Molecular Biology and Evolution

MBE Advance Access originally published online on January 29, 2008
Molecular Biology and Evolution 2008 25(4):778-786; doi:10.1093/molbev/msn024

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Research Articles

Divergence Times in Caenorhabditis and Drosophila Inferred from Direct Estimates of the Neutral Mutation Rate

Asher D. Cutter

Department of Ecology and Evolutionary Biology and the Centre for the Analysis of Genome Evolution and Function, University of Toronto, Toronto, Canada

E-mail: asher.cutter{at}utoronto.ca.

	Abstract

TOP Abstract Introduction Materials and Methods Results and Discussion Conclusions Supplementary Material Acknowledgements References

 
Accurate inference of the dates of common ancestry among species forms a central problem in understanding the evolutionary history of organisms. Molecular estimates of divergence time rely on the molecular evolutionary prediction that neutral mutations and substitutions occur at the same constant rate in genomes of related species. This underlies the notion of a molecular clock. Most implementations of this idea depend on paleontological calibration to infer dates of common ancestry, but taxa with poor fossil records must rely on external, potentially inappropriate, calibration with distantly related species. The classic biological models Caenorhabditis and Drosophila are examples of such problem taxa. Here, I illustrate internal calibration in these groups with direct estimates of the mutation rate from contemporary populations that are corrected for interfering effects of selection on the assumption of neutrality of substitutions. Divergence times are inferred among 6 species each of Caenorhabditis and Drosophila, based on thousands of orthologous groups of genes. I propose that the 2 closest known species of Caenorhabditis shared a common ancestor <24 MYA (Caenorhabditis briggsae and Caenorhabditis sp. 5) and that Caenorhabditis elegans diverged from its closest known relatives <30 MYA, assuming that these species pass through at least 6 generations per year; these estimates are much more recent than reported previously with molecular clock calibrations from non-nematode phyla. Dates inferred for the common ancestor of Drosophila melanogaster and Drosophila simulans are roughly concordant with previous studies. These revised dates have important implications for rates of genome evolution and the origin of self-fertilization in Caenorhabditis.

Key Words: divergence time • Caenorhabditis • Drosophila • molecular evolution

	Introduction

TOP Abstract Introduction Materials and Methods Results and Discussion Conclusions Supplementary Material Acknowledgements References

 
A major problem in understanding the history of life is the inference of appropriate dates of common ancestry among species—the difficulty of which is compounded in taxa that lack fossil or biogeographic reference points (Bromham and Penny 2003; Kumar 2005). Paleontological evidence is absent for the nematode model organism Caenorhabditis elegans and its relatives, and the Melanogaster subgroup of Drosophila relies on biogeographic calibration of molecular divergence with distantly related Hawaiian congeners (Rowan and Hunt 1991; Russo et al. 1995; Tamura et al. 2004). In these and other taxa with poor fossil records, it would be desirable to infer dates of common ancestry without relying on distant external calibration that might inappropriately reflect evolution in the focal taxon. External calibrations are particularly problematic in Caenorhabditis, which appears to experience a higher mutation rate than other metazoans (Mushegian et al. 1998; Denver et al. 2004), leading to valid concerns (Félix 2004; Kiontke et al. 2004) about published divergence date estimates (Coghlan and Wolfe 2002; Stein et al. 2003). Explicit incorporation of local or relaxed molecular clock models that accommodate rate variation provide one valuable avenue for inferring divergence times for taxa with evidence of rate differences among them (Thorne et al. 1998; Huelsenbeck et al. 2000; Sanderson 2002; Drummond et al. 2006). In this study, however, I derive dates of divergence by applying internal molecular clock calibration from direct estimates of the rate of neutral mutation in contemporary laboratory populations (Denver et al. 2004; Haag-Liautard et al. 2007) to neutral substitution rates inferred for thousands of orthologs from 6 species in each of these genera.

A key prediction of the neutral theory of molecular evolution holds that the rate of substitution of neutral mutations will be independent of population size and will be equal to the rate of neutral mutation (Kimura 1968). This follows as a consequence of a diploid population producing 2Nµ new mutations per generation (neutral mutation rate µ and effective population size N), each with fixation probability 1/2N via genetic drift; the substitution rate at equilibrium is therefore 2Nµ x 1/2N = µ neutral substitutions per generation and is independent of population size. This framework forms the theoretical basis of the notion of the molecular clock (Zuckerkandl and Pauling 1962), in which the time to the most recent common ancestor (TMRCA) (T) of a pair of lineages may be inferred from DNA sequences, assuming that the neutral divergence (K) between them will simply be the product of the substitution rate (µ) and the duration of divergence, summed across both lineages: K = 2µT. Heterogeneity in divergence among lineages might be caused by differences in the per generation mutation rate or by different generation times. Note that, strictly speaking, the molecular clock prediction applies only to neutral substitutions, although it is often successfully employed to date common ancestors using protein evolution (Kumar and Hedges 1998). Typically, the TMRCA is inferred using one or more paleontological or biogeographic reference points to calibrate the relative divergence between a given set of lineages (Bromham and Penny 2003; Kumar 2005). This standard approach requires that a fossil record exists for the taxa under consideration and that historical dates of divergence can be inferred accurately. A limitation of this traditional method is that taxa with poor fossil preservation must rely on calibrations from distantly related organisms, which may experience drastically different mutation rates or generation times and therefore compromise estimates of divergence time.

Here, I demonstrate the utility of an alternative approach to calibrating a taxonomically local molecular clock that exploits the neutral mutation rate measured directly in species for which this parameter can be estimated in contemporary laboratory populations. A concern of applying contemporary rates of mutation to the deeper timescale of substitution is that comparative estimates of mutation rates generally are lower than those based on pedigrees or mutation accumulation experiments (Ochman et al. 1999; Ochman 2003; Ho et al. 2005; but see Emerson 2007; Bandelt 2008). Consequently, it is necessary to correct measures of divergence to account for potential effects of selection. By focusing on divergence at synonymous sites, I can account for weak selection on codon usage in order to make measures of both divergence and mutations most accurately reflect a neutral process. A second concern is that assumption of a universal molecular clock may be unjustified in analyses of deep timescales (Thorne et al. 1998; Ochman et al. 1999); indeed, this is the root of criticism for current divergence dates of Caenorhabditis species (Félix 2004; Kiontke et al. 2004). I minimize the potential impact of mutation rate heterogeneity among taxa by focusing on collections of closely related species and large samples of orthologous loci. By computing lineage-specific neutral substitution rates for thousands of orthologous groups of genes, I apply estimates of µ from mutation accumulation experiments in the nematode Caenorhabditis elegans (Denver et al. 2004) and the fruit fly Drosophila melanogaster (Haag-Liautard et al. 2007) to infer times to common ancestors for closely related members of these groups for which fossil calibration is lacking or limited.

	Materials and Methods

TOP Abstract Introduction Materials and Methods Results and Discussion Conclusions Supplementary Material Acknowledgements References

 
I obtained a distribution of estimates of neutral divergence from multiple sequence alignments for putative coding sequence orthologs of 6 Caenorhabditis species (Caenorhabditis japonica DF5081, C. elegans, Caenorhabditis brenneri CB5161, Caenorhabditis remanei, Caenorhabditis briggsae, and Caenorhabditis sp. 5 JU727) and 6 Drosophila species (Drosophila ananassae, Drosophila yakuba, Drosophila erecta, D. melanogaster, Drosophila sechellia, and Drosophila simulans). The Caenorhabditis divergence values derive from a combination of public genome sequence gene annotations and an expressed sequence tag (EST) sequencing effort, described elsewhere (Cutter et al. 2008). Briefly, coding sequences for C. elegans, C. remanei, and C. briggsae were obtained from Wormbase release WS170, whereas coding sequences for the remaining species of Caenorhabditis were inferred from ESTs (GenBank accession numbers for C. japonica: FD512256–FD513938; C. brenneri: FD509784–FD512255; and C. sp. 5: FD513939–FD517806). The raw EST sequences were clustered into unique sequence objects with the PartiGene data pipeline (Parkinson, Anthony, et al. 2004), from which appropriate coding sequence translations were inferred with prot4EST (Wasmuth and Blaxter 2004) and stored in NemBase (Parkinson, Whitton, et al. 2004). The phylogeny relating these Caenorhabditis species was established previously (Cho et al. 2004; Kiontke et al. 2004, 2007), and divergence estimates assume this topology (fig. 1). Full-genome coding sequences for D. melanogaster were extracted from Flybase release 5.1; coding sequences for the remaining Drosophila species were obtained from the supplementary online datafiles reported by Pollard et al. (2006). I follow the previously derived phylogenetic topology for Drosophila in calculations of lineage-specific divergence (O'Grady and Kidwell 2002; Pollard et al. 2006; fig. 1).

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 1.— Phylogenetic relationships used in calculations of divergence for Caenorhabditis (A) and Drosophila (B). Phylogenetic topologies are derived from (Cho et al. 2004; Kiontke et al. 2004, 2007) and from (O'Grady and Kidwell 2002; Pollard et al. 2006), respectively. Divergence estimates for a lineage are calculated from extant species at the tips to the most recent common ancestral node (e.g., for the C. elegans lineage, from the tip to node C1; for the D. melanogaster lineage, from the tip to node D3).

 
Based on multiple sequence alignments in each of the focal genera, I used the codeml program in PAML to compute branch-specific synonymous-site divergence (d_S) as a measure of neutral divergence (Goldman and Yang 1994; Yang 1997). For Drosophila, I simply used the ortholog designations described by Pollard et al. (2006). The canonical peptide translations of these nuclear-encoded loci were used for multiple sequence alignment with ClustalW using default parameters (Thompson et al. 1994) in an automated fashion with custom Bioperl-based scripts (http://www.bioperl.org), from which branch-specific synonymous-site divergence values were calculated (permitting branch-specific d_N/d_S ratios, i.e., model = 1; codon model F3 x 4). This provided 8088 orthologous groups with representatives in all 6 species of Drosophila. Because the lineage-specific divergence for the outgroup species D. ananassae cannot be easily defined, 2 versions were computed: 1) the D. ananassae divergence values reported by codeml (excluding those with d_S 0.0001 or summed d_S > 5) and 2) summed values for D. ananassae and the internal branch leading from D. ananassae to the common ancestor of the remaining taxa (i.e., the distance between nodes D0 and D1 in fig. 1). The true lineage-specific divergence for D. ananassae probably lies between these extremes. Exclusion of D. ananassae loci with d_S 0.0001 or summed d_S > 5 led to 7397 and 7889 loci for the 2 approaches to reporting D. ananassae divergence.

As described elsewhere (Cutter et al. 2008), for Caenorhabditis species, putative orthologs were inferred with OrthoMCL (Li et al. 2003) through its reciprocal best-hit Blast procedure on canonical peptide translations of the genes (and EST gene fragments). The same procedure described above for Drosophila species was then followed for alignment and calculation of branch-specific divergence. The EST collections do not contain representatives for every gene in the genome, so I checked the putative orthologs for evidence of inappropriate orthology classification, based on instances of exceptionally high divergence (d_N > 0.5 or d_S > 5), and removed such cases from further analysis. This procedure yielded 63 orthologous groups with representatives in all 6 Caenorhabditis species, 6,398 orthologous groups specific to C. elegans–C. remanei–C. briggsae, and 1,244 orthologous groups with other configurations of 3 or more taxa. For groups of orthologs with incomplete species membership, I used lineage-specific divergence values for only those lineages that led to an ancestral node in the full 6 species phylogeny (e.g., in the C. elegans–C. remanei–C. briggsae comparison, only the C. remanei divergence values are retained). As for the outgroup species in Drosophila, divergence for the outgroup species C. japonica was computed with or without summation with the internal branch leading from C. japonica to the common ancestor of C. elegans and other taxa (i.e., from C. japonica to C0 plus the distance between nodes C0 and C1 in fig. 1), excluding loci with values of d_S = 0. The true lineage-specific divergence for C. japonica probably lies between these extremes. It should be noted that sequence divergence at synonymous sites among Caenorhabditis species is saturated in pairwise comparisons (i.e., >1 substitution is expected to have occurred at each synonymous site). To the extent that multiple hits corrections over- or undercompensate for the nonlinear relationship between the number of sequence differences and the number of substitutions, the inferences of divergence time will be directly affected. However, the codon-based maximum likelihood method used here for estimating synonymous-site divergence (Goldman and Yang 1994) performs well (see simulation procedure and results, below).

Because selection for codon bias is evident in both Caenorhabditis (Stenico et al. 1994; Duret and Mouchiroud 1999; Cutter and Charlesworth 2006; Cutter et al. 2006) and Drosophila (Akashi 1995; Duret and Mouchiroud 1999), the calculations of d_S might not accurately reflect the neutral substitution rate. Therefore, I checked for correlations between lineage-specific d_S and codon bias (effective number of codons, N_c, Wright 1990; J. Peden's program codonW). Most Drosophila species demonstrated weak, albeit statistically significant, associations between d_S and N_c (Spearman's nonparametric correlations: D. sechellia = –0.08***, D. simulans = 0.03*, D. melanogaster = –0.11***, D. erecta = 0.07***, D. yakuba = 0.09***, and D. ananassae = –0.16***; ***P < 0.0001, *P < 0.05). However, because of the small magnitude of the correlations, their inconsistent sign (a positive correlation is expected under selection for codon bias), and previous discussion of how this codon-based method for measuring divergence is generally uncorrelated with codon bias in D. melanogaster (Goldman and Yang 1994; Bierne and Eyre-Walker 2003), I do not consider the potential for selection on synonymous sites further in the analysis of Drosophila species. Codon bias is relevant to this study only in its potential to lead to artificially low estimates of synonymous-site divergence for loci that experience selection on codon usage. However, this maximum likelihood method generally produces higher divergence estimates than other approaches (Yang 2006), and the lack of strong association with measures of codon bias therefore makes these calculations conservative with respect to underestimating sequence divergence along lineages, as compared with other methods of estimating divergence.

In contrast to Drosophila sequences, divergence values for the orthologous groups in Caenorhabditis demonstrated strong correlations with codon bias (Spearman's nonparametric correlations: C. japonica + internal branch = 0.50, C. japonica = 0.24, C. elegans = 0.55, C. brenneri = 0.60, C. remanei = 0.26, C. briggsae = 0.58, and C. sp. 5 = 0.51; all P < 0.0001). This strong effect could be in part due to EST collections disproportionately containing genes with high expression and correspondingly high codon bias due to selection for translational efficiency and/or accuracy (Shields and Sharp 1987). Consequently, synonymous divergence values were adjusted for each species by adding the residuals of a least-squares regression of branch-specific d_S on N_c to the expected d_S for N_c = 61 (where codon bias is absent). This adjusted measure of synonymous-site divergence (d_S') should reflect neutral patterns of substitution by removing the effects of selection for preferred codons at synonymous sites.

To confirm the accuracy of the method of calculating divergence for Caenorhabditis, because the inferred estimates of d_S' imply saturation in pairwise comparisons, I simulated 1,000 orthologous groups of sequences for 6 species using the PAML program evolver. Simulation input parameters were selected to mimic the empirical data set. Each sequence contained 300 codons (median length for the empirical data set = 294 codons) and was simulated assuming a transition/transversion ratio of 2.0 and a d_N/d_S ratio of 0.029 (the average d_N/d_S' across species), with codon frequencies calculated according to the distribution observed for the coding genome of C. elegans version WS170. The tree topology of figure 1 was applied, with branch lengths defined as the number of substitutions per codon for branch i as (Anisimova et al. 2001), where p_S is the fraction of synonymous sites, which was assumed to be 0.25. Observed d_N and d_S' for each species were used to calculate L for its respective lineage. Because the true estimate of d_S' for C. japonica is unknown, I arbitrarily used a value intermediate between the extremes calculated in this study (table 1). The simulated sequence alignments were then passed through codeml, in the same way as for the empirical sequences, to estimate synonymous-site and replacement-site divergence values.

View this table: [in this window] [in a new window]   Table 1 Divergence Estimates for Caenorhabditis Lineages from Extant Species to Their Most Recent Common Ancestor

 
I calculated a range of TMRCA values for species of Caenorhabditis and Drosophila given the putatively neutral divergence at synonymous sites (d_S' or d_S, respectively), incorporating uncertainty in generation time and standard errors for estimates of the neutral mutation rate. Under a standard neutral, infinite sites model of mutation, the mutation rate (µ) equals the substitution rate (K) (Kimura 1968). Thus, the TMRCA for a pair of sequences is simply K/(2µ) and for a single branch of a phylogeny the divergence time is K/µ. For Caenorhabditis species, the mutation rate estimate (µ) for C. elegans of µ = 9.0 x 10^–9 single nucleotide mutations per site per generation on average (standard error of the mean [SEM] = 6 x 10^–10) (Denver et al. 2004; Denver D, personal communication) was used to calibrate the molecular clock for the genus. For Drosophila, I applied the single nucleotide mutation rate estimate from D. melanogaster of µ = 5.8 x 10^–9 mutations per site per generation on average (SEM = 6 x 10^–10) (Haag-Liautard et al. 2007). Both of these mutation rate estimates derive from direct measurement of new nucleotide sequence differences that accrued over the course of several hundred generations in mutation accumulation lines of C. elegans and D. melanogaster, respectively. Although there is good reason to expect these laboratory-based estimates of average mutation rate to closely reflect mutation rates in nature, it is important to recognize that the average mutation rate in nature might differ. In particular, longer generation times (more opportunity for oxidative damage to DNA) and environmental insults could contribute to higher average per-generation mutation rates in nature, which would lead to overestimation of divergence time based on laboratory mutation rates. Median values of the distribution of selection-adjusted synonymous-site divergence were used as point estimates of K, and the 10th and 90th percentiles were used as lower and upper bounds. Upper and lower bounds on the mutation rate estimates were taken as ±1 SEM, the results of which are given in supplementary tables 1 and 2 (Supplementary Material online). The resulting TMRCA estimates measure time in units of generations, so a range of plausible generation times was applied to infer TMRCA in units of years. Under benign laboratory conditions, C. elegans generation time is 2–6 days, depending on temperature (Wood 1988). However, Caenorhabditis are generally found in nature as quiescent dauer larvae (Barrière and Félix 2005; Barrière and Félix 2007), which may persist for months; the true number of generations passed through each year in these species is not known. I conservatively propose an average generation time of 60 days (6 generations per year) but provide results for calculations of divergence time that assume a 14-day and 30-day turnover. Calculations based on even slower generation times are given in supplementary table 1 (Supplementary Material online). It is commonly assumed that Drosophila species experience approximately 10 generations per year, although they may undergo >20 generations per year in laboratory culture. It is likely that Drosophila pass through at least 5 generations per year, which I use as a lower limit in calculations of divergence time.

	Results and Discussion

TOP Abstract Introduction Materials and Methods Results and Discussion Conclusions Supplementary Material Acknowledgements References

 
I estimated neutral divergence from lineage-specific rates of synonymous-site substitution (d_S' or d_S) for orthologous groups of genes in Caenorhabditis and Drosophila (table 1; figs. 2 and 3). Lineage ages (T) were inferred by applying median synonymous-site divergence values and direct measures of the average per-site mutation rate in C. elegans (µ = 9.0 x 10^–9 mutations per generation) (Denver et al. 2004) and D. melanogaster (µ = 5.8 x 10^–9 mutations per generation) (Haag-Liautard et al. 2007) to the prediction from the neutral theory of molecular evolution that d_S = µT (table 1; figs. 2 and 3). Contemporary measures of the mutation rate in these 2 species provide the most proximate calibration of molecular divergence in these groups, given the phylogenetic distance to other taxa for which fossil or biogeographic calibrations could be made. For Caenorhabditis, I adjusted the divergence values upward, accounting for selection on codon usage in order to accurately reflect patterns of neutral substitution; this correction was unnecessary in Drosophila.

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 2.— Divergence along lineages leading to extant species of Caenorhabditis (A) and Drosophila (B). The lengths of the thick, external branches correspond to median dS' (A) or dS (B) values, and the gray boxes indicate the bounds of the 10th and 90th percentiles. Internal branches serve only to graphically connect the external branches in the figure; angled lines that connect lineages simply reflect different lineage-specific median divergence values given that branch tips are fixed at zero (the present day). In (A), timescale in generations assumes equal substitution and mutation rates of 9 x 10–9 per generation; timescale in years further assumes 60 days per generation. In (B), the timescale in generations assumes equal substitution and mutation rates of 5.8 x 10–9 per generation; timescale in years further assumes 10 generations per year.

 

View larger version (30K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 3.— Distribution of lineage-specific synonymous-site substitution rates and their corresponding divergence times for species of Caenorhabditis (A) and Drosophila (B). In (A), timescale in generations assumes equal substitution and mutation rates of 9 x 10–9 per generation; timescale in years further assumes 60 days per generation. In (B), the timescale in generations assumes equal substitution and mutation rates of 5.8 x 10–9 per generation; timescale in years further assumes 10 generations per year. The few points corresponding to 0.5 < dS < 5 are excluded from (B) for clarity. Note the different scales for Drosophila ananassae relative to the other Drosophila. Sample size, n, is indicated in each panel.

 
For C. elegans, the median selection-adjusted, lineage-specific divergence (d_S' = 1.017; table 1) translates to an estimated common ancestry date of 113.0 x 10⁶ generations ago (table 1). Although generations time in nature is unknown for Caenorhabditis species, the long-lived dauer stage likely predominates, leading to few generations per year relative to the laboratory (Barrière and Félix 2005; Cutter 2006; Barrière and Félix 2007). I conservatively propose a 60-day average generation time (6 generations per year), which yields a point estimate of the TMRCA for C. elegans and its sister clade (including C. briggsae) of 18 MYA and for the 2 closest relatives (C. briggsae and C. sp. 5) of 14 MYA (supplementary table 1). Calculations using values of µ ± 1 SEM and extreme deciles of d_S' span a range of TMRCAs 11.6–29.9 MYA for the C. elegans lineage (table 1); however, ruling out any time 5–30 MYA is difficult given uncertainty in generation time, with faster generation times consistent with shorter TMRCAs (fig. 4). Median divergence values for other lineages suggest dates of common ancestry of 81.1–114.3 x 10⁶ generations ago, depending on the lineage (table 1; figs. 2 and 3). The times to common ancestry among taxa suggest that speciation occurred in their history over a relatively short interval, as proposed previously (Fitch et al. 1995). Some TMRCAs could be overestimated if certain lineages experienced higher mutation rates (Baer et al. 2005) or for species inhabiting warmer climates (e.g., C. briggsae and C. brenneri, Kiontke and Sudhaus 2006; Sudhaus and Kiontke 2007) that might turn over generations faster than cool region species.

View larger version (10K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 4.— Calculated divergence times as a function of the assumed generation time for the Caenorhabditis elegans lineage leading to the most recent common ancestor with other species. Solid diamonds correspond to median dS' values; open squares and circles correspond to, respectively, 10th and 90th percentiles with mutation rate +1 SEM and –1 SEM.

 
These Caenorhabditis divergence dates are substantially more recent than reported for analyses dependent on nonnematode calibration (30–180 MYA, Prasad and Baillie 1989; Kennedy et al. 1993; Coghlan and Wolfe 2002; Stein et al. 2003) (but see Heschl and Baillie 1990, 23–32 MYA) but accord with arguments that a 100 MYA TMRCA is too long ago (Félix 2004; Kiontke et al. 2004). Because nematodes appear to experience a higher mutation rate than other eukaryotes (Mushegian et al. 1998; Denver et al. 2004), also reflected in long-branch lengths typical for nematode lineages in metazoan phylogenies (Aguinaldo et al. 1997; Peterson and Eernisse 2001; Wolf et al. 2004; Philippe et al. 2005), one must assume an implausibly slow 1 generation per year to reconcile a 100 MYA TMRCA (fig. 4). Furthermore, given the conservation of chromosomal synteny between different Caenorhabditis species (Hillier et al. 2007), partial development of interspecific hybrids (Baird et al. 1992; Geldziler et al. 2006), and conservation of female-secreted mate attraction signals (Chasnov et al. 2007), the dates suggested here are more biologically plausible than very ancient common ancestry. However, these revised TMRCA estimates imply that rates of genome rearrangement (Coghlan and Wolfe 2002; Stein et al. 2003), intron gain and loss (Gotoh 1998; Robertson 1998; Cho et al. 2004; Coghlan and Wolfe 2004; Kiontke et al. 2004), and gene family dynamics (Robertson 1998; Lynch and Conery 2000, 2003; Katju and Lynch 2003; Cho et al. 2004; Thomas et al. 2005; Thomas 2006) are even greater than previously realized. These revised date estimates also narrow the maximum potential duration of self-fertilization in the lineages leading to C. elegans and to C. briggsae.

Because synonymous-site divergence for the Caenorhabditis species shows evidence of saturation (i.e., pairwise d_S' > 1), I conducted simulations to assess the robustness of the method of estimating sequence divergence. Using input parameter values matched to those observed for Caenorhabditis, the simulations of DNA sequence evolution indicate that the method of calculating divergence generally performs well (table 2). Simulated synonymous-site divergence values tend to be slightly underestimated, but all median estimates for within-group taxa fall within 10% of the true simulated value. Thus, the 5-fold more recent divergence times reported here relative to other studies are not an artifact of extreme underestimation of neutral substitution rates.

View this table: [in this window] [in a new window]   Table 2 Results of Simulation Estimates of Divergence

 
Based on 8088 orthologs shared between 6 Drosophila species, I calculate from a median d_S = 0.068 that D. melanogaster diverged from its common ancestor with D. simulans and D. sechellia approximately 11.7 x 10⁶ generations ago (table 3; figs. 2 and 3). Other Drosophila lineages have TMRCAs of 3.6–18.9 x 10⁶ generations ago (except for the much more divergent D. ananassae; table 3). The nominally longer median lineage-specific divergence for D. yakuba relative to its sister species D. erecta (fig. 2) implies that D. yakuba might pass through more generations per year or potentially experience an elevated mutation rate, although it is conceivable that widespread incomplete lineage sorting could contribute to a similar pattern (Pollard et al. 2006). Assuming 10 generations per year leads to a comparable or somewhat more recent TMRCA between D. melanogaster and D. simulans (1.17 MYA) than is generally believed (0.8–5.4 MYA; Caccone et al. 1988; Russo et al. 1995; Li et al. 1999; Tamura et al. 2004). However, the disparity with other studies in date estimates is greater for the common ancestor of D. melanogaster and D. yakuba. Discrepancy could result from several factors: 1) slower generation turnover than assumed, 2) unaccounted selection on synonymous sites, 3) upwardly biased mutation rate estimate, and/or 4) mutation rate difference between melanogaster group species and taxa used for external molecular clock calibration. Issue 1) cannot fully compensate for the difference (table 3) and 2) is unlikely here because the method of calculating d_S should preclude such an effect by accounting for selection on codon bias and by yielding higher divergence estimates than other methods (Goldman and Yang 1994; Bierne and Eyre-Walker 2003); also, we observe no strong, consistent association between d_S and codon bias in Drosophila (see Materials and Methods). In addition, synonymous sites in Drosophila experience less selective constraint than noncoding sites (Andolfatto 2005). Issue 3) could arise if mutation rates differ at coding and noncoding sites, perhaps as a by-product of nucleotide-content differences, although there is no evidence for such an effect (Haag-Liautard et al. 2007). The lower bound on mutation rate (Haag-Liautard et al. 2007) is consistent with a D. melanogaster–D. simulans divergence of up to 4.3 MYA (table 3), which reconciles easily with traditional dates. Note also that the estimates of the mutation rate and neutral divergence reported here are somewhat higher than assumed in previous studies (Li et al. 1999; Tamura et al. 2004); these 2 features partially cancel out, leading to the roughly similar divergence date estimates for D. melanogaster–D. simulans. Determining whether contemporary mutation rates or biogeographic calibrations from Hawaiian taxa are most appropriate for the Melanogaster subgroup remains a challenge.

View this table: [in this window] [in a new window]   Table 3 Divergence Estimates for Drosophila Lineages from Extant Species to Their Most Recent Common Ancestor

 
How confident can we be in the accuracy of divergence dates inferred from calibration of a molecular clock based on contemporary rates of mutation? It has been argued that de novo mutation rates generally exceed substitution rates inferred from phylogenies (Ochman et al. 1999; Ochman 2003; Ho et al. 2005), so population-based measures of mutation should be applied cautiously to deeper timescales (Ho and Larson 2006). I have minimized the potential problems in this context by focusing on synonymous sites for which the molecular clock hypothesis is most appropriate (rather than amino acid substitutions), by correcting synonymous-site divergence for the effects of selection, by using the median values for thousands of orthologous genes, and by limiting the scope of taxa to close relatives.

	Conclusions

TOP Abstract Introduction Materials and Methods Results and Discussion Conclusions Supplementary Material Acknowledgements References

 
For taxa that lack divergence time landmarks from fossils or biogeography, such as nematodes, direct estimates of the mutation rate from contemporary laboratory populations provide the best available tool for calibrating molecular clocks. With the technical ability to determine contemporary mutation rates at hand, this approach is now feasible in a variety of organisms. Caenorhabditis exemplifies this method's merits, in which dates of common ancestry derived from genome-scale data sets suggest that Caenorhabditis species diverged from one another much more recently than inferred from external calibration points and the dubious assumption of a universal molecular clock (e.g., TMRCA of mammals, insects, and nematodes; Coghlan and Wolfe 2002). This result is conservative with respect to assumptions about generation time and methods of calculating divergence and corresponds more plausibly with a variety of biological phenomena.

	Supplementary Material

TOP Abstract Introduction Materials and Methods Results and Discussion Conclusions Supplementary Material Acknowledgements References

 
Supplementary tables 1 and 2 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).

	Acknowledgements

TOP Abstract Introduction Materials and Methods Results and Discussion Conclusions Supplementary Material Acknowledgements References

 
I thank the Washington University School of Medicine Genome Sequencing Center for making C. remanei genome sequences publicly available. I am grateful to A. Eyre-Walker, M.A. Félix, and several anonymous reviewers for critical comments on the manuscript. This work was supported by funds from the National Science Foundation (Doctoral Dissertation Improvement Grant and International Research Fellowship Program grant 0401897) and by startup funds from the Department of Ecology and Evolutionary Biology at the University of Toronto.

	Footnotes

 
John H. McDonald, Associate Editor

	References

TOP Abstract Introduction Materials and Methods Results and Discussion Conclusions Supplementary Material Acknowledgements References

 

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Accepted for publication January 21, 2008.

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