PHYLOGENY OF PRIMATES AND OTHER EUTHERIAN ORDERS: A CLADISTIC ANALYSIS USING AMINO ACID AND NUCLEOTIDE SEQUENCE DATA MORRIS GOODMAN’. *. 3, JOHN CZELUSNIAK’, *, AND JUDITH E. BEEBER’, 3 ‘Depadmmt of Amlomy, Wayne Stafe Universily Schwl ofMedicine, Lktmit M I 48201 ZDepartmmt of Biologics[ Scamces, Wayne State Uniuersav, Detroit M I 48202 3Depadmmt o f Anthmpolop, Wayne State Uniumiy2 Delroit M I 48202 Abstract - Genealogical reconstructions carried out by the parsimony method on protein amino acid and DNA nucleotide sequenre data are providing fresh evidence on cladistic branching patterns at taxonomic levels from the classes of Vertebrata and orders of Eutheria to the genera of Hominoidea. Minimum length trees constructed from amino acid sequence data group Mammalia with Archosauria (i.e., Aves plus Crocodilia), Arnniota with Amphibia, and Tetrapoda with Teleostei. Within Mammalia, Edentata and Paenungulata (e.g., Proboscidea) appear as the mast anciently se p a t e d from othereutherians. Another superordinaleutherian clade consists of Artiodactyla, Cetacea, and Peridactyla. A third consistently contains Primates, Lagomorpha, and Epaia. The cladistic positions of such orders as Carnivora, Chiroptera, Insectivora, and Rodentia are not well resolved by the currently still sparse body of sequence data. However, recent dramatic progress in the technology of gene doning and nudeotide sequencing has opened the way for so enlarging the body of sequence data that it should become possible to solve almost any problem concerning the phylogenetic systematics of extant mammals. An example is provided by hominoid genera. Minimum length t m constmcted from mitochondrial DNA nudeotide sequence data very strongly group fin, Homo, and Gorilla into Homininae and then join Homininae and Ponginae (Bngo) into Hominidae as the sister family of Hylobatidae (HyLobafa). Resolution of the hominine trichotomy into two dichotomous branchings should be forthcoming as kilobase sequencing of nuclear genes progresses. Introduction In a masterful cladistic analysis of the paleontological and comparative anatomical evidence on higher eutherian phylogeny, Novacek (1982) found this evidence currently incapable of converting the phylogenetic “bush”traditionally portrayed for the 15 or so extant eutherian orders into a consistently bifurcating tree. Orders such as Primates and Chiroptera illustrate the problem. Some of the evidence reviewed by Novacek (e.g., that of McKenna, 1975)would group Primates, Scandentia (Tupaioidea or tree shrews), Dermoptera (flying lemur), and Chiroptera (bts) into a superordinal clade Archonta Other evidence (that favored by Novacek himself) would keep Chiroptera with Dermoptera, while grouping Primates with lipotyphlous Insectivora (soricoid shrews, moles, and hedgehogs) and then have these two putative superordinal clades plus three to four others and Scandentia all diverge simultaneously from the same ancestral node. Furthermore, Novacek noted (but rejected) the cladistic hypothesis of Smith (1976) of a diphyletic Chiroptera: Megachiroptera in Archonta (with Primates, Scandentia, and Dermoptera) and Microchiroptera grouped with lipotyphlous Insectivora. Clearly, when other equally conflicting views on the cladistic positions of the remaining eutherian orders (Pholidota, Edentata, Carnivora, etc.) are also considered, only a bush could be drawn to depict the consensus of cladistic arrangements proposed for eutherian phylogeny from the traditional paleontological and comparative anatomical evidence. A new source of evidence which ultimately may resolve such “bushes” comes from the developing molecular biological analysis of phylogeny, in particular from genealogical reconstructions carried out by the parsimony method on protein amino acid sequence data and on DNA nucleotide sequence data. Indeed these data have already helped produce a more accurate picture of primate phylogeny and also information for working out the course of branching of the i nfda s s Eutheria into major monophyletic clades. The present paper deals with these cladistic results and indicates some promising avenues of research in continuing the search for the correct inter- and intia-ordinal branching pattern of eutherian mammals.
Transcript
PHYLOGENY OF PRIMATES AND OTHER EUTHERIAN ORDERS: A CLADISTIC ANALYSIS USING AMINO ACID
AND NUCLEOTIDE SEQUENCE DATA
MORRIS GOODMAN’. *. 3, JOHN CZELUSNIAK’, *, AND JUDITH E. BEEBER’, 3
‘Depadmmt of Amlomy, Wayne Stafe Universily Schwl ofMedicine, Lktmit M I 48201 ZDepartmmt of Biologics[ Scamces, Wayne State Uniuersav, Detroit M I 48202
3Depadmmt of Anthmpolop, Wayne State Uniumiy2 Delroit M I 48202
Abstract - Genealogical reconstructions carried out by the parsimony method on protein amino acid and DNA nucleotide sequenre data are providing fresh evidence on cladistic branching patterns at taxonomic levels from the classes of Vertebrata and orders of Eutheria to the genera of Hominoidea. Minimum length trees constructed from amino acid sequence data group Mammalia with Archosauria (i.e., Aves plus Crocodilia), Arnniota with Amphibia, and Tetrapoda with Teleostei. Within Mammalia, Edentata and Paenungulata (e.g., Proboscidea) appear as the mast anciently sepated from othereutherians. Another superordinal eutherian clade consists of Artiodactyla, Cetacea, and Peridactyla. A third consistently contains Primates, Lagomorpha, and Epaia. The cladistic positions of such orders as Carnivora, Chiroptera, Insectivora, and Rodentia are not well resolved by the currently still sparse body of sequence data. However, recent dramatic progress in the technology of gene doning and nudeotide sequencing has opened the way for so enlarging the body of sequence data that it should become possible to solve almost any problem concerning the phylogenetic systematics of extant mammals. An example is provided by hominoid genera. Minimum length t m constmcted from mitochondrial DNA nudeotide sequence data very strongly group fin, Homo, and Gorilla into Homininae and then join Homininae and Ponginae (Bngo) into Hominidae as the sister family of Hylobatidae (HyLobafa). Resolution of the hominine trichotomy into two dichotomous branchings should be forthcoming as kilobase sequencing of nuclear genes progresses.
Introduction
In a masterful cladistic analysis of the paleontological and comparative anatomical evidence on higher eutherian phylogeny, Novacek (1982) found this evidence currently incapable of converting the phylogenetic “bush” traditionally portrayed for the 15 or so extant eutherian orders into a consistently bifurcating tree. Orders such as Primates and Chiroptera illustrate the problem. Some of the evidence reviewed by Novacek (e.g., that of McKenna, 1975) would group Primates, Scandentia (Tupaioidea or tree shrews), Dermoptera (flying lemur), and Chiroptera (bts) into a superordinal clade Archonta Other evidence (that favored by Novacek himself) would keep Chiroptera with Dermoptera, while grouping Primates with lipotyphlous Insectivora (soricoid shrews, moles, and hedgehogs) and then have these two putative superordinal clades plus three to four others and Scandentia all diverge simultaneously from the same ancestral node. Furthermore, Novacek noted (but rejected) the cladistic hypothesis of Smith (1976) of a diphyletic Chiroptera: Megachiroptera in Archonta (with Primates, Scandentia, and Dermoptera) and Microchiroptera grouped with lipotyphlous Insectivora. Clearly, when other equally conflicting views on the cladistic positions of the remaining eutherian orders (Pholidota, Edentata, Carnivora, etc.) are also considered, only a bush could be drawn to depict the consensus of cladistic arrangements proposed for eutherian phylogeny from the traditional paleontological and comparative anatomical evidence. A new source of evidence which ultimately may resolve such “bushes” comes from the developing molecular biological analysis of phylogeny, in particular from genealogical reconstructions carried out by the parsimony method on protein amino acid sequence data and on DNA nucleotide sequence data. Indeed these data have already helped produce a more accurate picture of primate phylogeny and also information for working out the course of branching of the infdass Eutheria into major monophyletic clades. The present paper deals with these cladistic results and indicates some promising avenues of research in continuing the search for the correct inter- and intia-ordinal branching pattern of eutherian mammals.
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Procedure
The maximum parsimony method (Farris, 1970; Fitch, 1971; Moore et al., 1973; Goodman, 1981) accounts for evolutionary descent of amino acid sequences by minimizing homoplasy, i.e., by seeking a genealogical arrangement which maximizes genetic likenesses associated with common ancestry, while minimizing incidences of parallel and back mutations. This is done by using the genetic code to represent amino acid sequences as mRNA sequences and by then seeking a tree with a minimum number of nucleotide replacements, or lowest NR length. Since common ancestry, as opposed to convergent evolution, is most likely responsible for extensive matches of nucleotide sequences between species, the tree reconstruction approach based on maximum parsimony is a sensible means of arriving at a preferred genealogical hypothesis.
The amino acid sequence data for the parsimonious genealogical reconstructions came from 94 cytochromes c, 46 lens alpha crystallins, 47 combined fibrinopeptides A and B sequences, 21 carbonic anhydrases, 46 members of the protein family containing calmodulin, 321 globins, ofwhich those from the jawed vertebrates subdivide into myoglobin, ahemoglobin, and @-hemoglobin, and 36 ribonucleases. The vast majority of sequences (587 of 664) and a majority of species (209 of 273) are from vertebrates. The vertebrate class best represented by the sequences is the Mammalia (156 species), in particular its subclass Eutheria (148 species of 16 orders). The best sampled eutherian order is Primates (38 species). Catalogs of most of these amino acid sequences and the species from which they originated are given for ribo- nucleases in Bientema and Lenstra (1982) and for the remaining protein chains in Goodman (1981) and Goodman et al. (1982). Table 1 lists, for the eleven kinds of protein chains most widely sequenced in mammals, the orders of Mammalia represented.
Table 1 Protein sequences utilized in the study of ordinal Mammalian relationships: a = a-hemoglobin; 0 = 0- hemoglobin; m = myoglobin; I = lens a-crystallin A; P = fibrinopeptide A ; P = fibrinopeptide B ; c = cytochrome c; CaI = carbonic anhydrase I ; CaII =carbonic anhydrase 11; CaIII = carbonic anhydrase
111; r = ribonuclease.
EUTHERIA Primates x x x x x x x x x x Lagomorpha x x x x x x x x x Tupaioidea x x x x Rodentia x x x x x x X Insectivora x x x x
Carnivora x x x x x x x Pholidota X
Cetacea x x X X
Edentata x x X X Proboscidea x x x x x Sirenia X
Tubulidentata x x
Chiroptera x x X
Perissodactyla x x x x x x x x x X
Artiodactyla x x x x x x x x x x x
Hyracoidea X
METATHERIA
PROTOTHERIA Marsupialia x x x x x x x
Monotremata x x x
X
X
19851 PHYLOGENY OF PRIMATES 173
A genealogical reconstruction by our maximum parsimony method, modified for use with nucleotide sequences (Czelusniak et al., 1982), was also carried out on nucleotide sequence data from 44 hemoglobin genes and pseudogenes of 10 species (9 vertebrates and 1 plant). This reconstruction focused on the amino acid encoding regions (exons) of the genes and corresponding regions of the pseudogenes. Further maximum parsimony trees wre constructed for the nucleotide sequence data on corresponding sections of the mitochondrial (mt) DNA of hominoid genera (Brown et al., 1982), mouse (Bibb et al., 1981), and cow (Anderson et al., 1982).
Two kinds of most parsimonious genealogical trees were constructed: gem phylogaies for separate collections of sequences (each collection consisting only of evolutionarily related sequences, such as hemoglobin genes and pseudogenes); and speciesphylogenies from sequences of separate collections combined in extended tandem alignments as though encoded by giant genes, such combinations being analogous to multicharacter sets in morphology. While the sequence fiie for a gene phylogeny can have orthologous sequences aligned against paralogous sequences, the sequence file for a species phylogeny should have only orthologous sequences aligned against other orthologous sequences. Orthologous sequences represent gene-lineages which split from common ancestors simultaneously with separation of the species-lineages, whereas paralogous sequences represent gene-lineages which arose from gene duplication prior to species separation (Fitch, 1970).
It has been found (Goodman et al., 1979; Maeda and Fitch, 1981) that different proteins can yield lowest NR length trees that are non-concordant with respect to the same taxa (i.e., in these cases each such putative gene phylogeny can disagree with features of the species phylogeny supported by evidence from other proteins). This could be due to real differences in branching arrangements between gene and species phylogenies or, instead, indicate incorrect groupings of sequences that happen to have excesses of convergent residues causing these sequences to be represented as sharing a more recent common ancestor than they actually do. Or, as another possibility, the most parsimonious tree for those data sets may not yet have been found. In order to construct more accurate genealogical trees, the parsimony criterion was expanded to encompass not only NRs, but also those categories of genic changes which can account for a different branching order of the lineages in a putative gene phylogeny (e.g., a tree of lowest NR length) from the branching order of the species-lineages. As discussed in detail elsewhere (Goodman et al., 1979; Goodman, 1981; Goodman et al., 1982), these categories are gene duplications (GDs) and deletions and regulatory mutations affecting gene expression (i.e., gene expression events or GEs). By utilizingthis expanded parsimony criterion, in which the aim is to minimize the sum of NRs+GDs+GEs, we can then construct more accurate genealogies. GDs and GEs may be viewed as “extra-curricular genetic events” because the maximum parsimony programs employed in the repetitive computer searches for trees of lower length only count NRs.
When the computer search revealed a range of lowest length trees, consensus branching arrangements were constructed following the procedure of Adams (1972) and in some cases the procedure of Penny et al. (1982) as well.
Cladistic Findings
Evidence on branching patterns in higher level eutherian phylogeny was obtained from the lowest NR length trees found for 64 extant vertebrate taxa represented by up to 7 polypeptide chains combined in an extended tandem alignment. Each taxon was represented in the align- ment by at least two polypeptide chains and usually by three or four, but because of gaps in the available sequence data only 10 species could be represented by all seven chains. Figures 1 and 2 depict the Adams consensus tree derived from the trees of lowest NR length, each at 2811 NRs, of which 13 were found. The Adams consensus tree shows 7 disagreements with traditional evidence on monophyletic groupings in vertebrate phylogeny. Three of the 7 dis-
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agreements, however, are absent from the dichotomous 2811 NR tree (one of the 13 at lowest NR length) shown in Figure 3, which in contrast to the consensus tree groups duck with geese and swan, llama with camel, and hedgehog with mole and musk shrew; thus, this tree shows 4 rather than 7 disagreements. Despite these 4 remaining disagreements which we shall shortly single out, it may be observed that in the 2811 NR trees (Figs. 1-3) Mammalia unites with Archosauria (i.e., Aves plus Cmodilia), Amniota with Amphibia, and Tetrapoda with Teleostei. This pattern of branching among major vertebrate clades accords well with traditional evi- dence on vertebrate phylogeny. The disagreements with traditional evidence arise because the Therians (marsupials and placentals) fail to originate as a monophyletic group; instead, the monotremes occupy a less remote position than marsupials and are even less remote than the eutherian orders Proboscidea and Edentata. Also, within Eutheria artiodactyls do not emerge as a monophyletic group; instead, bovids are closest to equines, camelids to Cetacea, whereas Sus is the most distant from these lineages. Nevertheless the main features of the 2811 NR trees are also present in the trees of lowest NR+GD+GE length constructed under the expanded parsimony criterion. It may especially be emphasized that the remote divergence of Proboscidea and Edentata holds, in spite of the surrounding marsupials and monotremes; rabbits stay next to primates, whether immediately joined by Tupaiu or not; Cetacea remains with ungulates; and the Insectivora-Chiroptera branch consistently joins Carnivora. Since our “bat” (Chiroptera) is a hybrid taxon represented by a megachiropteran for myoglobin, a micro- chiropteran for lens a-crystallin A, and yet a different microchiropteran for cytochrome c, we are precluded from investigating the question of the monophyletic versus diphyletic origin of Chiroptera. However, once orthologously related proteins are sequenced in several or more species of these two suborders, such species will be treated as separate taxa in future reconstructions of mammalian phylogeny.
Aside from the broad cladistic pattern of vertebrate and mammalian branches noted above, other noteworthy features in the 2811 NR trees which do not change, even when the expanded parsimony criterion is applied, concern the primate branch. Within Primates, Strepsirhini (lorises and lemurs) separates from Haplorhini (tarsier and Anthropoidea). Within Anthro- poidea, Platyrrhini (marmosets and new world monkeys) separates from Catarrhini. The latter divides into Cercopithecoidea (Old World monkeys) and Hominoidea and within Hominoidea the two most closely related genera are Homo and Pun. This human-chimpanzee branch is joined by gorilla to form the genealogical subfamily Homininae (Goodman and Moore, 1971). Thus the two African ape genera are cladistically closer to Homo than to the Asiatic ape branches: Ponginae (Bngo or orangutan) and Hylobatinae (Hyhkztes or gibbon).
At least 18 extra-curricular genetic events (4 GDs + 14 GEs) are required for the 281 1 NR tree shown in Figure 3 if we choose not to challenge the substantial traditional evidence for the monophyletic origin of therian mammals and, later in descent, the monophyletic origin of artiodactyls. In other words, this tree then has a total length of 2829 genic events (281 1 NRs + 4GDs + 14GEs). Certain trees which do not oppose such well substantiated monophyletic groupings prove more parsimonious (under the expanded criterion) than the 281 1 NR trees. Indeed trees were found with scores of 2819 NRs but requiring no postulation of extracurricular genetic events. By the expanded parsimony criterion, these 2819 NR trees are in fact the lowest length trees which we found. Trees almost as parsimonious, requiring 2820 and 2821 NRs but no GDs and GEs, were also found. From the range of these most- and nearly-most parsimonious trees a dichotomous consensus tree was chosen by the procedure of Penny et al. (1982), i.e., a tree whose branching features were the ones repeated most often in the afore- mentioned range of trees. The mammalian portion of this “majority” consensus tree is shown in Figure 4, and salient features of the main most-parsimonious (the 2819
apmlf*fBc aBmlf*fBc a p m l - - - a8 - I f* - c ap---- C a8 - I f*fBc
a8----- a p m l - - - - -ml - - c a Bml f*fBc --mlf* - c aBmlf* - - aB-lf*fB- a p m l f*fBc a B - If*fBc aB--f*fBc --ml - - c aBm f*fB-
a p m l PfBc aBm f*fBc aBmlPfBc aBm-- -- aBm---- a B - l - - - -@mlf*fB- a Bml f*fBc a p m l - - - aB-- - - C
ap-----
--- --
aB--- - -
a p m l - - c SHARK
Fig. 1. Adams consensus tree derived from the 13 trees of lowest NR length (each requiring 281 1 NRs) found for 64 taxa using a tandem alignment of up to 7 polypeptide chains: branching arrangement of all taxa except for subbranching of Primates. The ordinate time scale in millions of years before the present (MyBP) was arrived at following procedures described in Goodman (1981). Each disagreement with a generally accepted grouping is indicated by a heavy dot at a taxon misplaced from the branch it is thought to belong to. The source of sequences and species for 49 of the 64 taxa are obtainable from Goodman (1981). Of the 15 additional taxa, 8 involve hemoglobin sequences not referenced in either Goodman (198l), Goodman et al. (1982), or Czelusniak et al. (1982), or in references cited therein. These hemoglobin sequences are from the following mammals and birds: Musk shrewlSuncus rnurinur (Maita et al., 1981); European molelTulpa curopaca (Kleinschmidt et al., 1981); ZebralEquus zebra and Wild asslEquus hemionus kufan (Mazur and Braunitzer, 1982); Armadjllolhjpus nourmcinctus (Kleinschmidt et al., 1982); Canada gooselBranta caMdmris and Mute swanlCygnus olor (Oberthiir et al., 1982); PheasantlPlasianus colchichus colchirus (Braunitzer and Godovac, 1982). Edentata is represented by armadillo hemoglobin sequences and by two-toed sloth lens a-crystallin A.
176 CLADISTICS [VOL. 1
a B m I f A f e c
a f3 m-fA f e c
af3m-fAfB-
aBm-fAfe-
af3m-fAfB-
a @ m - - - -
a@ - - f A f e -
a p m - - - -
a p m l f A f B c
a @ - - - - -
as - -f*fB-
a p m - - - - - Bm- - - -
a@m-fAfBc
a@m-fAfBc
a @ m - - - - a p - - - - -
0 50 100 (MyBP) I
C H I M PAN Z EE
ORANGUTAN
GIBBON TANDEM ALIGNMENT OF :
a HEMOGLOBIN (a)
MYOGLOBIN (m) LENS a CRYSTALLIN A ( I 1 FlBRlNOPEPTlDE A (1” )
PATAS FlBRlNOPEPTlDE B (1’) CYTOCHROME C ( C 1
LANGUR B HEMOGLOBIN ( 8 ) VERVET
R MACAQUE
J. MACAQUE
MANGABEY
BABOON
SAIMIRI
CEBUS
ATE L I N A E
MARMOSET
a8 - - - - - SLENDER LORIS
aBmI fA fBc SLOW LORIS
a p m l - - - LEMUROIDEA
Fig. 2 . Subbranching of Primates for the Adams tree shown in Figure 1.
NR length) trees of the range from which the “majority” tree was chosen are depicted in Figure 5 . The Adams tree for this data is shown in Figure 6; it depicts a similar arrangement.
Since almost all questions on cladistic relationships among orders of eutherian mammals have been wide open, any or all arrangements of bifurcations among the stem-lines descending to eutherian orders could be accepted without postulation of extra-curricular genetic events. Thus the Penny and Adam consensus trees and the range they represent (Figs. 4-6) provide further parsimony evidence on the ordinal groupings into larger
apmIfAfBc aBmI fA fBc a B m l - - - a@- I f A - c ap- - - - C
ap- IfAfBc a@--- - - a@--- - - a @ m l - - - - - m l - - c a@mlf*fBc - -ml fA - c a @ m l f * - - a B - - - - - aB-11418- a a m l f* fBc a B - I fAfBc aB- - f * f@c - - m l - - c a B m f*fB-
apmlP fBc a e m f*fBc a @m I f*fBc aBm- - -- a @ m - - -- ae-l --- -@mlf*fB- af3mlf*fBc a e m l --- a B - - - - C
a@--- - -
-- - -- are----- CANADAGOOSE a B - - - - - MUTE SWAN ore-- - - C OSTRICH a B m l f A - c CHICKEN are----- PHEASANT
ALLIGATOR m e - - - - - CAIMAN a @ - - - - - NILE CROCODILE - a------
0 100 200 // 300 400 450 (MyBP) I I I N I I 1
TANDEM ALIGNMENT OF:
a HEMOGLOBIN (a 1 B HEMOGLOBIN (8) MYOGLOBIN (m) LENS a.CRYSTALLIN A ( I ) FlBRlNOPEPTlDE A (fr) FIBRINOPEPTIDE B (1') CYTOCHAOME C ( c
MUSK SHREW
DUCK \
178
200-
100
0-
C LAD1 STIC S
(2820 NRs - No Extra Curricular Genetic Events)
-
[VOL. 1
iii n Fig. 4 . Mammalian portion ol“majority” tree derived from lowest and near-lowest length trees utilizing
the expanded maximum parsimony criterion. Portions not shown (the non-mammalian region and most subbranchings within mammalian orders) are identical to corresponding portions in the 281 1 N R lowrst length tree of Figure 2 ; the Senrral pattern of rnammalian hranchings is similar in the two trers.
eutherian clades. The main cladistic features at this interordinal level, already depicted by the trees in Figures 1-3, again reappear in the trees of Figures 4-6, namely, Probos- cidea’s outgroup position relative to most other extant eutherians, the position of Lago- morpha (and Tupuiu) close to Primates, Cetacea’s union with ungulates, and the joining of the Insectivora-Chiroptera branch to Carnivora. Although one of the 2819 N R length trees groups Edentata with Rodentia, maximum parsimony trees constructed for ribo- nucleases (Beintema and Lenstra, 1982) support the much more ancient separation of Edentata from Rodentia shown in the consensus trees.
Further evidence on the cladistic pattern of higher eutherian phylogeny comes from the individual genealogical trees, constructed separately for the different proteins listed in Table 1. In particular the most parsimonious lens a-crystallin trees (De Jong and Goodman, 1982) join Sirenia (sea cows), Hyracoidea (hyrax), and Tubulidentata (aardvark) with Proboscidea into an enlarged superordinal clade, Paenungulata, orig- inating with Edentata at the very base of the Eutheria. These a-crystallin trees also join Pholidota (pangolin) to Carnivora. This putative superordinal clade consisting of Chiroptera, Insectivora (i.e., Lipotyphla), and Carnivora (depicted in Figs. 1-6) might also include Pholidota. A close grouping of Primates to Lagomorpha, which is neither supported or opposed by any overriding paleontological views, has been one of the more consistent findings from maximum parsimony analysis of sequence data. Aside from the extended tandem alignment results (Figs. 1-6), which are reflected in the individual trees for amino acid sequences of hemoglobins, cytochromes c, fibrinopeptides A and B, and lens a-crystallins A, the nearness of Primates to Lagomorpha is also found in the genealogical reconstruction for carbonic anhydrases (Goodman et al., 1982; Tashian et al., 1983) and in the gene phylogeny constructed for nucleotide sequences of hemo-
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180 CLADISTICS [VOL. 1
globin genes and pseudogenes (Czelusniak et al., 1982; and current work on 44 sequences).
That we are far from resolving the eutherian “bush” into a consistently bifurcating tree is illustrated by the ambiguous placement of the suggested superordinal clade con- sisting of Carnivora, Insectivora, and Chiroptera (and possible Pholidota as well); this major branch was either placed next to ungulates and cetaceans (Figs. 1-3) or grouped with a primate-lagomorph-tupaiid-rodent assemblage (Figs. 4-6). Such ambiguity should be resolvable in that current interpretations of the fossil record suggest (e.g., Novacek, 1982) that the orders of extant eutherian mammals separated from one another over 15 to 30 million years of time over the later Cretaceous and Paleocene. Clearly the course of branching of Eutheria into its major monophyletic clades will be clarified as more extensive sequence data (as well as denser data in terms of taxa represented within each order) are gathered and used for the reconstruction of phylogenetic history.
Opportunities for Cladistic Analysis from DNA Nucleotide Sequencing
The recent dramatic progress in the technology of gene cloning and nucleotide sequencing has opened the way for so enlarging the body of sequence data that it should become possible to solve almost any problem concerning the phylogenetic systematics of extant mammals. An example of an intraordinal problem, recently solved by nucleo- tide sequence data, is provided by the hominoid region of the lowest NR length Adams consensus tree constructed for the 64 vertebrate taxa, using the extended tandem align- ment of the different protein amino acid sequences. As can be noted (Fig. 2), this A d a m tree depicts a trichotomy of Hylobatinae (gibbon), Ponginae (orangutan), and Homininae (human, chimpanzee, gorilla). Resolution of the trichotomy is readily achieved with presently available mtDNA nucleotide sequence data on mouse, ox, and hominoids. Taking mouse and ox as outgroups of the five hominoids (man, chimpanzee, gorilla, orangutan, and gibbon), the NR length was calculated for each of the 105 possible dichotomous branching orders among the five hominoid lineages. The results (a sum- mary appears in Fig. 7) provide strong evidence for dadistically dividing Hominoidea into two families, Hylobatidae (gibbons) and Hominidae, with the latter in turn dividing into subfamilies Ponginae (orangutan) and Homininae (man, chimpanzee, and gorilla). There are two lowest length trees, each at 750 NRs (Fig. 8): one subdivides Homininae into a Pan-Homo branch and a Gorilla branch, and the other subdivides Homininae into a Pan-Gorilla branch and a Homo branch. Thus the lowest NR length Adams tree depicts a Homo, Pan, Gorilla trichotomy. For 4 NRs more, i.e., a tree at 754 NRs, the third arrangement for Homininae is obtained: a Gorilla-Homo branch joining Pan, but still keeping intact Hominidae and within it Homininae (Pan, Homo, and Gorilla). At least 9 NRs (tree length 759 NRs) are added to the most parsimonious score by breaking up the hominid (human-chimpanzee-gorilla-orangutan) clade, and at least 19 NRs (tree length 769 NRs) by breaking up Homininae. Indeed, 22 and 41 NRs respectively (tree length 772 and 791 NRs) are added to the most parsimonious score by joining first only orangutan and then both orangutan and gibbon to a chimpanzee-gorilla clade not including man. Thus these mtDNA nucleotide sequence data provide by the parsimony criterion much stronger evidence than do the amino acid sequence data for the cladistic grouping of Homininae (Pan, Homo, and Gorilla) and Ponginae (Pongo) in Hominidae and this family’s sister grouping with Hylobatidae (Hylobates).
The small piece of amino acid sequence evidence for first grouping Pan and Homo together, a single synapomorphic amino acid substitution in a-hemoglobin chains, has not been corroborated by mtDNA sequence data. Thus the question remains of whether the correct dichotomous branching order within Homininae can be resolved or whether the Pan, Homo, Gorilla trichotomy shown in Figure 7 will continue indefinitely to best
19851
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PHYLOGENY OF PRIMATES
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181
,(r M C C . a L
nrw -. Fig. 7. Cladistic analysis of hominoid mitochondria1 nucleotide sequence data using mouse and ox as
outgroups. These nucleotide characters correspond to positions 11,680 to 12, 575 of Anderson et al. (1981) encompassing the 3' end of URF4, TRNAHis, TRNA"r(A"Y), TRNAL'U(''UN' , and the 5'end of URF5. (H = Homo sapiens, C = Pan sp., Go = Gorilla gorilla, 0 = Pongo pygmaeus, and Gi = Hylobates lar, M = Mu musculus, O x = Bos lourus.) (A) Left, Adams consensus tree for the two trees with nucleotide replacement scores (NR) equal to 750. Center, Adams consensus tree for the four trees with N R 5759. Right, Adams consensus tree for the ten trees with NR 1 7 6 9 . (B) Summary of the information obtained from the consensus trees. A tree with the orang removed from the human-chimp-gorilla clade adds at least 9 NRs to the most parsimonious NR score, and a tree with the human-chimp-gorilla clade broken up adds at least 19 NRs to the most parsimonious score. (C) Nucleotide replacement distribution for the 105 possible trees.
describe the phylogenetic evidence. There are portions of nuclear DNA which like mtDNA evolve at much more rapid rates than the amino acid sequence encoding regions of structural genes. Conceivably, extensive sequence data on such rapidly evolving DNA could settle this issue of branching order within Homininae. In collaboration with Dr. Alan Scott we have begun to sequence the y-hemoglobin genes of the gorilla and find that certain non-coding portions of these genes indeed evolve rapidly, not only accumu- lating point mutations but also insertions and deletions (Scott et al., 1982, 1984; Olson et al., 1982 . Moreover, our present evidence on two tandemly linked y-hemoglobin loci (called y and found in hominines indicates that from the time humans and gorillas last shared a common ancestor no conversion of one y gene by the other occurred
A G in the line of descent to gorillas whereas two conversions of the y locus by y occurred in the line to humans. Thus, the y-hemoglobin genes of humans and gorillas may be sufficiently different in structure and evolutionary history for this gene system to reveal, once the chimpanzee genes are sequenced, if Pan with regard to shared y-gene features is equally related to Homo and Gorilla or most related to only one of these two genera. In other words, this system may prove capable of resolving the Homo-Pan-Gorilla trichotomy.
As just indicated, sequence data on tandemly linked (non-allelic) nuclear genes may prove sufficiently discriminating to be especially useful for resolving phylogenetic bushes at low taxonomic levels (e.g., within subfamilies). However considerable care will be
L
182 CLADISTICS [VOL. 1
Alternative Hominoid Branching Orders from Nucleotide Sequences of a Section of mtDNA
Gl66ON I . I . I . , . I . , . I . I . , . I . I . I . I
0 10 20 30 40 50 60 70 80 90 100 110 120
Number of Nucleotide Replacements
Fig. 8. T h e hominoid portions of the two lowest N R length trees (each with score 750 NRs) found for thr rntDNA sequenrrs. T h e lcngth o f each branch equals the number of NRs calculated to have occurred along that line of c v ~ I u t i ~ n
needed to reliably distinguish orthologously related sequences of the different species from paralogously related ones. Failure to do so can result in incorrect species phylog- enies. Nucleotide sequencing of mtDNA reduces the risks of making such mistakes and thus should provide excellent data for reconstructing the cladistic pattern of higher level eutherian phylogeny. In contrast to nuclear genes which often duplicate to give rise to non-allelic paralogous loci (e.g., Gy- and *y-hemoglobin loci), each mitochondrial gene apparently occurs only once in a mammalian mitochondrial genome as indicated by the completely determined sequences of human (Anderson et al., 198l), mouse (Bibb et al., 1981), and ox (Anderson et al., 1982) genomes. Each ofthese genomes has about the same size (roughly 16,000 base pairs) and essentially the same number and ordering of their genes. Even the toad Xenopus and mammals have very similarly organized mito- chondrial genomes, as judged from RNA transcripts (Rasti and Dawid, 1979). Thus nucleotide sequences of mtDNA of different species, when matched for sequence homology, will always be from orthologous genes (genes with species phylogenies which mirror the cladistic relationships of species-lineages). Despite saturation of silent substi- tutions after only short periods of evolutionary separation (Brown et al., 1982), the amino acid changing base replacements in the protein-encoding mitochondrial genes accumulate at rates that seem suited to finding the correct branching pattern among major intra- and interordinal eutherian clades.
As the body of nucleotide sequence data on nuclear and mitochondrial genes of eutherian species increases, it will be possible to assess how well different types of sequence data and different optimality criteria support particular cladistic groupings. For example, we will be able to test the assumption that rapidly-accumulating, non- coding base replacements best delineate cladistic relationships at lower taxonomic levels whereas the amino-acid changing base replacements best delineate distant cladistic rela-
19851 PHYLOGENY O F PRIMATES 183
tionships. To carry out such assessments, and to thereby extract more accurate evolu- tionary and systematic information from sequence data, we are further developing the data handling system (illustrated by our work with mtDNA sequences) for tracking all trees examined during a search for optimal phylogenetic trees. A subroutine, used in conjunction with a branch-swapping program, writes a shorthand description of each tree along with its N R score onto a tape. Another program sorts through these trees, eliminates duplicate trees, and prints out a summary of trees examined so far. Further programs will be developed to construct consensus trees from the trees at the lowest and nearly lowest N R scores. Such processing and retrieval of information from the range of trees examined will be useful in (a) determining how strongly a given data set suggests a particular grouping; (b) showing how thorough a search was done by keeping track of the number of trees examined; and (c) comparing how different opti- mality criteria such as maximum parsimony, character compatibility, or mixtures of the two behave on different data sets.
Acknowledgments
This study was supported by NSF grant DEB 7810717.
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Note Added in Proof
Phylogenetic reconstructions by the maximum parsimony method have recently been carried out on nucleotide sequence data on the $7-globin and y-globin loci which are found in the chromatin region of linked @-related globin genes and which have been
19851 PHYLOGENY OF PRIMATES 185
sequenced in all three hominines (Pan, Homo, Gorilla) as well as in one or more outgroups. The parsimony results both for $7 sequences (Goodman et al., 1985) and for stretches of orthologous y sequences (Slightom et al., 1985) group Pun and Homo together first and then join Gorilla to their stem; the next most parsimonious arrangement (Pan grouped first with Gorilla) adds three more genic changes to the $7 reconstruction and four to the y reconstruction (each of these reconstructions involves about 2000 base pair positions for the aligned homologous sequences). It may be noted that this parsimony evidence from chromatin nucleotide sequences for a HomolPan monophyletic clade within Homininae considerably strengthens the finding from a-hemoglobin amino acid sequences and agrees with divergence phenetic findings from cellular DNA hybridization comparisons (Sibley and Ahlquist, 1984).
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