Molecular Clocks: Advanced article Determining the Age of Article Contents . Introduction . Considerations in Dating the Human–Chimpanzee the Human–Chimpanzee Divergence . Current Estimates, Conflicts and Resolution Divergence Online posting date: 30th April 2008

Michael I Jensen-Seaman, Duquesne University, Pittsburgh, Pennsylvania, USA Kathryn A Hooper-Boyd, Duquesne University, Pittsburgh, Pennsylvania, USA

The approximate clocklike nature of the accumulation of nucleotide substitutions (the ‘molecular clock’) allows for the estimation of the time of divergence between modern species, dependent on calibrating the clock with known divergence dates from the fossil record. The molecular clock gives dates of approximately 6–8 million years ago for the human–chimpanzee divergence, in general agreement with the palaeontological evidence.

Introduction differences among apes and humans (hominoids) were cal- ibrated with a fossil-based divergence date between ho- The idea that degrees of similarity among macromolecules minoids and Old World monkeys of 30 million years ago (deoxyribonucleic acid – DNA, ribonucleic acid – RNA (Mya), humans and chimpanzees diverged approximately 5 and protein) of different species could be used to construct Mya (Sarich and Wilson, 1967). Compared to interpreta- phylogenies of primates, to complement those from anat- tions of the fossil record at the time, which postulated a omy, is over 100 years old. When these differences were human–ape divergence in the middle Miocene (414 Mya; more accurately quantified and collected from numerous Pilbeam, 1968) or even earlier (Leakey, 1967), a human– species, it was suggested that changes in proteins may be chimpanzee divergence of only 5 Mya seemed remarkably occurring in a more or less regular manner, in proportion to recent. If Sarich and Wilson’s date was correct, then any evolutionary time (Zuckerkandl and Pauling, 1965); the fossil substantially older than this, including Ramapithecus same was suggested of DNA (Hoyer et al., 1965). Since, in (Pilbeam, 1968) and Kenyapithecus (Leakey, 1967), could principle, amino acid or nucleotide substitutions occur in not possibly be a hominin – that is, a species more closely direct proportion to time, this constant accumulation of related to humans than to chimps. This bold assertion that change was dubbed the ‘molecular clock’, and could be one must negate huge portions of the fossil record from used to date the time of divergence between extant species. consideration on the basis of molecular data led to the first The first attempt to use the molecular clock to date the major conflict between molecular and morphological data divergence between humans (Homo sapiens) and our clos- in the study of human evolution. Over the ensuing decade est relatives the chimpanzees (Pan troglodytes and Pan additional molecular data would support a recent date, paniscus) was by Vincent Sarich and Allan Wilson. In a while new fossil discoveries would reveal these early and series of papers in the late 1960s, they demonstrated that middle Miocene genera to be more ape-like than originally the amount of difference between serum albumin of described, supporting the possibility of a relatively recent primate species, as measured with quantitative cross- human–chimpanzee divergence (Greenfield, 1980). In reactions, was consistent with known phylogenies, that many ways this ‘victory’ for the molecular evolutionary the accumulation of differences occurs essentially ‘clock- biologists set the tone of confidence, if not arrogance, for like’ along different primate lineages, and that when the the ability of molecular data to inform – and indeed over- turn – interpretations based on the fossil record.

ELS subject area: Evolution and Diversity of Life Considerations in Dating the Human– Chimpanzee Divergence How to cite: Jensen-Seaman, Michael I; and, Hooper-Boyd, Kathryn A (April 2008) Molecular Clocks: Determining the Age of the Human–Chimpanzee General principle Divergence. In: Encyclopedia of Life Sciences (ELS). John Wiley & Sons, To estimate the time of divergence between humans and Ltd: Chichester. chimpanzees using the molecular clock, one needs an es- DOI: 10.1002/9780470015902.a0020813 timate of the evolutionary distance between these species,

ENCYCLOPEDIA OF LIFE SCIENCES & 2008, John Wiley & Sons, Ltd. www.els.net 1 Molecular Clocks: Determining the Age of the Human–Chimpanzee Divergence as well as an estimate of the substitution rate by calculating independent regions from different chromosomes. This the evolutionary distance between species whose time of also mitigates the possibility that selection is adversely divergence is known from the fossil record (Figure 1). The affecting the estimate of evolutionary distance, since it is evolutionary distance nowadays almost always comes unlikely to be acting to bias the estimate across all regions from comparing DNA sequences. If the molecular clock of the genome. The most pronounced intragenomic vari- ticks at the same rate in all relevant species, we can estimate ation in evolutionary distance between humans and chim- the nucleotide substitution rate (how fast the clock ticks) by panzees is seen in comparisons between the autosomes and dividing the evolutionary distance by the known time, and the sex chromosomes. The X-chromosome diverges more then apply this rate to the human–chimpanzee evolution- slowly than the autosomes, which in turn diverge more ary distance to calculate the unknown time. See also: slowly than the Y-chromosome (Innan and Watanabe, Molecular Clocks; Molecular Clocks 2006; Patterson et al., 2006); these differences are explained at least partly, but perhaps not entirely, by a higher mu- Estimating the evolutionary distance tation rate in the male germline (Makova and Li, 2002). Finally, the mitochondrial genome has a very high substi- There are several practical considerations in estimating the tution rate, but is almost entirely coding sequence. evolutionary distance between any two species. The first is Once nucleotide sequences are obtained from the rele- the choice of DNA sequences to use. Typically, noncoding vant species, they are aligned and an estimate of the evo- DNA (introns, intergenic regions or four-fold degenerate lutionary distance is made. The simplest measure would be sites) is used under the assumption that the rate of nucleo- to count the differences and divide by the total number of tide substitution reflects the underlying neutral mutation nucleotides to get the proportion of nucleotides that have rate, without being affected by natural selection which changed. This will, however, almost always be an under- could dramatically increase or decrease the rate of change estimate of the number of substations that have occurred in one lineage or another. Secondly, one must decide which because multiple substitutions at the same site may appear genes or what chromosomal regions to use. It is known that as a single change or no change. For this reason, many substantial variation in the local substitution rate exists different mathematical models have been developed to es- among regions of the human genome, perhaps due to var- timate the actual number of substitutions. In addition to iation in local GC (guanine–cytosine)-content, local re- correcting for multiple substitutions, increasingly complex combination rate and position relative to the telomere, models incorporate transition–transversion bias, unequal among other possible factors (CSAC, 2005). For this rea- nucleotide frequencies, and among site rate variation. son, sequences ideally would be chosen from many See also: Evolutionary Distance; Evolutionary Distance: Estimation Hominoid Cercopithecoid The final major consideration in estimating the evolu- tionary distance is whether the substitution rate is the same in all lineages; that is, does the molecular clock tick uni- Hominine Pongine formly across species? It is now well accepted that the nu- cleotide substitution rate does, in fact, vary among taxa. Hominin Old World monkeys, apes and humans (the catarrhines) have slower molecular clocks than the mammalian average Human Chimpanzee Orangutan Macaque (Yi et al., 2002), while rodents seem to have particularly fast molecular clocks. Within catarrhines, apes and humans (the hominoids) possess a slower molecular clock than Old World monkeys, an idea first proposed over four-and- ? a-half decades ago (Goodman, 1961) and now well- supported with large amounts of genomic sequence (Yi et al., 2002). The lack of a universal, or global, clock does not preclude the ability to estimate divergence dates since if Known date the clock ticks approximately uniformly in the relevant species (i.e. a ‘local clock’), it is still useful; furthermore, more complex models that incorporate substitution rate variation among lineages can be used to account for the Known date lack of a universal clock within primates. See also: Mole- cular Clocks; Molecular Clocks

Calibrating with the fossil record Figure 1 Estimating the time of divergence between human and chimpanzee relies on a calibration based on a known divergence time – in this In order to convert the estimated evolutionary distance case either the hominine–pongine split or the hominoid–cercopithecoid into an estimate of species divergence in geologic time, it split. Note the taxonomy used herein. needs to be calibrated with the evolutionary distance

2 ENCYCLOPEDIA OF LIFE SCIENCES & 2008, John Wiley & Sons, Ltd. www.els.net Molecular Clocks: Determining the Age of the Human–Chimpanzee Divergence between two or more species with a known divergence time 2007), but must be treated as a ‘soft’ maximum bound and from fossils. Ideally, this calibration point would have a our confidence in it may depend on the completeness of the rich and unambiguously interpreted fossil record. In many fossil record. Although the logic behind using fossils to cases, the divergence is a split with one subsequent lineage calibrate the phylogenetic tree is rather straightforward, its leading to humans, such as the hominoid–cercopithecoid misuse is pervasive in the primate molecular clock litera- split or the human–orangutan split shown in Figure 1. ture, predominantly in mistaking the minimum date for a However, it could also be based on two or more other pri- best point estimate of the divergence, and in the improper mate taxa, or even nonprimate taxa, or could use a global use of a dated fossil as a ‘hard’ maximum bound. substitution rate based on many mammalian divergences. Even if applied correctly, the calibration of the molecular As mentioned earlier, the lack of a mammal-wide global clock may be problematic due to the incompleteness of the clock, but the presence of a reasonably good local clock in fossil record and the inability to reliably place every fossil catarrhines, has led many researchers to use either the hu- on the phylogenetic tree with complete certainty. At the man–orangutan split or the hominoid–cercopithecoid split time of any split, the resulting daughter species will be as the calibration point. morphologically very similar, as similar as any modern In an ideal world, the date of divergence to be used as the sister species (e.g. the common chimpanzee and the pygmy calibration could be determined with great certainty from chimpanzee, or bonobo). Using the example in Figure 2, the fossil record. Unfortunately, phylogenies and diver- even with a perfect fossil record, fossils C and D may not gences do not fossilize; dead animals do. Fossils can never have yet evolved any synapomorphies that can link them provide a firm divergence date, but they can provide a with modern species 1 and 3. It may be millions of years minimum constraint on the divergence, with the following before a species (fossil E) evolves a synapomorphy that can logic (Figure 2). The earliest occurrence in the fossil record definitely link it with later extant species. This, combined of species on one or both descendent lineages (fossils C and with an incomplete fossil record, ensures that calibration DinFigure 2) provides a minimum date for the split, since dates will always be minima with the actual divergence date by definition the split must have happened before the ex- occurring some unknowable time prior. istence of the descendent lineages. The identification of these fossils and their correct phylogenetic placement relies Calibrating the human–chimpanzee on the occurrence of shared derived features (synapomorp- divergence hies) with their modern relatives. Note that the fossil record can never truly provide a maximum constraint on the di- Since there appears not to be a universal mammalian vergence date. The existence of fossils that morphologically molecular clock, most researchers have used divergences resemble the ancestral species – that is, that lack any within catarrhine primates to calibrate the human– synapomorphies with the descendent species – tell us little chimpanzee split, usually either the split between orang- chronologically since they could have died off before the utans on one hand and the African apes and humans on the split (fossil A) or persisted long after the split (fossil B). It other (that is, the pongine–hominine split; Figure 1), or the has been suggested that a reasonable maximum date can be split between Old World monkeys and apes (the cerco- inferred in a few different ways (see Benton and Donoghue, pithecoid–hominoid split; Figure 1). Calibrating from these splits requires the identification and dating of the earliest fossil clearly in the pongine clade or the hominine clade in Present Species 1 Species 2 Species 3 the first case, or a fossil that is clearly either a cercopithe- coid or a hominoid in the latter. In both cases, the afore- mentioned difficulties regarding the incompleteness of the fossil record and the lack of clear synapomorphies in the ? descendent species following the split make an accurate E dating of the divergence difficult. B Orangutans split from the African ape–human clade some time in the Miocene, when the diversity of apes was substantially greater than today. The earliest member of the pongine clade is typically cited as Sivapithecus,whosefirst D C appearance in the fossil record is dated to approximately 13 Calibration Mya. There is not a universal agreement that Sivapithecus is node ancestral to orangutans, leaving the possibility that it may be A broadly ancestral to all great apes (Greenfield, 1980), nor is there universal agreement over exactly which fossils belong in this genus (Pilbeam, 1968). Nonetheless, the consensus Past view today among palaeoanthropologists is that Si- vapithecus does indeed group with orangutans, following Figure 2 Hypothetical species divergence leading to extant taxa (species their split from the African ape–human clade, and therefore 1–3) and fossils used to date the calibration node (A–E). that the orangutan and human lineages were distinct by at

ENCYCLOPEDIA OF LIFE SCIENCES & 2008, John Wiley & Sons, Ltd. www.els.net 3 Molecular Clocks: Determining the Age of the Human–Chimpanzee Divergence

least 13 Mya. Although the fossil record cannot place a Human Chimpanzee maximum constraint on this calibration, a ‘soft’ maximum Present can be derived by taking the earliest date for the hominoid– cercopithecoid split, using the logic that the hominine– pongine split could not have occurred before this. A date of approximately 23 Mya can therefore be used as this soft maximum (see later), keeping in mind that this date is indeed very soft considering the paucity of catarrhine primate fos- 2 Mya Australopithecus sils between 23 and 30 Mya. garhi Evidence for a minimal date of the cercopithecoid– hominoid split comes in the form of both early Old World Au.afarensis monkeys (cercopithecoids) and early apes (hominoids). The most pronounced synapomorphy uniting the cercopithe- 4 Mya coids is the derived bilophodont molars, as compared to the simple and primitive flat molars of the apes. The most ob- Ardipithecus ramidus vious synapomorphy of the living hominoids is the lack of a tail. The common ancestor of the cercopithecoids and the Ar. kadabba hominoids would undoubtedly have had a tail and ape-like 6 Mya molars, and it is likely that the descendent sister species that Orrorin tugenensis resulted from the cercopithecoid–hominoid split would Sahelanthropus have maintained these primitive features, at least initially tchadensis immediately following the split, making the identification of early cercopithecoids and early hominoids as such difficult. The earliest evidence for cercopithecoids (Victoriapithecus) 8 Mya is found at approximately 19–20 Mya. The earliest evidence for hominoids is probably Morotopithecus, dated to 20.6 Originalrecalibrated molecular molecular dates dates Mya (Young and MacLatchy, 2004), although some spec- Recalibrated molecular dates imens of Proconsul have been dated to 23 Mya. A soft max- imum date would come from the oldest definitive catarrhine, 10 Mya as separate from the platyrrhines (New World monkeys). Conservatively, this would be 31.5 Mya, the date of the old- est Pliopithecus fossils in the early Oligocene, although this gets pushed back to the late Eocene (approximately 37 Mya), assuming the Oligopithecids (Oligopithecus and Cat- opithecus) are true catarrhines. 12 Mya To summarize, with our present understanding of the fossil record, the hominine–pongine split can be placed at between 13 and 23 Mya, and the hominoid–cercopithecoid split to 23–37 Mya. Within these ranges, there is no reason Figure 3 The current state of knowledge regarding the hominin fossil record (left) surrounding the human–chimpanzee divergence, compared to to assume the actual divergence date was near the middle of divergence dates from molecular data (right). Fossil species are taken from the range, or for that matter, that any date within the range Wood (2002), references therein, and the more recent primary literature. is more likely than any other date. Most molecular studies Molecular dates are largely taken from Steiper and Young (2006, p. 390 their seeking to date the human–chimpanzee split have used ei- Table 4), removing dates not based on DNA sequence data, and adding the ther a human–orangutan split of approximately 14 Mya, or dates from Hasegawa et al. (1985) and Patterson et al. (2006). Open circles represent divergence dates as estimated in the original publications (or a hominoid–cercopithecoid split of approximately 25 Mya means if ranges were given); closed circles are the same dates standardized by (see review of studies in Steiper and Young, 2006). It should assuming a 30.5-Mya divergence between hominoids and cercopithecoids be noted that the dates typically used are close to the min- or an 18.3-Mya divergence between humans and orangutans (Steiper and imal divergences, with rather large associated soft maxima. Young, 2006). Furthermore, these could change substantially with the discovery of new fossils. to pinpoint the time of the human–chimpanzee divergence using DNA sequences from the mitochondrial, autosomal and sex chromosomes, and including both coding and noncoding data. Estimated divergence dates have ranged Current Estimates, Conflicts and from less than 3 to over 11 Mya (Figure 3). The range of Resolution dates comes from different estimates of the evolutionary distance, as well as differences in the calibration. When the Over the past four decades since Sarich and Wilson’s (1967) latter is accounted for by standardizing the calibration, the groundbreaking work, numerous studies have attempted dates are in greater agreement (Figure 3). Here, a standard

4 ENCYCLOPEDIA OF LIFE SCIENCES & 2008, John Wiley & Sons, Ltd. www.els.net Molecular Clocks: Determining the Age of the Human–Chimpanzee Divergence calibration of 30.5 Mya for the hominoid–cercopithecoid among either geneticists or palaeoanthropologists with re- split is used, taken from a recent analysis of noncoding spect to the putative hominin status of any given fossil, with genomic data (Steiper and Young, 2006), with over half of present knowledge data from both fields converge on a late the dates falling between 6.5 and 7.5 Mya. These dates are Miocene date for the origin of hominins, some time approx- broadly consistent with the existing fossil record, consid- imately 6–8 Mya. Finally, it is worth noting the irony that, ering the uncertainty in calibrating the molecular clock although probably not true in 1967, today the fossil record (Figure 3). While this is a bit circular (the date of 30.5 Mya surrounding the human–chimpanzee divergence is better comes not from fossils, but from estimating the hominoid– represented than that surrounding either the human–orang- cercopithecoid split using the molecular clock calibrated utan or hominoid–cercopithecoid divergences that are used with an approximately 6.6-Mya human–chimpanzee split), to calibrate most molecular clocks purporting to more ac- the point here is that there is less disagreement among mo- curately date the human–chimpanzee divergence. As a re- lecular datasets than is apparent at first. With a younger sult, the greatest area for improving our estimate of this calibration date of 25 Mya, the majority of dates would fall divergence will come not from increasing precision by col- between 5 and 6 Mya, as estimates of the minimum date of lecting more DNA sequence data, but by increasing accu- divergence between human and chimpanzee. racy in the calibration from the fossil record. When the first estimates of the human–chimpanzee di- vergence based on a molecular clock were published (Sarich and Wilson, 1967), at 5 Mya they stood in stark References contrast to the consensus view of human origins at the time, which postulated the human lineage extending separately Benton MJ and Donoghue PCJ (2007) Paleontological evidence from the apes at least into the mid-Miocene (approximately to date the tree of life. Molecular Biology and Evolution 24: 15 Mya) and perhaps earlier (Leakey, 1967; Pilbeam, 1968). 26–53. Subsequent fossil discoveries and reinterpretations led to CSAC, The Chimpanzee Sequencing and Analysis Consortium the abandonment of these mid-Miocene fossils as putative (2005) Initial sequence of the chimpanzee genome and com- hominins. Over the years, molecular results have been seen parison with the human genome. Nature 437: 69–87. to conflict with existing interpretations of the fossil record. Goodman M (1961) The role of immunochemical differences in the phyletic development of human behavior. Human Biology For example, Hasegawa et al. (1985), after deriving a 2.7- 33: 131–162. Mya human–chimpanzee divergence date, proposed that Greenfield LO (1980) A late divergence hypothesis. American either Australopithecus afarensis (‘Lucy’) was not a ho- Journal of Physical Anthropology 52: 351–365. minin, but rather could be a bipedal ancestor to humans, Hasegawa M, Kishino H and Yano T (1985) Dating of the human- chimpanzees and gorillas, or that hybridization occurred ape splitting by a molecular clock of mitochondrial DNA. between the incipient humans and chimpanzees two million Journal of Molecular Evolution 22: 160–174. years after their initial divergence. It now seems that this Hoyer BH, Bolton ET, McCarthy BJ and Roberts RB (1965) The early date was due to the use of a very small data set, com- evolution of polynucleotides. In: Bryson V and Vogel HJ (eds) bined with the assumption of a universal mammalian mo- Evolving Genes and Proteins, pp. 581–590. New York: Aca- lecular clock, both a reflection of the times. Nonetheless, a demic Press. recent study proposed a nearly identical scenario – hy- Innan H and Watanabe H (2006) The effect of gene flow on the bridization between proto-humans and proto-chimpan- coalescent time in the human–chimpanzee ancestral popula- zees – to explain discrepancies between their dating and the tion. Molecular Biology and Evolution 23: 1040–1047. fossil record, as well as discrepancies among different chro- Leakey LSB (1967) An early Miocene member of Hominidae. mosomal regions (Patterson et al., 2006). This study esti- Nature 213: 155–163. mated the human–chimpanzee divergence at 56.3 Mya, or Makova KD and Li W-H (2002) Strong male-driven evolution of ‘a more realistic’ estimate of 55.4 Mya, which was inter- DNA sequences in humans and apes. Nature 416: 624–626. preted as inconsistent with Sahelanthropus, Orrorin or Patterson N, Richter DJ, Gnerre S, Lander ES and Reich D (2006) Ardipithecus kadabba being hominins, contrary to the con- Genetic evidence for complex speciation of humans and chim- sensus view of palaeoanthropologists. However, Patterson panzees. Nature 441: 1103–1108. et al. (2006) used unjustifiably young maximum bounds in Pilbeam DR (1968) The earliest hominids. Nature 219: 1335– 1338. the fossil calibration (18 Mya as the maximal human– Sarich VM and Wilson AC (1967) Immunological time scale for orangutan divergence) and furthermore treated these human evolution. Science 158: 1200–1203. maxima as absolute constraints. Steiper ME and Young NM (2006) Primate molecular divergence Although historically molecular evolutionary biologists dates. Molecular Phylogenetics and Evolution 41: 384–394. and palaeontologists have often had an antagonistic rela- Wood B (2002) Hominid revelations from Chad. Nature 418: tionship, in general the current molecular dates for the hu- 133–135. man–chimpanzee divergence are broadly congruent with Yi S, Ellsworth DL and Li W-H (2002) Slow molecular clocks in the fossil record, especially when the molecular dates are Old World monkeys, apes, and humans. Molecular Biology and seen as minimal estimates, and recognizing that they are Evolution 19: 2191–2198. highly dependent on an imperfect fossil record for calibra- Young NM and MacLatchy L (2004) The phylogenetic position of tion. Needless to say, while there is not complete unanimity Morotopithecus. Journal of Human Evolution 46: 163–184.

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Zuckerkandl E and Pauling L (1965) Evolutionary divergence and Elango N, Thomas JW, NISC Comparative Sequencing Program, convergence in proteins. In: Bryson V and Vogel HJ (eds) Evolv- Yi SV (2006) Variable molecular clocks in hominoids. Proceed- ing Genes and Proteins, pp. 97–166. New York: Academic Press. ings of the National Academy of Sciences of the USA 103: 1370– 1375. Steiper ME, Young NM and Sukarna TY (2004) Genomic data Further Reading support the hominoids slowdown and an Early Oligocene es- timate for the hominoid-cercopithecoid divergence. Proceed- Donoghue PCJ and Benton MJ (2007) Rocks and clocks: cali- ings of the National Academy of Sciences of the USA 101: 17021– brating the Tree of Life using fossils and molecules. Trends in 17026. Ecology and Evolution 22: 424–431.

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