimana. Thomas Huxley, as cited in Darwin (18711, brought Homo sapiens back into the order Primates but separated our species from other primates by placing it in a whole suborder reserved for humans alone. Darwin, who had pointed out that genealogical relationships provide the truly real basis for classifying species, was much bolder than Huxley. After reviewing the evidence from comparative anatomy and behavior that group humans with anthropoid apes, as a branch of the Catarrhines, Darwin (1871) concluded that from a genealogical point of view “man ought to form merely a Family or possibly even only a Sub- Family.” Now, near the end of the 20th century, the molecular evidence from DNA sequences supports a genealogical classification ;hat would at most separate the human species at the genus level from its near- est living ape relatives.

Simpson (1945) in his authoritative classification of mammals separated humans from apes at the family level, Hominidae for humans and Pongidae for apes, and then united these two families in the superfamily Hominoidea. Later, when studies of serum proteins (Goodman, 1963), hemoglobins (Zuckerkandl, 19631, and chromosomes (Klinger et al., 1963) showed humans and each of the African “great” apes (chimpanzees and gorillas) to be more closely related to one another than to the Asian apes (the “great” ape orangutan and the “lesser” ape gibbon), Simpson (1963) accepted this new evidence but still insisted on the validity of the formal classification of hominoids gradistically dividing Hominoidea into Pongidae for all apes and Hominidae for humans. However, Simpson’s hierarchical treatment of the apes themselves in fact does portray their cladistic relationships to one another. In a revision of his 1945 classi-


fication, he eliminated the genus Gorilla and placed gorillas with chimpanzees in the genus Pan and then, as in his 1945 classification, grouped the African apes with orangutan (Pongo) in the subfamily Ponginae and then grouped this great ape subfamily with the gibbon subfamily Hylobatinae to yield the ape family Pongidae (Simpson, 1963).

A large body of evidence, both morphological and molecular, has established that gorillas and chimpanzees are more closely related to each other than to orangutans but more closely related to orangutans than to gibbons. This evidence (reviewed in Cio- chon, 1983; Groves, 1986; Andrews, 1987; Goodman et al., 1989, 1990; Bailey et al., 1991, 199213) also established that humans should be included in the African ape clade. The molecular evidence provides especially strong support for the monophyletic grouping of humans, chimpanzees, and gorillas (Goodman et al., 1989, 1990; Bailey et al., 1991, 1992b). In contrast some parts of the morphological evidence suggest that Simp- son’s Ponginae is a truly monophyletic great ape clade (Schultz, 1963; Kluge, 1983) and some other parts group orangutans specifi- cally with humans (Schwartz, 1984), although it seems that a majority of the morphological evidence depicts a closer kinship of African apes to humans than to orangutans (Ciochon, 1983; Groves, 1986; An- drews, 1987). The issue is further confused in that Fleagle (1988) and Martin (1990), in their up to date authoritative works on primate evolutionary history, both agree that the combined morphological and molecular evidence clearly groups the African apes closest to humans rather than to orangutans; yet their classification of hominoids, like Simpson’s, places the African apes with Pongo in family Pongidae and places Homo in the separate family Hominidae. A cladistic classification, as we shall elaborate on later in this paper, would group all the living hominoids and those of their fossil relatives descended from their most recent common ancestor into the family Hominidae, would subdivide Hominidae into subfamily Hylo- batinae for gibbons and subfamily Homini- nae for the clade grouping orangutan, gorilla, chimpanzee and human lineages, and would then divide Homininae into tribe

Pongini for orangutans and tribe Hominini for gorillas, chimpanzees, and humans (Goodman, 1989).

With regard to the phylogenetic branching pattern of gorilla, chimpanzee, and human lineages, a good portion of the cladistic evidence from morphological studies depicts gorillas and chimpanzees as sharing the most recent common ancestor (Ciochon, 1983; Martin, 1986; Andrews, 1987) but another portion depicts humans and chimpanzees as sharing the most recent common ancestor (Groves, 1986; Andrews, 1987). In fact in the tabulation of morphological characters by Groves (1986) a majority are in favor of the human-chimpanzee clade, whereas in the tabulation by Andrews (1987) a majority are in favor of the gorilla- chimpanzee clade. The molecular evidence from DNA sequences, especially that accu- mulating in recent years, is much less equiv- ocal and is much more in favor of the human-chimpanzee clade than of the gorilla- chimpanzee clade.

The focus of our review will be on these recent DNA studies, of which some of the most extensive have been carried out by our- selves. Drawing upon the studies already published or in press, we shall summarize the main results and conclusions that deal with the major clades of primates and the overlapping issues of the sister group of primates and the diphyly or monophyly of bats. Then we shall review in some detail the DNA evidence on hominoid phylogeny. As our review will show, this evidence strongly supports the placement of chimpanzees and humans as the sister groups of subtribe Hominina in a cladistic classification of hominoids.

METHODS FOR PHYLOGENETIC ANALYSIS OF SEQUENCE DATA

The DNA studies that have provided the results for our review have employed three seemingly quite different methods for phylogenetic analysis of sequence data: parsimony, the neighbor-joining method (Saitou and Nei, 19871, and a maximum likelihood method based on a Markov chain model (Felsenstein, 1983). The maximum likelihood and neighbor-joining methods use explicit stochastic models to estimate phy-

8 M. GOODMAN ET AL.

logeny. The maximum likelihood method calculates a goodness of fit measure between observed and expected patterns of nucleotides on a tree and chooses the tree with the greatest agreement. The observed patterns refer to the distribution of patterns of the four nucleotides at homologous positions among the aligned contemporary DNA sequences. The expected patterns refer to what this distribution would be when made to conform to a stochastic model of nucleotide substitution on a tree in which the terminal taxa are the contemporary sequences. The likelihood value used to assess goodness of fit is similar to the chi-squared statistic commonly used in introductory genetics. Both measure the discrepancy between the data and a model. The tree chosen as the maximum likelihood tree for these aligned sequences has an 1nL value (in this case, the natural logarithm of L which is the probabil- ity of getting the expected patterns of nucleotides) that is closest to the 1nL value of the observed patterns. In being closest, this maximum likelihood tree will have a higher 1nL value than any of the other trees examined.

The neighbor-joining method assumes that all phylogenetic information can be obtained through maximum likelihood esti- mates of pairwise distances by a simple stochastic model of evolution. Such a model converts the observed proportion of differing nucleotides for each pair of aligned sequences into a pairwise distance “corrected for the superimposed substitutions pre- dicted by the model. If these distances are accurately estimated, they will be additive on the true phylogenetic tree, i.e., the distance between two contemporary sequences will be the sum of the lengths of the branches connecting the two sequences on the tree. If the assumed model of evolution is true and there are enough homologous positions in the dataset, then the maximum likelihood solution tree of this simple model and the neighbor-joining tree will be the same. The results from the neighbor-joining analyses by themselves are questionable since it is difficult to assess the number of positions needed to give accurate results.

Parsimony chooses as the best estimate of the true phylogeny that tree which requires

the least number of necessary changes. This choice is based on the assumption that simi- larities are due more to common ancestry than to convergence. Parsimony makes no explicit assumptions about stochastic models of evolution, although there are sugges- tions that it may implicitly make stochastic assumptions (Goldman, 1990). Two significance tests on parsimony trees with primates and bats are given later whose values should be taken as indications or trends rather than exact probabilities since they do not exclusively measure departure from phylogeny (J.C. unpublished data).

As indicated above, the first step in the phylogenetic analysis of DNA sequence date is to align homologous (i.e., evolutionarily related) sequences against one another in a manner that reveals the common ancestry shared by these sequences. In arranging the data sets of aligned sequences, molecular ev- olutionists have found it necessary to distin- guish between two types of homologous sequences, those that are orthologous and those that are paralogous. Orthologous sequences arise from speciation events, thus are found at corresponding genomic locations in different species and can reveal the common ancestry of these different species. Paralogous sequences arise from gene duplication events, thus are found at different genomic locations in the same species and at noncorresponding genomic locations in different species and can reveal the common ancestry of duplicated genes. (Here the term gene refers broadly to any DNA segment that can not only replicate during meiosis to give rise to daughter alleles but can also tandemly duplicate or multiply within a genome to give rise to related but nonallelic segments.) If the object of a DNA study is to deduce the cladistic relationships that exist among species, the deductions should be based on the phylogeny found for orthologous sequences.

P-GLOBIN GENE CLUSTER SEQUENCES: A SOURCE OF PHYLOGENETIC

INFORMATION Extensive comparative sequence data ex-

ist for the region in mammalian genomes called the p-globin gene cluster. The alignments of orthologous sequences in these


- e Y 9 b s

Eutherian ancestor

Fig. 1. Schematic representation of the gene loci in galago, lemur, tarsier, and human P-globin gene clusters. In each cluster and among clusters, the nonallelic loci (e,y,q,6,@) are paralogously related to one another. However, E sequences in the four species are orthologously related; this is also true for y sequences, for (~q sequences, for 6 sequences, and for p sequences.

data (Figs. 1-3) have provided evidence on primate phylogeny (Koop et al., 198913; Goodman et al., 1990; Bailey et al., 1991, 1992a,b) and also, with one of the alignments (that for eglobin gene sequences [Fig. 311, on the question of whether bats are monophyletic or instead whether megabats are flying primates (Bailey et al., 1992a). The aligned orthologues of these comparative sequence data represent not only Homi- noidea, Cercopithecoidea, Ceboidea, Tarsio- idea, Lorisiformes, and Lemuriformes (i.e., the six major primate groups), but also Scandentia, Dermoptera, Megachiroptera, Microchiroptera, Lagomorpha, and Artio- dactyla.

The genes of a primate or other mammalian P-globin gene cluster all trace back to a

single progenitor P-type globin gene that ex- isted in the ancestral therian mammals who lived 150-200 million years ago (MYA) (Efstratiadis et al., 1980; Czelusniak et al., 1982). Two paralogous loci arose from a tandem duplication of this progenitor p-globin gene, and by the time of the last common ancestor of eutherian and metatherian mammals (about 110-135 MAY), these two loci had differentiated into a proto-c gene expressed solely in embryonic life (the 5’ locus) and an ontogenetically later expressed proto+ gene (the 3’ locus) (Koop and Good- man, 1988). In the early eutherian mammals (about 85-100 MYA) further tandem duplications led to five loci (5’-e-y-~y&-p-3’) in which E,Y, and 7 originated from the proto-e locus while 6 and P originated from

10 M. GOODMAN ET AL.

HsaA

HsaB

Ptr

4 8

Ggo

PPY

Hla

MmU

Age Cd An

Tsy

Gcr

LfU

Cme

OCU

Chi

I - H H I I

I I I

I I __ ~

1 - I

I I I I

I I

M H H H

Fig. 2. Schematic representation of the P-globin gene cluster sequenced regions (y and $q loci and their flanking DNA) from two composite human alleles and 15 additional mammalian species that were the source of the three data sets of aligned orthologous sequences in the study of Bailey et al. (1992b). The horizontal bars to the right of the species codes span the regions sequenced for these species. To insure that only orthologous sequences were compared in reconstructing the phylogeny of the simian species lineages, Bailey et al. (1992b) removed from the alignment those sequence regions with a history of gene conversions. Those regions were the y1 and yz gene proper sequences of y1 and y2 as well as approximately 0.6 kb of 5’ flanking DNA for each gene of all the simian primates. Orthologous rabbit, galago, and tarsier sequences were aligned against the nonconverted noncoding flanking sequences of the simian y1 genes. Very little y gene flanking sequence was

the proto-@ locus (Goodman et al., 1984). The q-globin locus became a pseudogene (i.e., a silent nonfunctional gene) in primates and was deleted in rodents and lago- morphs. In turn the y-globin locus was deleted in artiodactyls, while it changed from an embryonically expressed gene to a fetally expressed one in the stem of the simian primates (Goodman et al., 1987; Tagle et al.,

H H H H

H

available for the brown lemur and dwarf lemur and thus, in a separate analysis designed to test the affinities of the dwarf lemur, Bailey et al. included y1 gene proper sequences for all the simian primates, tarsier, galago, brown lemur, dwarf lemur, and rabbit. Species codes are as follows: HsaA, HsaB, human (Homo sapiens) composite alleles A and B; Ptr, common chimpanzee (Pan trogolodytes), Ppa, pygmy chimpanzee (Pan panis- cm); Ggo, gorilla (Gorilla gorilla); Ppy, orangutan (Pongo pygmaeus); Hla, gibbon (Hylobates lar); Mmu, rhesus monkey (Macaca mulatta); Age, spider monkey (Ateles geoffroyi); Cal, capuchin monkey (Cebus albi- from); Atr, owl monkey (Aotus triuirigatus); Tsy, tarsier (Tarsier syrichta); Gcr, galago (Galago crassicaudatus); Lfu, brown lemur (Lemur fuluus); Cme, dwarf lemur (Cheirogaleus medius); Ocu, rabbit (Oryctolagus cunicu- lus); Chi, goat (Capra hircus).

1988); it also tandemly duplicated in this stem to give rise to the two paralogous loci (5’-y1-y2-3’) found in both simian in- fraorders (Platyrrhini and Catarrhini) of Anthropoidea (Fitch et al., 1991; Hayasaka et al., 1992a). This duplication involved a 5 kilobase (kb) long DNA segment containing the y-globin gene near its middle and two members of the primate L1 family of repeti-


K 4.1 kb >I

I & - Globin Locus ExNl EXNZ FxN3

I l { capuchin

1-1 Tarsier

k Galago

1-1 Lemur

H Flying Lemur

H Tree Shrew

Rabbit

H Megabat

H Microbat

1-1 Goat

Fig. 3. Schematic representation of the e-globin locus orthologues from mammals in the study of Bailey et al. (1992a). Scientific names for these mammals are as follows: flying lemur, Cynocephalus uariegatus; tree shrew, Tupaia glis; megabat, Cynopterus sphinx; microbat, Megaderma lyra; see legend to Figure 2 for scientific names of remaining mammals.

tive sequence elements, one member (Lla) at the 5' border and the other (Llb) at the 3' border of the 5 kb segment. An unequal homologous cross-over between Lla and Llb in misaligned chromatids then duplicated this 5 kb DNA segment (Fitch et al., 1991; Ha- yasaka et al., 1992a).

The size of the promoter and the transcriptional unit of each gene locus in a mam-

malian p-globin gene cluster is about 1,700 base pairs (bp) or 1.7 kb, of which the sequence encoding the globin polypeptide is distributed in three exons totaling 438 bp. Thus, about 1.3 kb of the gene is noncoding sequence distributed in introns 1 and 2, promoter, and 5' and 3' untranslated regions. As intron 2 (the intervening sequence between exons 2 and 3) is relatively large (in


the range of 850-900 bp), its functional sequence elements, such as those coding for RNA sites involved in the splicing of pre- messenger RNA, probably account for only a small percent of the total sequence. More- over, each gene is surrounded by extensive flanking and intergenic noncoding sequences. In simian primates, the p-globin gene cluster has the gene order 5’+y1-y2-(p q-643-3’ and spans about 50-60 kb, starting just 5‘ of the E locus and ending just 3’ of the p locus. The exons of the five functional genes (E, yl, y2, 6, p) account for 2,190 bp. The cis-acting regulatory sequence elements associated with the functional genes probably account for less than 1 kb of the 60 kb. Thus about 95% of this genomic region con- sists of noncoding DNA (intron, flanking, and mainly intergenic sequences) with no known sequence-specific functions. Such noncoding, nonfunctional DNA, compared to coding DNA, accumulates sequence changes that escape both the constraining effects of purifying selection and the directional effects of positive selection. Therefore this noncoding DNA offers advantages in resolving cladistic relationships over the short to moderate phylogenetic distances found within Primates. Such noncoding DNA not only evolves more rapidly than coding DNA but also its shared changes in descending lineages more frequently reflect changes shared through common ancestry rather than through selected convergences.

In their cladistic study, Bailey et al. (1991) sequenced the cpq-globin locus and its flanking DNA (an 8.4 kb segment of noncoding DNA) from the common gibbon (Hylobates lar) and also completed sequencing the orthologous DNA from galago (Galago crassicaudatus), a 3.3 kb segment that was trun- cated by a deletion in the ancestry of the galago genome that removed most of the 3’ end of the cpq locus and most of the intergenic sequence between cpq and 6 loci (Tagle et al., 1992). Bailey et al. (1991) then updated a previously established data set of orthologous noncoding sequences spanning about 12.5 kb of aligned nucleotide positions representing the cpq-globin locus and its flanking DNA (Miyamoto et al., 1988; Good- man et al., 1989; Koop et al., 1989b) and aligned the gibbon and galago sequences

along with the published orthologous sequences from ten other species. These sequences represented two pairs of four human alleles (a 5’ pair and a 3’ pair), four apes (chimpanzee, gorilla, orangutan, and gibbon), an Old World monkey (rhesus monkey), two New World monkeys (spider and owl monkeys), tarsier, two strepsirhines (galago and brown lemur), and goat. The owl monkey, tarsier, and goat sequences spanned only the cpq locus proper and small stretches of 5’ and 3‘ DNA, and thus spanned only about 2 kb of the alignment, whereas most of the anthropoid sequences spanned the full 12.5 kb alignment. The lemur sequence at only about 0.8 kb was the shortest. It spanned only the 5’ half of the cpq locus; its 3’ half had been deleted by an unequal homologous cross-over between cpq and 6 of misaligned chromatids during the evolutionary history of the lemur lineage (Harris et al., 1984). Recently, Bailey et al. (199213) updated this data set of aligned cpq and flanking noncoding sequences by addition of 4.9 kb of sequence from a pygmy chimpanzee (Pan paniscus) and 6.4 kb of sequence from a third New World monkey (a capuchin monkey, Cebus albi- from), the latter sequenced by Hayasaka et al. (1992b).

Bailey et al. (1992b) also established a data set of orthologous noncoding sequences representing up to 8.3 kb of aligned nucleotide positions flanking the y-globin genes for orangutan, gorilla, common chimpanzee, pygmy chimpanzee, and two human alleles. These sequences were also aligned against gibbon (Fitch et al., 19911, rhesus (Fitch et al., 19911, spider and capuchin monkeys (Fitch et al., 1991; Hayasaka et al., 1992a,c), tarsier (Hayasaka et al., 1992~1, galago (Tagle et al., 19921, and rabbit (Margot et al., 1989). The full sequenced region spanning the duplicated y1 and y2 loci was more than 11 kb in length and required an alignment of close to 13 kb of nucleotide positions to ac- commodate the gaps in different sequences. However, in data sets in which homologous y1 and y2 sequences were aligned against one another, portions of these paralogues were shown to have a history of gene conversions in all the platyrrhine and catarrhine lineages that were examined; these portions


spanned the gene proper (promoter and transcriptional unit) of each y locus and typ- ically about 0.6 kb of 5’ flanking DNA (Fitch et al., 1990, 1991; Hayasaka et al., 1992a; Bailey et al., 1992b). As the gene conversions were not always in the same direction and not always as frequent or as extensive in some species lineages and in some of the subregions of homology as in other species lineages and subregions, segments of the aligned y2 sequences might actually represent y1 sequence in some of the species and conversely segments of the aligned y1 sequences might represent y2 sequence in some of the species. Thus, Bailey et al. (1992b) removed from the alignment those portions with a history of gene conversions in the simian lineages in order to ensure that only orthologous sequences would be compared in reconstructing the phylogeny of the simian species lineages. The pygmy chimpanzee insert in the lambda clone that was sequenced (Ppa-Ch35-14) extended from the 3’ end of exon 2 of y’ to 1.3 kb 3‘ of the cpq poly-A site (Bailey et al., 199213). Thus in each of the two data sets of noncoding sequences (the set containing nonconverted flanking and intergenic sequences from the duplicated y loci and the set spanning the cpq locus and its flanking sequences), the pygmy chimpanzee was not as fully represented as the other catarrhines. Also in the data set of noncoding and nonconverted y orthologues, rabbit, galago, and tarsier with their single rather than duplicated y loci were represented only over half the full 5’-y1-y2-3’ alignment of the simian sequences; the nonsimian sequences were aligned against the 5’ and 3’ nonconverted flanking sequences of the simian y1 genes. The published y-globin gene sequences of brown lemur and dwarf lemur (Harris et al., 1986) contained so little of the flanking sequences that they did not warrant inclusion in the data set of nonconverted, noncoding y sequences. Instead, to address the question of whether the dwarf lemur is a lemuriform or a lorisiform, Bailey et al. (1992b) established a data set of y sequences that spanned primarily the gene locus proper (promoter and transcriptional unit). This data set included the gene proper y1 sequences from simians and the gene proper y

sequences from tarsier, galago, brown lemur, dwarf lemur, and rabbit.

Bailey et al. (1992a) used e-globin gene sequences to investigate whether bats have monophyletic or diphyletic origins and whether megabats, if not all bats, are close cladistic relatives of primates. The data set consisted of aligned orthologues from 17 species: 11 primates, tree shrew (Tupaia glis), flying lemur (Cynocephalus uariegatus), a megabat (Cynopterus sphinx), a microbat (Megaderma lyra), rabbit, and goat. The full alignment spanned 4.3 kb of aligned nucleotide positions extending from 2.2 kb 5’ of the initiation codon to about 0.2 kb 3’ of the polyadenylation site. Six sequences (human, orangutan, gibbon, capuchin, galago, and rabbit), each about 4.1 kb in length, spanned the full alignment; seven sequences (common chimpanzee, pygmy chimpanzee, gorilla, rhesus, tarsier, lemur, and goat), each about 1.8 kb in length, spanned all or most of the region starting upstream in the proximal promoter and ending a short distance downstream of the polyadenylation site; and four sequences (tree shrew, flying lemur, megabat, and microbat), each about 1.2 kb in length, spanned the region starting at the 3‘ end of exon 1 and ending at the 5’ end of exon 3. Thus, the e-globin sequences that provided the most insight on the question of bat monophyly or diphyly spanned intron 1, exon 2, and intron 2 and therefore consisted mainly of noncoding DNA.

PHYLOGENETIC RELATIONS FROM SUPERFAMILIAL TO SUPERORDINAL

LEVELS The most parsimonious (MP) trees found

by Bailey et al. (1992a)b) for their data sets of aligned orthologous sequences (Fig. 2, 3) yielded highly congruent results. These MP trees divided the primates into a strepsirhine branch and a haplorhine branch, the haplorhines into a tarsier branch and a simian or anthropoid branch, the simians into a platyrrhine or ceboid branch and a catarrhine branch, and the catarrhines into a cer- copithecoid branch and a hominoid branch. The parsimony procedure used in this work to find an MP or lowest length tree also found the series of trees that broke up the clades in the MP tree with minimal addi-


tions of length over the MP length; in such a series the weakest supported clades broke first, and the strongest supported clades broke last. These strength of grouping results provided strong evidence for the cladistic validity of Hominoidea, Catarrhini, Ce- boidea, Anthropoidea, and Strepsirhini. On putting together the results from the four data sets (E, y, nonconverted y, cpq), a mini- mum of 138 and 333 additional sequence changes over those in the MP trees were required to break up Catarrhini and Anthro- poidea, respectively. For the three data sets on which strength of grouping results were obtained on Hominoidea and its subdivi- sions (E, nonconverted y, cpq), it took 115 additional sequence changes to break up the monophyly of Hominoidea. For the two data sets in which Ceboidea was represented by more than one genus (nonconverted y, cpq), it took 97 additional sequence changes to break up this ceboid clade. For the three data sets in which both galago and lemur represented Strepsirhini (E, y, qq), it took 70 additional sequence changes to break up this strepsirhine clade.

Even though tarsier, simians, and strepsirhines were represented in all four data sets (E, y, nonconverted y, cpq), haplorhines received the weakest support from the strength of grouping results. It took only 20 additional sequence changes to break up the monophyly of Haplorhini. Nevertheless the MP tree for each of the four data sets did group tarsier with Anthropoidea. Moreover the MP trees for data sets of 6- and p-globin gene sequences also supported the division of primates into strepsirhines and the haplorhine clade of tarsier and simians (Koop et al., 1989a,b). Comparisons in primates and other mammals of nucleotide sequence data for a coding region of the involucrin gene consisting of tandem repeats further corrob- orate the strepsirhine-haplorhine division of primates (Djian and Green, 1991). In addition, the DNA-DNA hybridization results of Bonner et al. (1980) grouped tarsier with the simians. On the other hand, Hayasaka et al. (1988) analyzed by the neighbor-joining method (Saitou and Nei, 1987) a data set of orthologous DNA sequences from a range of primates for a 0.9 kb region of the mt genome and obtained a neighbor-joining tree

that when rooted by the midpoint of the longest path depicted tarsier as closer to lemur than to simians. However, when Ha- yasaka et al. (1988) included mouse and cow orthologues in their phylogenetic analysis, they were not able to support the monophyly of Primates, and they concluded that mtDNA sequences (known to evolve at a much faster rate than nuclear DNA sequences) are poorly suited for resolving phylogenetic relationships among very an- ciently separated primate branches.

In agreement with the earlier results of Tagle et al. (1988) and Koop et al. (1989b1, the results of Bailey et al. (1992b) for y-globin gene proper sequences provided strong evidence that Cheirogaleidae (as represented by the dwarf lemur, Cheirogaleus medius) is a lemuriform not a lorisiform. In the MP tree for these y gene proper sequences, dwarf lemur and brown lemur (Le- mur fuluus) first joined each other and then their common stem joined the lorisiform lineage to galago. It took 63 additional sequence changes over those in the MP tree to break up the lemuriform clade of Cheirogaleus and Lemur. The DNA-DNA hybridization data of Bonner et al. (1981) also strongly supported the taxonomic placement of Cheirogaleidae in Lemuri- formes. The DNA sequence divergence values (measured in AT50H units) between Le- mur and each of the cheriogaleid genera (Cheirogaleus and Microcebus) were just half the divergence between lemuriform and lorisiform branches. With labeled Lemur DNA, the divergence values for comparisons with lemuriform DNAs from Lemur mongoz, Lemur catta, Varecia, Propithecus, Cheirogaleus, and Microcebus were 0, 6, 7, 10,12, and 12, respectively, and for comparisons with lorisiform DNAs from Galago, Nycticebus, and Loris were 22, 23, and 23, respectively (Bonner et al:, 1980, 1981). With labeled galago DNA, the divergence values for comparisons with lorisiform DNAs from Galago, Nycticebus, and Loris were 0, 11.5, and 11.3, respectively, and for comparisons with lemuriform DNAs from Loris mongoz, Loris catta, Propithecus, and Microcebus were 22.0, 22.4, 22.0, and 25.0, respectively (Bonner et al., 1980, 1981). Fi- nally, with labeled slow loris (Nycticebus)


c chimp

p. chimp

human

gorilla

orangutan

gibbon

rhesus

capuchin

tarsier

Len&-3614

galago

lemur

tree shrew

rabbit

flying lemur megaht

microht

goat

Fig. 4. Maximum parsimony tree with strength of grouping results for E-globin gene sequences, (Reproduced from Bailey et al., 1992a, with permission of the publisher.)

DNAs, the divergence values for comparisons with lorisiform DNAs from Nycticebus, Loris, and Galago were 0,6, and 10, respectively, and for comparisons with lemuriform DNAs from Loris catta, Varecia, Propithe- cus, Cheirogaleus, and Microcebus were 22, 23, 22, 24, and 24, respectively (Bonner et al., 1981; T. Bonner, unpublished data). The fact that these DNA-DNA hybridization comparisons involved a wide range of strepsirhine genera strengthens the conclusion that cheirogaleids are lemuriforms not lorisiforms.

The MP tree with strength of grouping results for E-globin gene sequences (Bailey et al., 1992a) is reproduced here in Figure 4. The two bats representing Megachiroptera and Microchiroptera form a clade that is as strongly supported as the strepsirhine clade of galago and lemur. It took 31 additional

sequence changes over those in the MP tree to break up Chiroptera and 37 to break up Strepsirhini. While the strength of grouping results indicate that tree shrew, rabbit, and flying lemur may be close relatives of the primates, they also indicate that the bats like the goat are distant relatives. It took 23 additional sequence changes to break up the group that included primates, tree shrew, rabbit, and flying lemur, but excluded bats and goats. Moreover the lowest scoring tree that tested the hypothesis of a Chiroptera- Dermoptera clade joined to a Primates- Scandentia clade to form the superorder Ar- chonta (Novacek, 1990) still required 52 additional sequence changes over those in the MP tree; also each of the lowest scoring trees that grouped primates, megabat, and flying lemur together in testing the flying primate or bat diphyly hypothesis of Petti-


grew (1986, 1991) required more than 100 additional sequence changes over those in the MP tree (Bailey et al., 1992a). A “win- ning sites” test provided further evidence for bat monophyly in that the number of positions (39) with synapomorphic (i.e., shared derived) sequence characters supporting bat monophyly was significantly greater (P = 0.000045) than the number (11) supporting the grouping of megabat with goat which was the arrangement that broke up Chiroptera with the least addition of sequence changes. Again, this test should not be taken as evidence of a conclusive statistical falsification but only a qualitative indi- cation that parsimony supports this group strongly. All but one of the 39 bat synapomorphic positions were located in the intron noncoding sequences of the E-globin gene. Thus selected convergences could not account for the high level of shared sequence similarity between megabat and microbat E-globin genes.

Several other recent DNA sequence studies have also focused on the issue of whether megabats are more closely related to primates and flying lemur than to microbats (Mindell et al., 1991; Adkins and Honeycutt, 1991; Stanhope et al., 1992; Ammerman and Hillis, 1992), and the results of each of these studies supported the monophyletic grouping of megabats with microbats. Stanhope et al. (1992) compared orthologues from a megabat (Pteropus hypomelanus), two microbats (Tonatia bidens and Tonatia silvi- cola), three primates (human, tarsier, galago), tree shrew, flying lemur, rabbit, mouse, cow, cat, and opossum for a 1 kb portion of exon 1 of the nuclear gene encoding interphotoreceptor retinoid binding protein (IRBP). Pteropus grouped with Tonatia to form the only well-supported clade in the MP tree for these IRBP nucleotide sequences. As the majority of the synapomorphic sequence characters that favored bat monophyly were synonymous substitutions, the relatively high similarity between Ptero- pus and Tonatia IRBP sequences could not be attributed to selected convergences but more plausibly was due to shared common ancestry. Regardless of the category of sequence changes examined (only transitions, only transversions, only nonsynonymous

substitutions, only synonymous substitutions), the MP solution invariably favored grouping megabats with microbats. As with eglobin gene sequences, trees that tested Novacek‘s Archonta hypothesis and Petti- grew’s flying primate hypotheses with IRBP sequences were very unparsimonious (Stan- hope et al., 1992). This same trend of results was also true in studies with mtDNA sequences and different representatives of Megachiroptera and Microchiroptera.

In a study employing portions of the mt 12s ribosomal RNA and cytochrome oxidase I genes, the MP solutions grouped the megabat Pteropus capestratus with the microbat Brachyphylla cavernarum rather than with human or mouse (the other mammals employed) but only when transversions were accorded more weight than transitions (Mindell et al., 1991). Ammerman and Hillis (1992) also used the mt 12s ribosomal RNA gene to test whether bats are monophyletic or diphyletic. Although they sequenced a smaller portion of the gene than did Mindell et al. (19911, they included in their analyses a wider range of mammals: two megabats (Pteropus scapulatus, Penthetor lucasi), two microbats (Tadarida brasiliensis, Glosso- phaga soricina), flying lemur, tree shrew, orangutan, mouse, cow, armadillo, and ko- ala. Their parsimony analyses indicated that bats were monophyletic and that flying lemur and three shrew were more closely related to primates than to bats. Adkins and Honeycutt (1991) tested Novacek’s Archonta hypothesis and Pettigrew’s flying primate hypotheses with mitochondria1 cytochrome oxidase I1 gene sequences from a megabat (Rousettus leschenaulti), a microbat (Phyl- lostomus hastatus), flying lemur, tree shrew, galago, human, mouse, cow, and armadillo. Their parsimony results strongly rejected these hypotheses but supported the grouping of flying lemur and tree shrew with primates and, when transversions were given more weight than transitions, the grouping of megabat with microbat in favor of a monophyletic Chiroptera.

CLADISTIC RELATIONSHIPS WITHIN HOMlNOlDEA

A very clear picture of the branching pattern of hominoid clades is provided by non-

DNA EVIDENCE ON PRIMATE PHYLOGENY

0.61

17

- HsaB 0.12

I

Fig. 5. A primate phylogenetic tree with the most likely branching arrangement for hominoids. This tree was constructed for noncoding nucleotide sequences from the nonconverted y and qq regions of the p-globin gene cluster. The tree’s InL (logarithm of likelihood) score, -58576, was calculated using a model similar to that of dnaml in PHYLIP (version 3.3). The numbers on the branches represent, as percent sequence divergence, amounts of nucleotide change between ancestral and descendant nodes. Terminal taxa are designated by ab- breviated species names of the primates represented by the contemporary sequences: Hsa A and HsaB for hu-

0.1 4

0.34 Ppa

coding P-globin gene cluster sequences in the nonconverted y and cpq data sets of Bai- ley et al. (1992b). Parsimony analysis of these noncoding sequences yielded for both data sets the same branching pattern. The

0.94

man (Homo sapiens) composite alleles A and B, Ppa for pygmy chimpanzee (Pan paniscus), Ptr for common chimpanzee (Pan troglodytes), Ggo for gorilla (Gorilla gorilla), Ppy for orangutan (Pongo pygmaeus), Hla for gibbon (Hylobates lar), Mmu for rhesus monkey (Macaca mulatta), Cal for capuchin monkey (Cebus albi- f rom) , Age for spider monkey (Ateles geoffroyi), Tsy for tarsier (Tarsius syrichta), and Gcr for galago (Galago crassicaudatus). The abscissa in units of millions of years ago places the nodes of the tree on a time scale inferred primarily from fossil evidence on extinct primates.

- Ptr 0.40 0.33

two MP trees depicted horninoid branching events that, on progressing from the most ancient to most recent, separated subfamily Hylobatinae (the lineage to gibbon or HyZo- bates Zar, Hla) from Homininae, tribe

0.52 0.84 Ggo

18 M. GOODMAN ET AL

human

chimp

gorilla

-33.1 , ppc orang

gibbon

Fig. 6. Strength of grouping by the likelihood criterion for the human-chimpanzee (H,C) and human-chimpanzee-gorilla (H,C,G) clades in the phylogenetic tree shown in Figure 1. A circled number on the stem of a clade is the difference in InL score between the tree of highest likelihood and the tree with highest score that does not contain that clade, i.e., does not contain (H,C) or (H,C,G). The three trees with the highest scores vary only in their branching arrangement within the (H,C,G) clade. The differences in 1nL between the tree of highest likelihood and itself and each of the two alternatives are listed in the bottom half of the figure; that the decreases in likelihood of these two alternatives are significant is also indicated. The standard deviations (S.D.) and significance levels were calculated using the assumptions of Felsenstein’s dnaml program, and are large sample normal approximations which may or may not be true on this data set, and should not be taken as conclusive.

Pongini (the lineage to orangutan or Pongo pygmaeus, Ppy) from Hominini, subtribe Gorillina (the lineage to Gorillagorilla, Ggo) from Hominina, genus Pan (chimpanzees) from Homo (humans), species Pan troglodytes (Ptr, common chimpanzees) from Pan paniscus (Ppa, pygmy chimpanzees), and human composite allele HsaA from HsaB. As the sequence data on humans involved three pairs of six alleles (each pair in a different region of the aligned sequences), a composite of three of the six alleles (one from each pair) was designated HsaA and a composite of the other three alleles was designated HsaB.

These monophyletic taxa or hominoid clades are well supported by strength of grouping results from the parsimony analysis of Bailey et al. (1992b) on their two data sets of noncoding orthologues (nonconverted y, cpq). This analysis required calculating the lengths of the trees that broke up the MP clades in the MP tree with minimal addi- tions of length over the MP length. The strongest support is for tribe Hominini (the clade of gorilla, human, and chimpanzee lineages); 145 extra sequence changes over those in the MP solution were required to break up this clade. The weakest support is for subtribe Hominina (the clade of human and chimpanzee lineages); nevertheless 18 extra sequence changes over those in the MP solution were required to break up Homin- ina. In the two sets of aligned noncoding orthologues, there are 25 derived sequence changes shared by human and chimpanzee sequences but not by any of the other hominoid sequences (Bailey et al., 1992b). In contrast there are only seven sequence changes shared by chimpanzee and gorilla sequences and only four shared by gorilla and human sequences. The statistical test of Williams and Goodman (1989) applied to these results shows that the human-chimpanzee clade is supported at approximately the 0.002% level of significance. This test should not be interpreted as being conclusive but merely indicative. Clearly these sequence data favor the sister grouping of humans and chimpanzees, i.e., the sequence characters grouping Homo and Pan can be attributed-at least a majority of these characters-to a period of shared common ancestry after the Gorilla divergence, whereas the sequence characters favoring the two alternative groupings must then be viewed as resulting from parallel or convergent changes that arose independently between gorilla and chimpanzee lineages and between gorilla and human lineages.

We further tested the significance of the sister grouping of chimpanzees with humans by these noncoding DNA orthologues by combining in an extended sequence alignment the nonconverted y and cp-q data sets for primates represented in both data sets, and then, using a computer program based on a model patterned after that of the dnaml

DNA EVIDENCE ON PRIMATE PHYLOGENY

TABLE 1. Numbers of putative synapomorphic nucleotide sequence positions supporting alternative human-chimpanzee (H,C!, chimpanzee-gorilla (C,G!, and human-gorilla (H,G! clades

19

Mitochondria1 genomic region'

12s rRNA 0.9 kb fragment' 4.8 kb fragment3

Total Nuclear

genomic region Immunoglobulin C,, pseudogene Involucrin 28SrRNA, ITS1 X,Y, pseudoboundary* al, 3GT Immunoglobulin Cal S-p intergenic P Nonconverted y Vl5

c

Total

Shared (kb)

0.9 0.9 4.8 6.6 -

2.4 1.2 3.5 1.2 0.4 2.6 3.9 1.3 1.7 7.1

10.1 35.4 -

Number of positions (H,C) (C,G) (H,G)

5 5 5 10 10 4

28 33 57 72 48 37 ~ - -

3 0

13 3 2 4 6 1 1

15 10 58 -

0 5 4 2 0 3 2 1 1 4 3

25 -

1 0 2 2 0 2 2 1 0 1 3

14 -

Source of aligned sequences

Hixson and Brown (1986) Hayasaka et al. (1988) Horai et al. (1992)

Ueda et al. (1989) Djian and Green (1989, 1990) Gonzalez et al. (1990) Ellis et al. (1990) Galili and Swanson (1991) Kawamura et al. (1991) Perrin-Pecontal et al. (1992) Perrin-Pecontal et al. (1992) Bailey et al. (1992a) Bailey et al. (199213) Bailey et al. (1992b)

'The location in the rnt genome of each sequenced region providing the synapomorphic information is shown in Figure 7. %is fragment contains genes for tRNA"", tRNAser, tRNALeU, and parts of ND4 and ND5. a n i s fra tRNAuC$%NAhP, tRNA'Y', and portions of ND1 and ATPase 6. 4A copy of these aligned sequences was provided to us by Nathan Eltis. 5The 3' 3/1 kb portion of these sequences is just upstream of the 8-globin gene; of these 3' sequences, the human was determined by Maeda et al. (1983) and the chimpanzee, gorilla, orangutan, rhesus monkey and spider monkey by Maeda et al. (1988).

ent contains enes for ND2, COI, COII, ATPase 8, tRNA'Ie, tRNAG'", tRNAMet , tRNAT'P, tRNAN", tRNA*'", tRNACYS, tRNAQ',

program in Felsenstein's Phylogenetic In- ference Package (PHYLIP, version 3.31, we calculated the 1nL scores for a series of trees that tested the monophyly of Hominina (human-chimpanzee clade). The tree shown in Figure 5 has the same branching pattern as the MP tree for these sequences and has a 1nL score of -58576. In calculating the 1nL scores of alternative trees, we restricted our analysis to the 15 possible trees formed by the four branches of Homininae genera (Homo, Pan, Gorilla, Pongo) and the out- group branch within which the positions of Hylobates, Macaca, ceboids, tarsier, and galago were fixed according to the arrangement in Figure 5. The tree with least difference in 1nL score that broke up Hominina or human-chimpanzee clade (H,C) did so by grouping chimpanzee with gorilla (C,G) at a score of -58609. The other tree that main- tained Hominini (H,C,G) but broke up the (H,C) clade did so by grouping human with gorilla (H,G) at score -58615. As summa- rized in Figure 6, these two alternatives- (C,G) and (H,G)-are much less likely and significantly worse than the tree with the (H,C) clade. The tree that broke Hominini or the (H,C,G) clade with least difference in

score did so by exchanging positions of gorilla and orangutan, i.e., by having a (((H,C)O)G) clade, at score -59073. The huge decrease in likelihood (-497) demon- strates that these sequence data ovenvhelm- ingly support the monophyletic grouping of humans, chimpanzees, and gorillas.

Several other recent studies at the DNA sequence level have provided corroborating evidence for an African ape-human clade with orangutan as its sister group and with gibbon as the sister group of this great ape- human clade. Some of the corroborating evidence was obtained by cross hybridization of total single copy DNAs from the nuclear genomes of the species being compared, i.e., by DNA-DNA hybridization comparisons (Sib- ley and Ahlquist, 1987; Caccone and Powell, 1989; Sibley et al., 1990). Other corroborating evidence was obtained from nucleotide sequence data representing the involucrin gene (Djian and Green, 1990) and the previously mentioned 0.9 kb region of the mt genome (Hayasaka et al., 1988). Corroborating evidence for humans and chimpanzees showing the longest common ancestry, i.e., for an (H,C) clade that has the gorilla lineage as its sister group, has also been ob-


Horai et al. (1992)

ND1

ND

Hayasaka et al (1988) I

I ATPase 6 ATPase 8

Fig. 7. A map of the mammalian mt genome, locat- ing the sequenced areas pertinent to our discussion of the human, chimpanzee, and gorilla clade. The outer circle indicates those genes transcribed from the heavy- strand DNA and inner circle indicates those genes transcribed from the light-strand DNA. 12s and 16s desig-

nate genes for ribosomal RNAs; genes for tRNAs are indicated as solid black circles; ND1-6 are genes for components for the NADH respiratory chain; CO1-3 are genes for cytochrome oxidase subunits; ATPase 6 and 8 are genes for subunits of the mt ATPase complex; and cyt. b is the gene for cytochrome b.

tained from the DNA-DNA hybridization studies (Sibley and Ahlquist, 1987; Caccone and Powell, 1989; Sibley et al., 1990) and from nucleotide sequence studies on the S-P intergenic region of the P-globin gene cluster (Perrin-Pecontal et al., 19921, the immunoglobulin Ce3 pseudogene (Ueda et al., 1989), 28s ribosomal genes (Gonzalez et al., 1990), the gene for a-l,3-galactosyltransferase (Galili and Swanson, 1991), the cytochrome oxidase I1 locus of the mt genome (Ruvolo et al., 1991; Horai et al., 1992), and 16 other mt genes (Horai et al., 1992). Of the nucleotide sequence studies concerned with the branching pattern within the African ape- human clade, only the study of the involu-

crin locus yielded opposing results (Djian and Green, 1989, 1990); the involucrin coding region of short sequence repeats has the chimpanzee and gorilla genes more closely related than either is to the human gene. In addition sequence data from some of the studies favored a trichotomy or very poorly supported one or another alternative; these sequence data represented the p-globin gene (Perrin-Pecontal et al., 19921, eglobin gene (Bailey et al., 1992a), short stretches of sex- specific and pseudoautosomal regions of X and Y chromosomes (Ellis et al., 1990), immunoglobulin Cal gene (Kawamura et al., 1991), the 0.9 kb region of the mt genome (Hayasaka et al., 19881, and the mitochon-

21 DNA EVIDENCE ON PRIMATE PHYLOGENY

drial gene for small (12s) ribosomal RNA (Hixson and Brown, 1986).

We have examined position by position the sequence characters of the aligned nucleotide sequences in the above studies concerned with the branching pattern within the (H,C,G) clade and have determined the number of putative synapomorphic positions that supports each of the alternative branching patterns for this clade. It can be seen from the results of this analysis (Table 1; Fig. 7) that for both mt and nuclear DNA sequences many more putative synapomorphic positions support the (H,C) clade over either the (C,G) or (H,G) clade. With mtDNA sequences the number of positions (72) supporting (H,C) is about 1.5 times greater than the number (48) supporting (C,G) and about 1.9 times greater than the number (37) supporting (H,G). With nuclear DNA sequences the number of positions (58) supporting (H,C) is about 2.3 times greater than the number (25) supporting (C,G) and about 4.1 times greater than the number (14) supporting (H,G). That the support for (H,C) provided by nuclear DNA sequences is stronger than that provided by mtDNA sequences may be due to a lower level of homoplasy among hominoids in their nuclear DNA orthologues than in their mtDNA orthologues. Nevertheless some homoplasy is still evi- dent in the nuclear DNA sequences, but this homoplasy does not seem large enough to argue for the separation of human, chimpanzee, and gorilla lineages from their common ancestor in a single trichotomous branching event. Rather we interpret the overall DNA sequence evidence as indicative of two separate dichotomous branching events that occurred close in time to one another such as depicted in Figure 2. To use the names of taxa in the cladistic classification (Goodman, 1989) that we advocate, the first branching event within Tribe Hominini separated subtribe Gorillina (gorilla lineage) from Hominina and the second branching event (that within Hominina) separated Pan (chimpanzee lineage) from Homo (human lineage).

ACKNOWLEDGMENTS We thank Dr. Alessandro Minelli for pro-

viding us with copies of those pages in the

Tenth Edition of Linnaeus’ Systema Natu- rae that deal with the classification of the order Primates. Assistance of Maria Iracilda C . Sampaio and Elaine Krobock in the prep- aration of this manuscript is appreciated. This research was supported by NSF grants BSR-9007056 and DEB 9116098 and NIH grant HL 33940.

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Molecular evidence on Primate phylogeny from DNA sequences