Rates of Nucleotide Substitution in Primates and Rodents and the Generation–Time Effect Hypothesis

MOLECULAR PHYLOGENETICS AND EVOLUTION

Vol. 5, No. 1, February, pp. 182–187, 1996 ARTICLE NO. 0012

Rates of Nucleotide Substitution in Primates and Rodents and the Generation–Time Effect Hypothesis WEN-HSIUNG LI, DARRELL L. ELLSWORTH, JULIA KRUSHKAL, BENNY H.-J. CHANG, AND DAVID HEWETT-EMMETT Human Genetics Center, SPH, University of Texas Health Science Center, P.O. Box 20334, Houston, Texas 77225 Received July 17, 1995

DNA sequence data from introns, flanking regions, and the h globin pseudogene region all show a significantly higher rate of nucleotide substitution in the Old World monkey lineage than in the human lineage after the separation of the two lineages, or, in other words, the data support the hominoid rate-slowdown hypothesis. Data from both protein sequences and DNA sequences show that the rate of evolution is significantly higher in the rodent lineage than in the primate lineage. Furthermore, DNA sequences from introns show that the rate of nucleotide substitution is at least two times higher in rodents than in higher primates. The male-to-female ratio of mutation rate is estimated to be between 3 and 6 in higher primates, whereas it is only 2 in mice and rats. These ratios are similar to the corresponding male-to-female ratios of germ cell divisions in higher primates and in rodents, suggesting that errors in DNA replication during germ cell division are the primary source of mutation, or, in other words, mutation is largely DNA replication-dependent. This conclusion provides further support for the generation–time effect hypothesis.  1996 Academic Press, Inc.

INTRODUCTION The molecular clock hypothesis has been controversial ever since its proposal in 1965 by Zuckerkandl and Pauling. This hypothesis postulates that the rate of evolution in any given protein or DNA sequence is approximately constant over time in all evolutionary lineages. One important aspect of the controversy is why the rate-constancy is on a per year rather than per generation basis. Classical genetic studies indicate that mutation rates are more comparable among organisms when measured in terms of generation than in terms of absolute time. For this reason, the molecular clock should run faster in organisms with a short generation time, for they will go through more generations per unit time than do organisms with a long generation. This has been known as the generation–time effect hypothesis. 1055-7903/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

The controversy over the generation–time effect hypothesis actually started with the proposal that the rate of molecular evolution has become slower in hominoids (humans and apes) after their separation from the Old World (OW) monkeys (Goodman, 1961, 1962, 1963; Goodman et al., 1971). This proposal has been known as the hominoid rate-slowdown hypothesis. We shall briefly review the controversy on this hypothesis and then provide data to support it. Another line of effort to test the generation–time hypothesis has been to compare the substitution rates in the primate and rodent lineages using another mammalian lineage as a reference. Such studies have also been controversial mainly because of the uncertainty as to whether the reference lineage used is really an outgroup to the primate and rodent lineages (see later). Using recent data and our new data, we shall provide evidence for a higher rate in rodents than in humans. The generation–time effect hypothesis implicitly assumes that an important source of mutation is DNA replication errors during germ cell division. That is, organisms with a short generation time should go through more rounds of DNA replication in germ cells per unit time and thus should have a higher mutation rate than do organisms with a long generation time. To see whether DNA replication errors are indeed a very important source of mutation, Chang et al., (1994) have compared the male-to-female ratio of mutation rate with the male-to-female ratio of the numbers of cell divisions in male and female germ lines. This study and our new data provide further support for the generation–time hypothesis. EVIDENCE FOR THE HOMINOID RATESLOWDOWN HYPOTHESIS The hominoid rate-slowdown hypothesis was originally based on rates estimated from immunological distance and protein sequence data (Goodman, 1961, 1962, 1963; Goodman et al., 1971). Sarich and Wilson (1967) and Wilson et al. (1977) contended that the slowdown was an artifact, owing to the use of an erroneous paleontological estimate of the ape–human divergence

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TABLE 1 Differences in the Number of Nucleotide Substitutions per 100 Sites and the Relative Rates of Substitution between the Old World Monkey (Species 1) and Human (Species 2) Lineages with the New World Monkey (Species 3) as a Reference Sequence η Globin pseudogenec Introns IGF2 ε Globin Insulin Mast cell carboxypeptidase A3 Carbonic anhydrase VII Interferon α/β receptor Apolipoprotein C-III Lipoprotein lipase Total Flanking and untranslated regions ε Globin Insulin Total

Nucleotides compared

K12a

K13a

K23a

K13 2 K23

Rate ratiob

8781

6.7

11.8

10.7

1.1 6 0.3**

1.4

1589 928 862 1275 501 885 1270 1168 8478

6.4 4.9 9.7 5.5 7.2 7.6 8.7 7.9 7.1

15.8 11.5 17.0 13.3 11.1 14.0 18.5 13.6 14.7

14.2 11.5 15.9 12.5 9.7 14.0 16.9 13.8 13.9

1.6 6 0.8* 0.0 6 0.8 1.1 6 1.3 0.8 6 0.8 1.5 6 1.4 0.0 6 1.1 1.6 6 1.0 20.3 6 1.0 0.8 6 0.3**

1.7 1.0 1.3 1.3 1.5 1.0 1.5 1.0 1.3

388 548 936

5.3 9.8 7.9

13.5 15.8 14.9

10.6 12.6 11.7

2.9 6 1.4* 3.2 6 1.5* 3.1 6 1.1**

3.4 2.0 2.3

Note. Data from Bailey et al. (1991), Porter et al. (1995), and Ellsworth et al. (1993, and unpublished). a Kij 5 number of substitutions per 100 sites between species i and j. b The ratio of the rate in the Old World monkey lineage to the rate in the human lineage. c Excluding Alu sequences. *Significant at the 5% level. **Significant at the 1% level.

time. They conducted relative rate tests using both immunological distance data and protein sequence data and concluded that there was no evidence for a hominoid slowdown. A similar conclusion was drawn from DNA hybridization studies (Kohne et al., 1972; Sibley and Ahlquist, 1984); however, more recent DNA hybridization studies have produced conflicting conclusions (Sibley and Ahlquist, 1987; Caccone and Powell, 1989). On the other hand, comparative analyses of DNA sequence data by Koop et al. (1986), Li and Tanimura (1987), and Li et al. (1987) provided strong support for the hominoid slowdown hypothesis and the hypothesis was accepted by many molecular evolutionists. However, Easteal (1991) has argued that the slowdown occurred only in the η globin pseudogene because when this pseudogene was removed from comparison, the rate of nucleotide substitution in the OW monkey lineage was no longer significantly higher than that in the human lineage. To resolve this controversy, Seino et al. (1992) and Ellsworth et al. (1993) obtained more sequence data. In Table 1, K13 represents the distance between an OW monkey and a New World (NW) monkey and K23 the distance between the human and a NW monkey. For the introns compared, K13 2 K23 is positive, with the exceptions that K13 2 K23 is 0 for the ε globin and interferon α/β receptor introns and is slightly negative for the lipoprotein lipase intron. However, when all introns are considered together, K13 2 K23 is significantly

greater than 0, implying that the rate of substitution in the OW monkey lineage is significantly faster than that in the human lineage. The same conclusion is obtained from the flanking sequence data (Table 1). Thus, there is indeed evidence for the hominoid slowdown hypothesis, even if the η globin pseudogene is excluded from comparison. The intron sequence data suggest that the OW monkey lineage evolves 1.3 times faster than the human lineage, which is similar to that (1.4) estimated from the η globin data. The flanking sequence data suggest that the rate ratio is more than 2 times. However, since the latter data set is small, the ratio estimated from this set may not be reliable. Further data are needed to see whether the ratio varies among different DNA regions. HIGHER RATES IN RODENTS THAN IN PRIMATES From DNA hybridization data, Laird et al. (1969) and Kohne (1970) estimated the substitution rates between mouse and rat and between human and chimpanzee and concluded that the former rate is much higher than the latter. They attributed the higher rate in rodents to a shorter generation time, i.e., the generation–time effect. Sarich and Wilson (1973) argued that this difference in rate was based on questionable assumptions about the divergence times between species.

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TABLE 2 Number of Amino Acid Substitutions Attributable to the Rodent Sequence (NR ) and to the Human Sequence (NH ) Number of proteins a NR . NH 35 P , 0.005

Substitutions

NR , NH

NR 5 NH

12

7

NR

NH

600 416 P , 0.001

Note. From Gu and Li (1992). a Total number of proteins compared 5 54; total number of residues compared 5 16.365.

In order to avoid the assumption of divergence times, Gu and Li (1992) used the relative rate test with the chicken as an outgroup. They used amino acid sequences instead of DNA sequences because the chicken and mammalian lineages diverged about 300 million years ago, making it difficult to obtain reliable divergence estimates at synonymous sites. Let NR (NH ) be the number of residues at which the human and rodent sequences are different but the human and chicken (rodent and chicken) sequences are identical. For example, if the residues at a particular site are alanine in human and chicken but valine in rodent, then NR 5 1 and N H 5 0 for that site. However, if the residues at a site are different for the three sequences (e.g., alanine in human, valine in mouse, and leucine in chicken), then that site is noninformative and is not included in the comparison. For each protein NR (NH ) is the sum of the NR (N H ) values over the informative sites of the sequences. Under the assumption of equal substitution rates in the human and rodent lineages, N R should be statistically equal to NH for each protein. Among the 54 proteins compared, 35 proteins show a faster rate in the rodent lineage (i.e., NR . NH ), 12 show a faster rate in the human lineage (NH . NR ), and the rest (7 sequences) show an equal rate (Table 2). A sign test indicates a significantly faster rate in rodents than in humans (P , 0.005). When all the proteins are pooled together, NR 5 600 is clearly significantly larger than NH 5 416 (P , 0.001). Therefore, there is strong evidence for an overall faster substitution rate, from the common primate–rodent ancestor, in the lineage to mouse or rat (the rodent lineage) than in the lineage to humans (the human lineage). Comparing the rates of nucleotide substitution in the human and rodent lineages using marsupial genes as references, Easteal and Collet (1994) concluded that the rate of silent substitution did not differ between the rodent and human lineages, though the rate of nonsynonymous substitution was significantly higher in the rodent lineage than in the human lineage. They took the near equality of estimated silent rates in rodents and humans as evidence that the mutation rate is the

same in rodents and primates, i.e., a molecular clock exists at silent sites. However, as silent distances between marsupial and rodent (or human) genes are large, estimates of these distances should be taken with caution. Table 3 shows a summary of their data for the coding regions. Easteal and Collet noted that the number of transition substitutions per site at twofold degenerate sites (A2 ) is similar to that at fourfold degenerate sites (A4 ) for the human–rodent and human–marsupial comparisons (27.4 vs 25.8 and 48.9 vs 44.2) but is much higher than the latter in the rodent–marsupial comparison (54.4 vs 40.6). For this reason, they omitted the data for A2 , arguing that these data were biased and not as suitable as those for A4 . However, note that for fourfold degenerate sites the number of transitions per site (A4 ) is about the same as the number of transversions per site (B4 ) for both the human–rodent comparison and the human–marsupial comparison but is much lower than the latter in the rodent–marsupial comparison. We note that A4 tends to be higher than B 4 in mammalian genes (Li et al., 1985; Li, 1993). Thus, in the rodent–marsupial comparison, what is peculiar is A4 rather than A2 because A2 is similar to B 4 , whereas A4 is much lower than B4 . Indeed, with the exception of A4 , all Ai and Bi for the rodent–marsupial comparison are larger than the corresponding values for the human–marsupial comparison, suggesting a higher rate in the rodent lineage than in the human lineage. Note that the number of transition substitutions per site (A) is estimated by the formula A 5 2(1/2) ln(1 2 2P 2 Q) 1 (1/4)ln(1 2 2Q), where P and Q are the proportions of transitional and transversional differences between the two sequences (Kimura, 1980; Wu and Li, 1985). Therefore, it is easier to obtain a reliable estimate of A when Q is small, which is the case for twofold degenerate sites, than when Q is large, which is the case for fourfold degenerate sites. For this reason, A2 is preferable to A4 and, as noted above, the A2 values point to a higher synonymous rate in the rodent lineage than in the human lineage. Moreover, since the number of transversional substitutions (B) is given by the simple formula B 5 2(1/2)ln(1 2 2Q), (Kimura, 1980; Wu and Li, 1985), it is easier to estimate B than A. Let us therefore consider B4 . For B4 , the difference between the rodent–marsupial and human– marsupial comparisons is 6.7%, which is significant because the standard error is 2.7%. In conclusion, the results in Table 3 actually point to a higher rate in the rodent lineage than in the human lineage for synonymous substitutions as well as for nonsynonymous substitutions.

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TABLE 3 Numbers of Transition Substitutions (Ai ) and Transversion Substitutions (Bi ) at Nondegenerate, Twofold Degenerate, and Fourfold Degenerate Sites in Comparisons between Human, Rodent, and Marsupial Genes Nondegenerate Taxa compared Human–rodent Human–marsupial Rodent–marsupial

Twofold

Fourfold

A0

B0

A2

B2

A4

B4

4.4 6 0.3 7.3 6 0.4 7.6 6 0.4

4.3 6 0.3 8.7 6 0.4 9.7 6 0.4

27.4 6 1.6 48.9 6 2.8 54.4 6 3.2

5.2 6 0.5 8.5 6 0.7 9.3 6 0.7

25.8 6 1.8 44.2 6 3.6 40.6 6 3.6

20.6 6 1.4 45.7 6 2.8 52.4 6 3.2

Note. Data from Easteal and Collet (1994).

Since at the early stage of the primate–rodent divergence the rates in the two lineages should have been very similar, the rate differences between the two lineages should have occurred mainly in more recent times. This appears to be the case. In order to estimate the substitution rate between two species we must know their divergence time. However, divergence times are usually not well established, so we consider a range of estimates and an intermediate date for every pair of species compared (Table 4). It is commonly thought (Gingerich, 1984; Pilbeam, 1984) that the divergence between the human and OW monkey lineages came after middle Oligocene times (some 30 Myr ago) and before early Miocene times (some 20 Myr ago), so we use a range of 20 to 30 Myr with an intermediate date of 25 Myr. The divergence time between the NW and OW monkey lineages has been generally thought to be between 35 and 45 Myr ago (Gingerich, 1984; Pilbeam, 1984; Fleagle et al., 1986) and we use a range of 30 to 45 Myr with an intermediate of 35 Myr ago. The divergence time between mouse and rat has been estimated to be between 8 and 14 Myr ago (Jacobs and Pilbeam, 1980), but Wilson et al. (1977) argued that it can be anywhere from 5 to 35 Myr ago. We use a range of 10 to 30 Myr but with an intermediate date of 15 Myr. Table 4 shows the estimates of the substitution rate in introns. The intermediate estimates are 1.4 3 1029, 2.1 3 1029, and 4.8 3 1029 for the human–OW monkey, human–NW monkey, and mouse–rat comparisons, im-

TABLE 4 Rates of Substitution per Site per Year in Introns Species pair Human vs OW monkeys Human vs NW monkeys Mouse vs rat

L (bp)

Percent divergence

Time (106 years)

Rate (1029 )

8478

7.1

25 (20–30)

1.4 (1.2–1.8)

8478

14.7

35 (30–45)

2.1 (1.6–2.5)

4038

14.4

15 (10–30)

4.8 (2.4–7.2)

Note. From Li, Ellsworth, and Krushkal (unpublished). Notation: OW, Old World; NW, New World.

plying that the rate may be two to four times higher in rodents than in higher primates. However, these comparisons should be taken with caution because they involve assumptions of divergence times and are based on limited sequence data. SEX RATIOS OF MUTATION RATE IN PRIMATES AND RODENTS Since Haldane (1935, 1947), it has been commonly believed that the mutation rate is much higher in the human male germ line than in the female germ line because the number of germ cell divisions per generation is much larger in males than in females. However, direct estimation of mutation rates was difficult and data supporting this view, mainly from X-linked genetic diseases, were limited (see Vogel and Motulsky 1986). Thus, it was unclear how high the ratio (α) of male to female mutation rates is. Another problem with this type of data is that no distinction between indels (insertions and deletions) and substitution mutations was made because the molecular defects causing the disease were not examined. Here we are interested in substitution mutations. With modern molecular techniques, one can determine the type of defect in a patient and may be able to trace the pedigree to determine the origin of mutation, i.e., whether the mutation occurred in a male or a female ancestor. From such data, the sex ratio (α) of mutation rate can be obtained. Applying this method to hemophilia B patients, Ketterling et al. (1993) obtained an estimate of α 5 3.5 A simpler alternative approach was proposed by Miyata et al. (1987), who considered the ratios of mutation rates in different types of chromosomes. For example, the ratio of the mutation rate in a Y-linked sequence to that in an X-linked sequence is Y/X 5 3α/(2 1 α). Since the substitution rate in a neutral sequence is equal to its mutation rate, the ratio Y/X can be estimated by sequencing a Y-linked neutral sequence and an X-linked homologous sequence from different species. Shimmin et al. (1993) applied this approach to the last introns of the zinc finger protein genes on the Y

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and X chromosomes (i.e., ZFY and ZFX genes) from humans, orangutans, baboons, and squirrel monkeys (a New World monkey) and obtained an estimate of α 5 6. Chang, Hewett-Emmett, and Li (unpublished data) extended the study to the colobus monkey (an Old World monkey) and the tamarin (a New World monkey) and found that the α value from these two species and the four species studied by Shimmin et al. remain unchanged. They also sequenced two introns from the SMC genes on the X and Y chromosomes in humans, orangutans, baboons, and squirrel monkeys and obtained an estimate of α < 4.2. Thus, the α value in higher primates is probably between 3 and 6. To see the effect of generation time on α, Chang et al. (1994) sequenced the last introns of Zfy and Zfx genes in mouse and rat and obtained an estimate of α 5 1.8. In addition, Chang and Li (1995) sequenced the Y- and X-linked Ube1 genes and pseudogenes and estimated α 5 2.0. These estimates suggest that the α value (,2) in mice and rats is considerably lower than that in higher primates. Therefore, there appear to be generation–time effects on α. THE GENERATION–TIME EFFECT HYPOTHESIS The data summarized above support the generation– time effect hypothesis. As noted, the rate of substitution is higher in monkeys than in humans and is even higher in rodents. These observations are consistent with the generation–time effect hypothesis because among these three groups of organisms, the generation time is highest in humans, intermediate in monkeys, and lowest in rodents. The hypothesis is further supported by the observation that the rate of substitution in the chloroplast genome is more than five times higher in grasses than in palms (Gaut et al. 1992). The generation–time effect hypothesis implicitly assumes that errors during DNA replication are the major source of mutation. This assumption can be tested by comparing the sex ratio of mutation rate (α) and the sex ratio of the number of germ cell divisions (c). Using published data on gametogenesis, Li (unpublished) has estimated the numbers of germ cell divisions per generation in females (nf ) and males (nm) and the ratio c 5 nm /nf . In the development of a female mouse, the number of DNA replications in the germ line (i.e., from zygote to mature egg) is nf < 27. In the development of male mice, the number of germ cell divisions from zygote to the formation of stem spermatogonia is <29. Spermatogenesis requires 10 additional DNA replications to produce spermatids, initiating on Day 6 after birth and occurring on average every 8.6 days in adult male mice. The span of high reproductivity in laboratory male mice is approximately from 2 to 8 months of age. If we assume that the average reproductive age of male mice in the wild is 5 months, then a stem spermatognia would have gone through 5 3 30/8.6 < 17 divi-

sions and the total number of DNA replications from zygote to age 5 months is nm < 29 1 10 1 17 5 56. Therefore, c < 56/27 5 2.1. If the average reproductive age is 2 or 8 months, c becomes 1.6 or 2.4, respectively. For the rat, nf < 28. The number of cell divisions in the male germ line to form stem spermatogonia is <31, the spermatogenesis cycle occurs every ,12.9 days, and the period of maximal fertility of laboratory rats occurs between 100 and 300 days after birth. If we assume that the average reproductive age of males in the wild is 7 months, a stem spermatogonium would have gone through 7 3 30/12.9 5 16 divisions, and the total number of DNA replications in the male germ line is nm < 31 1 10 1 16 5 57 and c < 57/28 5 2.0. In humans, nf < 33. The data for n m are more scanty than those for rodents and so the estimate of nm is less reliable. The number of cell divisions from zygote to stem spermatogonia at puberty was estimated to be ,40. Spermatogenesis requires five further DNA replications and the spermatogenesis cycle occurs every ,16 days or 23 cycles per year. If the average reproductive age of males is 20 years, then the number of DNA replications for stem spermatogonia from puberty (age 13) to 20 is (20 2 13) 3 23 < 160, nm < 40 1 5 1 160 5 205, and c < 6.2. If the average reproductive age of males is 15, then c < 2.8. Although these estimates are crude, they are similar to the estimates of α 5 2 in mice and rats and α 5 3 to 6 in higher primates. This rough agreement suggests that errors in DNA replication during germ cell divisions are indeed the primary source of mutation and that the contribution of replication-independent factors such as oxygen free radicals to mutation is considerably less important. In summary, both the comparison of substitution rates in higher primates and in rodents and the sex ratios of mutation in these organisms support the generation–time effect hypothesis. ACKNOWLEDGMENTS This study was supported by NIH grants.

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