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Oct 18, 1990 - showing a large separation of Homo/Pan from Gorilla and those showing little ... Go- rilla, siamang, and macaque cytochrome oxidase subunit II.
Proc. Nail. Acad. Sci. USA Vol. 88, pp. 1570-1574, February 1991 Evolution

Resolution of the African hominoid trichotomy by use of a mitochondrial gene sequence (hominoid phylogeny/mitochondrial DNA/cytochrome oxidase subunit H gene/molecular anthropology)

MARYELLEN RUVOLO*t, TODD R. DISOTELL*, MARC W. ALLARDt§, WESLEY M. BROWN¶, AND RODNEY L.

HONEYCUTT11

Departments of *Anthropology and *Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138; IDepartment of Biology, University of Michigan, Ann Arbor, MI 48109-1048; and I'Department of Wildlife and Fisheries Science, Texas A&M University, College Station TX 77843-2258

Communicated by Charles G. Sibley, October 18, 1990 (received for review August 18, 1990)

between successive divergence events which produced the three lineages, similar to that seen with DNA hybridization results and unlike most other DNA sequence comparisons.

Mitochondrial DNA sequences encoding the ABSTRACT cytochrome oxidase subunit H gene have been determined for five primate species, siamang (Hylobates syndactylus), lowland gorilla (Gorilla gorilla), pygmy chimpanzee (Pan paniscus), crab-eating macaque (Macacafascicularis), and green monkey (Cercopithecus aethiops), and compared with published sequences of other primate and nonprimate species. Comparisons of cytochrome oxidase subunit II gene sequences provide clear-cut evidence from the mitochondrial genome for the separation of the African ape trichotomy into two evolutionary lineages, one leading to gorillas and the other to humans and chimpanzees. Several different tree-building methods support this same phylogenetic tree topology. The comparisons also yield trees in which a substantial length separates the divergence point of gorillas from that of humans and chimpanzees, suggesting that the lineage most immediately ancestral to humans and chimpanzees may have been in existence for a relatively long time.

MATERIALS AND METHODS DNA Samples. Tissues were collected from Pan paniscus, Gorilla gorilla, Hylobates syndactylus, Macacafascicularis, and Cercopithecus aethiops. mtDNA was isolated by cesium chloride/propidium iodide gradient centrifugation (9) and resuspended in 10 mM Tris'HCl, pH 8.0/1 mM EDTA. Amplification and Sequencing of Single-Stranded DNA. Gorilla, siamang, and macaque cytochrome oxidase subunit II (COII) genes were amplified from total mtDNA by the polymerase chain reaction (10). Five nanograms of mtDNA underwent 30 cycles of amplification in a 50-gl reaction volume with 2 units of Taq DNA polymerase (Perkin-Elmer/Cetus) to form double-stranded DNA. Each cycle consisted of 1 min at 950C for denaturation, 1 min at 50-570C (depending on species) for annealing, and 1 min at 720C for extension. Five microliters of the product was reamplified by the unbalanced primer method (11) for 35 cycles as above, producing single-stranded DNA. Oligonucleotide primers for amplification and sequencing were located in the tRNAASP (A7552, 5'-AACCATTTCATAACTTTGTCAA-3') and tRNALYS (B8321, 5 '-CTCTTAATCTTTAACTTAAAAG-3') genes, flanking the COII gene. Other primers internal to the COI1 gene were also used for sequencing. After centrifugation three times with 10 mM Tris-HCI, pH 8.0/0.1 mM EDTA in Centricon-30 tubes (Amicon) according to manufacturer's directions, 7 jul of DNA was used for nucleotide sequencing (12) with 2'-deoxyadenosine 5'-[a-[35SJthio]triphosphate and Sequenase (United States Biochemical) following the manufacturer's protocol. For Cercopithecus and Pan, unamplified mtDNA fragments containing COII genes were cloned into plasmid vector pUC8 and subcloned into bacteriophage M13. Single-stranded phage DNA from recombinant M13 plaques was sequenced using "universal" M13 primers (13) and internal oligonucleotide primers as above. Products were electrophoresed through 6% polyacrylamide/7 M urea sequencing gels for 2.5, 4, and 6 hr at 50 mA. Gels were dried and exposed to x-ray film for 16-36

One of the most contentious and seemingly intractable problems in mammalian systematics has been the phylogenetic resolution of humans (H, Homo sapiens), chimpanzees (C, Pan troglodytes and Pan paniscus), and gorillas (G, Gorilla gorilla). Four hypotheses are possible: three paired associations, [(C, G), H], [(C, H), GI, and [(G, H), C], and a trichotomy [C, H, GI. Morphological characters can be found to support all four (1), although a consensus appears to favor [(C, G), H] (2). Specific outcomes of molecular studies also vary, but paired associations are significantly favored over the trichotomy by both maximum-parsimony and distance analyses (e.g., see ref. 3), and a developing consensus favors the hypothesis [(C, H), GI. Most molecular analyses fall into two categories-those showing a large separation of Homo/Pan from Gorilla and those showing little or no separation. The former include single-copy DNA hybridization studies (4-7) and analysis of ribosomal gene sequences (8), while the latter include nuclear and mitochondrial DNA sequence analyses. Variation among molecular data sets in this internal branch length (internode) has become an issue for debate. If comparisons of total single-copy nuclear DNA are valid, why have DNA sequence data failed to confirm branching proportions inferred from DNA hybridization data? Stated generally, why is there extensive variability in branch lengths among topologically congruent molecular phylogenetic trees? The mitochondrial DNA (mtDNA) sequence data presented here are important for resolving African ape phylogenetic relationships in providing evidence for a Homo/Pan clade.** They are also significant in suggesting a relatively long time

hr.

RESULTS AND DISCUSSION Sequence Analyses and Phylogenetic Trees. DNA sequences from five primate COII genes were determined and compared Abbreviation: COI, cytochrome oxidase subunit II. tTo whom reprint requests should be addressed. §Present address: Department of Zoology, University of Florida, Gainesville, FL 32611. **The sequences reported in this paper have been deposited in the GenBank data base (accession nos. M58005-M58009).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1570

with published sequences of human, four rodents, one artiodactyl, and one amphibian. Overall, Homo and Pan show 9.6% difference in COI0 sequences, compared to 13. 1% for Homo-Gorilla and 12.1% for Pan-Gorilla (Fig. 1). Three methods of phylogenetic analysis yield congruent topologies containing a Homo/Pan clade (Fig. 2). With maximum parsimony, 14 inferred synapomorphies define a Homo/Pan clade (at positions 60, 177, 204, 210, 213, 267, 268, 342, 450, 525, 549, 627, 637, and 684). The next most parsimonious trees are longer by 7 or 8 events with a Homo/Gorilla (7 inferred synapomorphies, at positions 75, 117, 165, 228, 345, 478, and 621) or a Pan/Gorilla (6 inferred synapomorphies, at positions 96, 99, 436, 474, 519, and 675) clade, respectively. By the maximum-likelihood method, the Homo/Pan grouping is significantly better than alternative trees [by the criterion of Kishino and Hasegawa (28), as implemented by Felsenstein (27)]. The common ancestral branch of Pan and Homo is significantly positive (P < 0.01 by approximate confidence limits). When only relatively rare transversions are used, three inferred synapomorphies support a Homo/Pan clade; alternative trees (longer by three events) show no putative synapomorphies. To test whether polymorphism occurs at phylogenetically significant sites, sequences were reanalyzed after removing 10 known human polymorphic restriction enzyme sites (39 bases) (29). The most parsimonious tree shows a Homo/Pan clade also with 14 inferred synapomorphies. Inferred amino acid sequences, however, do not significantly resolve the Homo-Pan-Gorilla trichotomy. Of 227 amino acid residues, there are 6 differences (2.6%) between Homo and Pan, versus 7 (3.1%) between either Homo and Gorilla or Pan and Gorilla (Fig. 1). A previously reported (30) M. fascicularis COIl sequence is 40 bp different from our newly generated sequence and shows an unusually high number of inferred synapomorphies A

Homo

9.6

Homo

Pan Gorilla Hylobates

Macaca Cercopithecus Muf

Rattus norv. 1 Rattus norv.2

Rattus rattus Bos Xenopus B

Pan

62 83 106 1 20 124 196 199 195 192 204 220

21 9

Homo

Pan

Gorilla

Hylobates

Macaca Cercopithecus Mus

4 8 27 41 37 104

99

124 11 8 201 201 194 200

1 34 1 25 1 97 209 204 1 95 200 220

101

8 27 41 35 106 103

98

100

102

104 96 118

Rattus norv. I Rattus norv.2 Rattus rattus Bos

94

Xenopus

116

13.1 12.1

77

Homo

Pan

Gorilla Hylobates Macaca

100

199

1571

Proc. Natl. Acad. Sci. USA 88 (1991)

Evolution: Ruvolo et al.

17.4 16.4 16.1

20.3 21.0 22.9 20.2

1 20 116 186 206 201 206 21 2 210

92 200 207 203 205 207 213

Gorilla Hylobates Uacaca 7 7

13 14 14

23 39 31 100 99 98 102 92 114

36 34 99 102 101 105 91 105

27 29 28 25

with the human sequence, unlike other Old World monkey COII sequences (T.R.D., unpublished data). This suggests that the previously published sequence contains errors, and it was therefore not included in the analyses. Hommnoid Tree Topologies and Internodes Estimated by Molecular Data Sets. Immunological studies which indirectly measure amino acid differences have been unable to resolve the African hominoid trichotomy (31, 32). Amino acid sequences (33, 34) and some DNA sequences only barely favor a Homo/Pan grouping. For example, the immunoglobulin e pseudogene (35) shows a short internode (10%) for the separation of a Homo/Pan clade (Table 1). Another nuclear, 10.8-kilobase DNA segment containing the i7-globin pseudogene resolves the trichotomy with a short internode (13-17%)

(36).

Previously reported mtDNA sequences also suggest a short internode for the Homo/Pan clade when resolution is possible. Three major regions, 20%o of mtDNA, have been studied among hominoids. The 896-bp region containing partial ND-4 and ND-5 (NADH dehydrogenase complex) genes and three tRNA genes (40) has been analyzed many times with differing results (38, 40-45). In general, parsimony methods show too few inferred synapomorphies for resolution, and distance methods such as neighbor joining yield a Homo/Pan clade with relatively short internode (17%, Table 1). The 12S rRNA sequences from Homo, Pan, Gorilla, and Pongo (3) offer little phylogenetically useful information, presumably due to their slow rate of change. All three alternative African hominoid trees are equally likely with maximum parsimony. Phylogenetic analysis of the control region (displacement loop) of Homo, Pan, and Gorilla (46) suffers from lack of an outgroup to the African hominoids. Additionally, the Gorilla control region is shorter than in Homo and Pan. Rattus

Rattus

Xonopus

norv.2

Rattus rattus

Bos

norv. 1 38.8 40.1 38.9 36.2 39.6 37.4

39.4 40.1 41.8 41.2 41.2 42.0 18.1

209 203 208 206 226

108 102 106 151 179

38.2 38.1 40.4 39.9 40.1 40.3 17.0 1.3

9 61 142 195

37.7 39.8 38.4 41.3 40.8 41.8 17.7 9.5 8.3

54 138 1 91

40.2 39.1 39.1 42.0 40.6 40.4 27.2 25.3 24.5 24.6

138 195

45.6 45.5 45.5 42.4 43.6 46.7 34.4 38.6 37.6 38.4 37.3

192

Cercopithecus

Mus

24 26 24 20 13 99 102 101 105 89 109

Cercopithecus

Mus

21.0 19.8 21.0 19.4 14.7 191

14 101

98 97 101 89 113

Rattus

Rattus

Xenopus

norv.2

Rattus rattus

Bos

norv. 1 65 67 65 65 73 69

69 71 69 68 74 70 7

43 42 44 68 96

65 67 65 65 71 67 3 4

3 9 67 103

64 66 64 64 72 68 2 7 3

6 68 104

62 64 62 62 71 68 21 25 21 21

70 102

72 74 76 70 76 77 57 63 59 58 55

94

FIG. 1. Sequence comparisons of the COII gene of six primates, four rodents, one artiodactyl, and one amphibian. Species abbreviations: Homo, H. sapiens (human) (14); Pan, P. paniscus (pygmy chimpanzee); Gorilla, G. gorilla (lowland gorilla); Hylobates, H. syndactylus (siamang); Macaca, M. fascicularis (crab-eating macaque); Cercopithecus, C. aethiops (green monkey); Mus (mouse, species not specified) (15); Rattus norv. I and 2, R. norvegicus (Norway or brown rat, two different individuals) (16, 17); Rattus rattus (black rat) (16); Bos, B. taurus (domestic cattle) (18); Xenopus, X. laevis (African clawed toad) (19). (A) Observed number of nucleotide differences (pairwise comparisons) for the 684-base-pair (bp) COII gene sequences, below the diagonal, and percent sequence difference [corrected for multiple substitutions (20)], above the diagonal. (B) Observed (pairwise) number of nucleotide transversional differences, below the diagonal, and observed number of COII amino acid differences, above the diagonal.

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Evolution: Ruvolo et al. A

~.15Pan

Proc. Natl. Acad. Sci. USA 88 (1991)

Xenopus

67

192bus 2 _~~~~~~~~1

.21

.13

B

0

34

(+3) ~

atu rfu

wHSnorv. 2 Rfs norv. 1

use&:&

(B8

u

032 02C) ~

pi

C

FIG. 2.

Primate relationships derived from analyses of aligned mitochondrial COIl gene sequences (684 bp) by three tree-building methods

(21-23). Species abbreviations are as in Fig. 1. (A) Maximum-parsimony tree (22) constructed using PAUP version 3.0 (24) and HENNIG 86 version 1.5 (25) with a nonmammalian outgroup (Xenopus), showing minimum possible branch lengths. Total tree length is 828. Numbers in parentheses are augmentation values (26) to correct for node-density effects, using an average of 5.4 additional substitutions per node found for all species in this data set. The tree above is scaled in proportion to the unaugmented values. (B) Neighbor-joining tree (21) using distances corrected for multiple substitutions [two-parameter correction method (20)] performed in DNAdist of PHYLIP version 3.2 (27). (C)

Maxcimum-likelihood

tree

(23) using empirically observed base frequencies as equilibrium frequencies under the substitution process [DNAml of PHYLIP version 3.2, F option (27)].

Estimates of nucleotide sequence divergence for combined data sets have fared better in resolving the trichotomy. For instance,

reanalysis

of coding and noncoding

regions of

nuclear DNA and mtDNA provide statistically significant

support for a Homo/Pan relationship (47), showing a short internode. Only one DNA sequence study has been interpreted as

supporting a Pan/Gorilla

dlade,

and this conclusion rests

Table 1. Relative internodes for the Homo-Pan-Gorilla trichotomy among molecular phylogenies

Neighbor joining

Maximum likelihood COIl* 0.058 0.043

Maximum

parsimony

4t qnt 8% bp§ COII* COIl* DNA.DNAt Branch Igoe' 27 76 0.0079 0.044 0.74 0.79 0.052 (a) Homo to Homo/Pan ancestor 21 0.0060 92 0.050 0.90 0.044 0.80 (b) Pan to Homo/Pan ancestor (c) Homo/Pan ancestor to 14 14 0.033 0.0007 0.008 0.11 0.39 0.015 Homo/Pan/Gorilla ancestor 0.17 0.58 0.66 0.10 0.17 0.13 0.31 0.49 Relative internode [= c/0.5 (a + b)] (21-23). algorithms tree-building by phylogenetic generated lengths are inferred branch For rows a-c, values *From this study, 684 bp of COIl gene [values corrected for multiple substitutions (20) in neighbor-joining analysis (21)]. tSingle-copy DNA hybridization data [from data in table 4 of ref. 7; our analysis using neighbor joining (21)]. t-Globin pseudogene region [from data in figure 3 of ref. 36; distance values corrected as in Jukes and Cantor (37)]. §mtDNA region [from data in table 4 of ref. 38; values corrected as in Gojobori et al. (39); our analysis using neigbor-joining algorithm (21)]. llmmunoglobulin e pseudogene [from data in figure 3 of ref. 35; values corrected as in Jukes and Cantor (37)].

Evolution: Ruvolo et al.

solely on sequence information in a tandemly duplicated repeat region of the involucrin gene, found to be polymorphic in gorillas, owl monkeys, humans, and other primates (48, 49, 60). Since involucrin is probably highly polymorphic in many species and represents the one exception to a growing number of molecular data sets supporting a Homo/Pan clade, it may be an example of the "gene tree-species tree" incongruency theoretically predicted by Pamilo and Nei (50). The strongest support for a Homo/Pan association, in the sense that a long internode is inferred, comes from singlecopy nuclear DNA hybridization studies. This method appears to provide reproducible results, even when different experimental protocols are used (4-7). Resulting internode estimates are larger than those inferred from almost all nucleotide sequence studies, =50% (Table 1). In COII molecular phylogenies (Fig. 2), the internal branch separating the Homo/Pan clade from the last common ancestor with Gorilla is relatively long, ranging from 31% to 66% of averaged Homo/Pan terminal branch lengths, de-

pending on phylogenetic tree-building algorithm (Table 1).

This range of values contains those found by DNA hybridization data analysis (49%o) and by sequence analysis of 28S rRNA gene and ribosomal internal transcribed spacer region [36%-66% depending on which and how many outgroup species are used (8)]. Until recently, the overall lack of consistency between DNA sequence results and DNA hybridization results in terms of internode length supporting a Homo/Pan clade seemed paradoxical. It was even argued that the discrepancy was reason for considering the DNA hybridization results "extremely unlikely" (51). However, the mitochondrial COII gene sequence analysis presented here and a recent nuclear DNA sequence study of the 28S rRNA gene and ribosomal internal transcribed spacer region (8) rebut that argument, since they also resolve the African hominoid trichotomy with relatively long internodes. Generally, molecular studies tend to support a Homo/Pan clade but vary in the amount of phylogenetic separation between the clade and its last common ancestor with Gorilla. This branch length variation can be interpreted in terms of evolutionary divergence times. Assuming molecular rates are clock-like and Homo and Pan lineages diverged, for example, about 6 million years ago (52), the Gorilla lineage divergence would be dated at 7.9-10.0 million years according to COII and 28S rRNA gene sequences and DNA hybridization data, but at 6.6-7.0 million years by previous DNA sequence data sets.

Possible Causes of Internode Variation. Several factors

might contribute

to branch length variation. Accelerated molecular rates could produce longer internodes. Although COII gene rate differences between primates and nonprimates are evident in cladistic and phenetic analyses [confirming earlier observations (16, 53)], rates within hominoids are approximately equal. The maximum-parsimony tree appears to show unequal branch lengths within primates, but after correction for number of nodes to terminal taxa (26), branches are equal within primates and 40%o longer than in nonprimates. The distance tree also has these characteristics (Fig. 2). Therefore molecular rate variation can probably be ruled out as an explanation. Variation in internal branch lengths is not correlated with type of molecular data. In particular, it is not true that DNA hybridization measurements generally give longer internodes than do DNA sequence analyses, as judged from other internodes on the hominoid molecular phylogenetic tree

(M.R., unpublished data).

Variation among data sets is most likely not ascribable to type of gene regions studied. Noncoding DNA sequences evolve more rapidly than coding regions (54), yet analyses of extensive noncoding regions (36, 55) show less separation

Proc. Natl. Acad. Sci. USA 88 (1991)

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among Homo-Pan-Gorilla than either DNA hybridization studies, which measure both single-copy coding and noncoding DNA regions, or COIl coding sequences. Different molecular rates among DNA regions also cannot explain variability in relative internode lengths. After all, two DNA regions with different but constant molecular rates produce sequence differences which, after correction for multiple substitutions (assuming nonsaturating conditions), are linearly scaled with respect to one another, giving the same relative branch lengths. However, statistical artifacts due to small numbers of observed events might explain part of the variability, for example in the case of a slowly evolving gene showing few substitutions among species. Gene tree-species tree discrepancies may be a source of variation among data sets. Given that gene trees and species trees can disagree topologically due to ancestral polymorphisms (50), less radical disagreement in branch lengths is also possible. Ancestral polymorphisms are gene divergences preceding species divergences; therefore, gene trees can only overestimate branch lengths in comparison to species trees. However, gene trees can have either longer or shorter internodes than species trees, depending on when the ancestral polymorphism originated. Because comparing many unlinked genes reduces the probability of gene tree-species tree discordances (50), one might predict that a DNA hybridization "gene tree" should show the least discordance since it measures all single-copy DNA differences between two genomes. Alternatively, since DNA hybridization is an averaging technique, a DNA hybridization "gene tree" might always overestimate species-tree branch lengths by an amount representing the average genetic divergence due to ancestral polymorphism. The possibility remains then that observed variation among data sets is due partly to gene tree (as opposed to species tree) variation arising from ancestral polymorphisms.

CONCLUSION Hominoid primates have been unusually well studied compared with other groups, and this has revealed interesting variation in tree topology and, more important in this case, tree proportions. Variation among hominoid molecular phylogenetic trees may be an example of a previously unobserved general phenomenon, unobserved because for most systematic problems only one or two molecular data sets are commonly collected. Alternatively, there may be some unusual feature, as yet undescribed, of African hominoid speciation patterns contributing to branch length variation in phylogenetic trees. In order to clarify why this variation exists and what are the most probable correct tree proportions, still more systems need to be studied and the theory of gene tree-species tree relationships further developed. Difficulty in resolving African hominoid phylogeny in some data sets has been interpreted as evidence for trichotomous speciation and has stimulated consideration of this type of speciation as a biological possibility (51, 56, 57). While a trichotomous divergence event may not be inconsistent with some models of speciation, the need to invoke one for the African apes is diminished, given the mitochondrial sequence data from the COII gene presented here. The COII gene sequence comparisons clearly favor a Homo/Pan clade. These mtDNA data add strong support to the results of both recent DNA sequence studies (8, 36, 38, 47, 58, 59) and DNA hybridization studies (4-7) demonstrating a Homo/Pan clade to the exclusion of a Pan/Gorilla clade. The mitochondrial COII gene trees suggest a separate ancestry for the common Homo/Pan dade longer than that seen in any other mtDNA analysis and comparable to that seen in DNA hybridization and nuclear rRNA sequence studies. Assuming an approximate divergence time of 6

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Evolution: Ruvolo et al.

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