Mutation rates vary between species across several orders of magnitude, with larger organisms having the highest per-generation mutation rates. Hypotheses for this pattern typically invoke physiological or population-genetic constraints imposed on the molecular machinery preventing mutations []. However, continuing germline cell division in multicellular eukaryotes means that organisms with longer generation times and of larger size will leave more mutations to their offspring simply as a byproduct of their increased lifespan []. Here, we deeply sequence the genomes of 30 owl monkeys (Aotus nancymaae) from six multi-generation pedigrees to demonstrate that paternal age is the major factor determining the number of de novo mutations in this species. We find that owl monkeys have an average mutation rate of 0.81 × 10per site per generation, roughly 32% lower than the estimate in humans. Based on a simple model of reproductive longevity that does not require any changes to the mutational machinery, we show that this is the expected mutation rate in owl monkeys. We further demonstrate that our model predicts species-specific mutation rates in other primates, including study-specific mutation rates in humans based on the average paternal age. Our results suggest that variation in life history traits alone can explain variation in the per-generation mutation rate among primates, and perhaps among a wide range of multicellular organisms.

Results and Discussion

−10 per base in Archaea to more than 1 × 10−8 in mammals [ 1 Lynch M. Evolution of the mutation rate. 4 Drake J.W. A constant rate of spontaneous mutation in DNA-based microbes. 5 Kondrashov A.S. Modifiers of mutation-selection balance: general approach and the evolution of mutation rates. 6 Lynch M. The origins of eukaryotic gene structure. 7 Lynch M. The Origins of Genome Architecture. 8 Lynch M. The cellular, developmental and population-genetic determinants of mutation-rate evolution. 9 Damuth J. Population-density and body size in mammals. The rate at which new mutations arise is a key parameter of life on Earth, contributing to both individual disease risk and the evolution of novel traits. The mutation rate per generation varies among taxa, from as low as 1 × 10per base in Archaea to more than 1 × 10in mammals []. Two classes of models have been proposed to explain this variation. In one, the physiological and biochemical costs of increased fidelity during DNA replication limit the minimum mutation rate achievable []. Selection for faster replication in smaller organisms constrains the accuracy with which the cellular machinery can copy DNA, resulting in an inverse relationship between body size and mutation rate. Alternatively, a population-genetic model invokes the limits to natural selection in organisms with smaller population sizes []. This model posits a higher rate of mutation in larger organisms because of their generally smaller population size [].

2 Crow J.F. The origins, patterns and implications of human spontaneous mutation. 10 Weinberg W. Zur vererbung des zwergwuchses. 3 Kong A.

Frigge M.L.

Masson G.

Besenbacher S.

Sulem P.

Magnusson G.

Gudjonsson S.A.

Sigurdsson A.

Jonasdottir A.

Jonasdottir A.

et al. Rate of de novo mutations and the importance of father’s age to disease risk. 11 Michaelson J.J.

Shi Y.

Gujral M.

Zheng H.

Malhotra D.

Jin X.

Jian M.

Liu G.

Greer D.

Bhandari A.

et al. Whole-genome sequencing in autism identifies hot spots for de novo germline mutation. 12 Wong W.S.

Solomon B.D.

Bodian D.L.

Kothiyal P.

Eley G.

Huddleston K.C.

Baker R.

Thach D.C.

Iyer R.K.

Vockley J.G.

Niederhuber J.E. New observations on maternal age effect on germline de novo mutations. 13 Goldmann J.M.

Wong W.S.

Pinelli M.

Farrah T.

Bodian D.

Stittrich A.B.

Glusman G.

Vissers L.E.

Hoischen A.

Roach J.C.

et al. Parent-of-origin-specific signatures of de novo mutations. 14 Besenbacher S.

Sulem P.

Helgason A.

Helgason H.

Kristjansson H.

Jonasdottir A.

Jonasdottir A.

Magnusson O.T.

Thorsteinsdottir U.

Masson G.

et al. Multi-nucleotide de novo mutations in humans. 15 Girard S.L.

Bourassa C.V.

Lemieux Perreault L.P.

Legault M.A.

Barhdadi A.

Ambalavanan A.

Brendgen M.

Vitaro F.

Noreau A.

Dionne G.

et al. Paternal age explains a major portion of de novo germline mutation rate variability in healthy individuals. 16 Rahbari R.

Wuster A.

Lindsay S.J.

Hardwick R.J.

Alexandrov L.B.

Turki S.A.

Dominiczak A.

Morris A.

Porteous D.

Smith B.

et al. UK10K Consortium

Timing, rates and spectra of human germline mutation. 17 Jónsson H.

Sulem P.

Kehr B.

Kristmundsdottir S.

Zink F.

Hjartarson E.

Hardarson M.T.

Hjorleifsson K.E.

Eggertsson H.P.

Gudjonsson S.A.

et al. Parental influence on human germline de novo mutations in 1,548 trios from Iceland. 3 Kong A.

Frigge M.L.

Masson G.

Besenbacher S.

Sulem P.

Magnusson G.

Gudjonsson S.A.

Sigurdsson A.

Jonasdottir A.

Jonasdottir A.

et al. Rate of de novo mutations and the importance of father’s age to disease risk. 12 Wong W.S.

Solomon B.D.

Bodian D.L.

Kothiyal P.

Eley G.

Huddleston K.C.

Baker R.

Thach D.C.

Iyer R.K.

Vockley J.G.

Niederhuber J.E. New observations on maternal age effect on germline de novo mutations. 13 Goldmann J.M.

Wong W.S.

Pinelli M.

Farrah T.

Bodian D.

Stittrich A.B.

Glusman G.

Vissers L.E.

Hoischen A.

Roach J.C.

et al. Parent-of-origin-specific signatures of de novo mutations. 14 Besenbacher S.

Sulem P.

Helgason A.

Helgason H.

Kristjansson H.

Jonasdottir A.

Jonasdottir A.

Magnusson O.T.

Thorsteinsdottir U.

Masson G.

et al. Multi-nucleotide de novo mutations in humans. 15 Girard S.L.

Bourassa C.V.

Lemieux Perreault L.P.

Legault M.A.

Barhdadi A.

Ambalavanan A.

Brendgen M.

Vitaro F.

Noreau A.

Dionne G.

et al. Paternal age explains a major portion of de novo germline mutation rate variability in healthy individuals. 17 Jónsson H.

Sulem P.

Kehr B.

Kristmundsdottir S.

Zink F.

Hjartarson E.

Hardarson M.T.

Hjorleifsson K.E.

Eggertsson H.P.

Gudjonsson S.A.

et al. Parental influence on human germline de novo mutations in 1,548 trios from Iceland. 18 Goriely A. Decoding germline de novo point mutations. 19 Ségurel L.

Wyman M.J.

Przeworski M. Determinants of mutation rate variation in the human germline. 20 Venn O.

Turner I.

Mathieson I.

de Groot N.

Bontrop R.

McVean G. Nonhuman genetics. Strong male bias drives germline mutation in chimpanzees. One difficulty in teasing apart the forces driving the evolution of the mutation rate among multicellular organisms is the fact that lifespan varies as much as the per-generation mutation rate. In multicellular organisms, the number of mutations passed on to offspring in a single generation is a combination of the errors made in each round of germline replication and the accumulation of unrepaired DNA damage. One hundred years after the first observation of increased disease incidence in the children of older parents [], whole-genome sequencing in humans revealed the precise contribution of parental age to the number of de novo mutations in their offspring []. In particular, the number of mutations passed on to the next generation is largely dependent on the age of the father [], though there is a non-negligible contribution from the age of the mother []. This is a consequence of the fact that after a set number of germline mitoses during development in both males and females, the male germline resumes cell division at puberty []. A similar effect of paternal age has been found in chimpanzees [], suggesting that the age of reproduction may generally be an important determinant of the per-generation mutation rate.

21 Dixson A.F.

Gardner J.S.

Bonney R.C. Puberty in the male owl monkey (Aotus trivirgatus griseimembra): A study of physical and hormonal development. 22 Rowe N. The Pictorial Guide to the Living Primates. 23 Huck M.

Rotundo M.

Fernandez-Duque E. Growth and development in wild owl monkeys (Aotus azarai) of Argentina. Figure 1 Pedigree Structures and Mutation Rates in Owl Monkeys Show full caption (A) We used six multi-generation pedigrees in these two formats. Four families have a single F2 offspring (left), and two families have two F2 offspring (right). In total, 14 independent trios can be constructed from these pedigrees. (B) Mutation rate estimates from the 14 owl monkey trios (purple points). A simple linear regression has been fit to these points (solid purple line; shaded area indicates 95% confidence interval) to show that the number of mutations increases with the father’s age. Our model of reproductive longevity (dashed purple line) is not significantly different from the fit of the linear regression. The rate of non-replicative mutations, such as those that occur at CpG sites (blue dots), are not correlated with reproductive longevity (blue line). The dotted vertical gray line indicates expected age of puberty. See also Data S1 and Figure S1 Studying closely related primates offers a unique opportunity to examine the role that life history traits—such as age of puberty and average generation time—may play in determining mutation rates. We sequenced the genomes of 30 owl monkeys (Aotus nancymaae) within six multi-generation pedigrees ( Figure 1 A; Data S1 A) in order to estimate the effect of parental age on the mutation rate. Owl monkeys reach sexual maturity at ∼1 year of age [] and can live up to 20 years in captivity []. Our sample includes individuals conceived by sires ranging from 3–13 years old and dams ranging from 3–12 years old, with an average age of 6.64 and 6.53 for sires and dams, respectively ( Data S1 A). These ages are comparable to those observed in the wild, as owl monkeys are solitary for some time before joining a mating group at around age 4 []. The genomes of all parents and offspring were sequenced to an average of 37× coverage (range: 35×–38×) using paired-end Illumina reads. Sequencing multi-generation pedigrees allows us to determine whether de novo mutations arose in either sires or dams, as well as to validate mutations transmitted to the next generation.

−8 per site per generation (2 = 0.25, d.f. = 12, p = 0.040). Also as expected, we find no effect of age on CpG mutations (2 = 0.58, d.f. = 4, p = 0.048). We did not find an increasing number of mutations with maternal age (R2 = 0.07, d.f. = 4, p = 0.307) or age of the offspring (R2 = −0.02, d.f. = 12, p = 0.388). This is the first direct observation of the paternal age effect outside of apes. We observe 283 de novo mutations across 14 trios ( Data S1 B) and estimate an average mutation rate for owl monkeys of 0.81 × 10per site per generation ( Data S1 C). In addition to stringent quality filters (see STAR Methods ), the average transmission frequency of de novo mutations passed from F1 individuals to F2 individuals across families was 0.502, giving us high confidence in our final set of mutations. As in humans, we find a strong association between paternal age and the number of de novo mutations ( Figure 1 B), with 2.92 additional mutations accumulating per year (R= 0.25, d.f. = 12, p = 0.040). Also as expected, we find no effect of age on CpG mutations ( Figure 1 B, blue points and line), as these are not associated with replication errors. We were able to assign phase to 105 of the 283 de novo mutations via transmission to the third generation in our pedigrees ( Data S1 B). We find that 71 of these 105 phased mutations are paternal, with the number of mutations passed on increasing with the age of the father (R= 0.58, d.f. = 4, p = 0.048). We did not find an increasing number of mutations with maternal age (R= 0.07, d.f. = 4, p = 0.307) or age of the offspring (R= −0.02, d.f. = 12, p = 0.388). This is the first direct observation of the paternal age effect outside of apes.

3 Kong A.

Frigge M.L.

Masson G.

Besenbacher S.

Sulem P.

Magnusson G.

Gudjonsson S.A.

Sigurdsson A.

Jonasdottir A.

Jonasdottir A.

et al. Rate of de novo mutations and the importance of father’s age to disease risk. 3 Kong A.

Frigge M.L.

Masson G.

Besenbacher S.

Sulem P.

Magnusson G.

Gudjonsson S.A.

Sigurdsson A.

Jonasdottir A.

Jonasdottir A.

et al. Rate of de novo mutations and the importance of father’s age to disease risk. 14 Besenbacher S.

Sulem P.

Helgason A.

Helgason H.

Kristjansson H.

Jonasdottir A.

Jonasdottir A.

Magnusson O.T.

Thorsteinsdottir U.

Masson G.

et al. Multi-nucleotide de novo mutations in humans. 24 Schrider D.R.

Hourmozdi J.N.

Hahn M.W. Pervasive multinucleotide mutational events in eukaryotes. 14 Besenbacher S.

Sulem P.

Helgason A.

Helgason H.

Kristjansson H.

Jonasdottir A.

Jonasdottir A.

Magnusson O.T.

Thorsteinsdottir U.

Masson G.

et al. Multi-nucleotide de novo mutations in humans. 24 Schrider D.R.

Hourmozdi J.N.

Hahn M.W. Pervasive multinucleotide mutational events in eukaryotes. Figure 2 Comparison of Mutational Spectra from Owl Monkeys, Humans, and Chimpanzees Show full caption χ 2 = 25.7, d.f. = 4, p < 0.05), but otherwise no difference between mutational spectra for these three species. Human data were averaged across four studies (see 20 Venn O.

Turner I.

Mathieson I.

de Groot N.

Bontrop R.

McVean G. Nonhuman genetics. Strong male bias drives germline mutation in chimpanzees. There is a slight but significant difference in the frequency of A→T mutations between owl monkeys and humans (= 25.7, d.f. = 4, p < 0.05), but otherwise no difference between mutational spectra for these three species. Human data were averaged across four studies (see Data S1 D for references), and chimpanzee data were extrapolated from Figure 3A in Venn et al. []. Mutation categories include their reverse complement. See also Data S1 B. Inspection of the types of mutations found in the genomes of owl monkeys shows a transition:transversion (Ts:Tv) ratio of 1.97. This is in close agreement with the observed human Ts:Tv ratio of 2.10 []. In fact, the overall mutational spectrum between humans, chimpanzees, and owl monkeys appears almost identical, with the only difference being a slightly higher proportion of A→T mutations in owl monkeys ( Figure 2 ). We also observe that 12.0% of mutations in owl monkeys occur at CpG sites, with CpG sites having a much higher Ts:Tv ratio (4.67), similar to observations in humans []. Multinucleotide mutations (MNMs) are mutations that occur in close proximity to one another (<20 bp apart), most likely caused by a single mutational event []. Here, we find six MNMs consisting of two mutations each, indicating that 2.1% of de novo mutations in owl monkeys are the result of MNMs ( Data S1 B). This fraction is also in agreement with that observed within humans [].

−8 mutations per site per generation [ 3 Kong A.

Frigge M.L.

Masson G.

Besenbacher S.

Sulem P.

Magnusson G.

Gudjonsson S.A.

Sigurdsson A.

Jonasdottir A.

Jonasdottir A.

et al. Rate of de novo mutations and the importance of father’s age to disease risk. 25 Scally A. The mutation rate in human evolution and demographic inference. 19 Ségurel L.

Wyman M.J.

Przeworski M. Determinants of mutation rate variation in the human germline. 25 Scally A. The mutation rate in human evolution and demographic inference. 26 Thomas G.W.C.

Hahn M.W. The human mutation rate is increasing, even as it slows. 3 Kong A.

Frigge M.L.

Masson G.

Besenbacher S.

Sulem P.

Magnusson G.

Gudjonsson S.A.

Sigurdsson A.

Jonasdottir A.

Jonasdottir A.

et al. Rate of de novo mutations and the importance of father’s age to disease risk. 11 Michaelson J.J.

Shi Y.

Gujral M.

Zheng H.

Malhotra D.

Jin X.

Jian M.

Liu G.

Greer D.

Bhandari A.

et al. Whole-genome sequencing in autism identifies hot spots for de novo germline mutation. 12 Wong W.S.

Solomon B.D.

Bodian D.L.

Kothiyal P.

Eley G.

Huddleston K.C.

Baker R.

Thach D.C.

Iyer R.K.

Vockley J.G.

Niederhuber J.E. New observations on maternal age effect on germline de novo mutations. 13 Goldmann J.M.

Wong W.S.

Pinelli M.

Farrah T.

Bodian D.

Stittrich A.B.

Glusman G.

Vissers L.E.

Hoischen A.

Roach J.C.

et al. Parent-of-origin-specific signatures of de novo mutations. 14 Besenbacher S.

Sulem P.

Helgason A.

Helgason H.

Kristjansson H.

Jonasdottir A.

Jonasdottir A.

Magnusson O.T.

Thorsteinsdottir U.

Masson G.

et al. Multi-nucleotide de novo mutations in humans. 15 Girard S.L.

Bourassa C.V.

Lemieux Perreault L.P.

Legault M.A.

Barhdadi A.

Ambalavanan A.

Brendgen M.

Vitaro F.

Noreau A.

Dionne G.

et al. Paternal age explains a major portion of de novo germline mutation rate variability in healthy individuals. 16 Rahbari R.

Wuster A.

Lindsay S.J.

Hardwick R.J.

Alexandrov L.B.

Turki S.A.

Dominiczak A.

Morris A.

Porteous D.

Smith B.

et al. UK10K Consortium

Timing, rates and spectra of human germline mutation. 17 Jónsson H.

Sulem P.

Kehr B.

Kristmundsdottir S.

Zink F.

Hjartarson E.

Hardarson M.T.

Hjorleifsson K.E.

Eggertsson H.P.

Gudjonsson S.A.

et al. Parental influence on human germline de novo mutations in 1,548 trios from Iceland. The mutation rate we observe in owl monkeys is 32.5% lower than the average human estimate of 1.2 × 10mutations per site per generation []. Although traditional models of mutation rate evolution invoke changes to the underlying replication machinery as the main cause of such differences, we asked whether a shift in reproductive timing could explain the lower rate in owl monkeys. The effects of paternal age on per-generation mutation rates have previously been modeled by combining estimates of the rate of mutation from different life stages []. The germline in males and females undergo a fixed number of divisions before birth, but the male germline continues dividing upon reaching sexual maturity. This phenomenon suggests that the length of time between puberty and the conception of offspring in an individual—which we define here as the “reproductive longevity” of males—plays a key role in determining the number of mutations passed on to the next generation. Although paternal age is sufficient for predicting mutation rates within a species [], the concept of reproductive longevity makes it possible to predict mutation rates between species with varying ages of puberty. We modeled the owl monkey mutation rate as a linear combination of the mutations accumulated as a result of a constant number of germline divisions in utero and those accumulated during continued germline divisions post-puberty. The rate of mutation in these two stages was estimated from human studies, and sexual maturity was set at 1 year of age ( STAR Methods ).

Our minimal model provides an excellent fit to the observed owl monkey data ( Figure 1 B, dashed line). In fact, a linear regression of the observed number of mutations with paternal age at conception is not significantly better than the predictions provided by our model (F = 0.996, d.f. = 13, p = 0.994). The main determinant of the mutation rate is reproductive longevity in sires, which determines the number of mitotic germline divisions before spermatogenesis. For instance, a 13-year-old owl monkey male (who reached sexual maturity at 1) will have the same reproductive longevity as a 25-year-old human male (who reached sexual maturity at 13). Our model therefore predicts the same estimated mutation rate if de novo mutations are sampled from offspring of these individuals, and this is what was observed ( Figure 1 B). Because reproductive longevity reflects replicative mutations, we observed no effect of father’s age on non-replicative mutations, such as those found at CpG sites ( Figure 1 B, blue).

27 Dixson A.

Anderson M. Sexual selection and the comparative anatomy of reproduction in monkeys, apes, and human beings. 28 Presgraves D.C.

Yi S.V. Doubts about complex speciation between humans and chimpanzees. 25 Scally A. The mutation rate in human evolution and demographic inference. 26 Thomas G.W.C.

Hahn M.W. The human mutation rate is increasing, even as it slows. 29 Yi S.V. Morris Goodman’s hominoid rate slowdown: the importance of being neutral. 30 Moorjani P.

Amorim C.E.G.

Arndt P.F.

Przeworski M. Variation in the molecular clock of primates. The association between mutation rates and reproductive longevity implies that changes in life history traits rather than changes to the mutational machinery are responsible for the evolution of these rates. Species that have evolved greater reproductive longevity will have a higher mutation rate per generation without any underlying change to the replication, repair, or proofreading proteins. The similarities between the mutational spectra of humans, chimpanzees, and owl monkeys ( Figure 2 ) are further evidence that the molecular mechanisms responsible for mutation have not changed between these species. Many differences in the details of germline cell division may exist between these primates, but these differences do not appear to affect either pre-birth or post-puberty mutation accumulation. For instance, varying levels of sexual selection between species in the form of sperm competition leads to variation testis and ejaculate size []. This sort of variation most likely also affects mutation rates through changing the germline replication rate [], which can be accommodated in our model (see STAR Methods ). The underlying consistency of mutation rates must also be reconciled with variation in the long-term substitution rate among primates [], as mutation rates are mechanistically tied to substitution rates (see STAR Methods and Figure S3 ). Nevertheless, the close fit between the observed and expected mutation rates suggests that reproductive longevity is the major determinant of variation in mutation rates.