Multiple Evolutionary Rate Classes in Animal Genome Evolution

Christopher Oldmeadow,1,3 Kerrie Mengersen,1 John S. Mattick,2 and Jonathan M. Keith*,1,3

1 2

School of Mathematical Sciences, Queensland University of Technology, Brisbane, QLD, Australia Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia 3 School of Mathematical Sciences, Monash University, Melbourne VIC, Australia *Corresponding author: E-mail: j.mattick@imb.uq.edu.au, j.keith@qut.edu.au. Associate editor: Jeffrey Thorne

Abstract
The proportion of functional sequence in the human genome is currently a subject of debate. The most widely accepted gure is that approximately 5% is under purifying selection. In Drosophila, estimates are an order of magnitude higher, though this corresponds to a similar quantity of sequence. These estimates depend on the difference between the distribution of genomewide evolutionary rates and that observed in a subset of sequences presumed to be neutrally evolving. Motivated by the widening gap between these estimates and experimental evidence of genome function, especially in mammals, we developed a sensitive technique for evaluating such distributions and found that they are much more complex than previously apparent. We found strong evidence for at least nine well-resolved evolutionary rate classes in an alignment of four Drosophila species and at least seven classes in an alignment of four mammals, including human. We also identied at least three rate classes in human ancestral repeats. By positing that the largest of these ancestral repeat classes is neutrally evolving, we estimate that the proportion of nonneutrally evolving sequence is 30% of human ancestral repeats and 45% of the aligned portion of the genome. However, we also question whether any of the classes represent neutrally evolving sequences and argue that a plausible alternative is that they reect variable structurefunction constraints operating throughout the genomes of complex organisms. Key words: noncoding, conservation, neutral evolution, constraints.

Research article

Introduction
Despite the vast amount of analysis of the human genome, it is unclear what proportion is functional. Protein-coding regions account for only ;1.2% (Taft et al. 2007), but human mouse and humandog comparisons suggest that ;5% is subject to purifying selection (Waterston et al. 2002; Lindblad-Toh et al. 2005). This gure is supported by the recent nding that 5% of bases are condently predicted as being under constraint in mammals by two of three algorithms employed in the ENCODE project (Birney et al. 2007). On the other hand, the third algorithm indicated not only a much higher gure but also that a substantial fraction of selection occurs outside of canonically dened blocks of conserved nongenic elements in highly fragmented patches that are diffusely distributed across the genome (Asthana, Noble, et al. 2007; Asthana, Roytberg, et al. 2007). In addition, the 5% estimate was based only on the ;25% of the mouse and human genomes that are alignable, with no insight into whether and what fraction of the remainder has also been subject to (lineage-specic) evolutionary selection. It is understood that conservation imputes function; thus, these estimates imply that at least 5% of the genome is functional. However, conservation is a relative term, and the amount of recognizably conserved sequence is only a lower bound on the amount of functional sequence, because lack of relative conservation does not imply lack of function (Pang et al. 2006). Indeed, the ENCODE pilot study found

many functional elements that are seemingly unconstrained across mammalian evolution, indicating either that these elements are evolving neutrally (Birney et al. 2007) or that current indexes of the true underlying rate of neutral evolution are incorrect (Pheasant and Mattick 2007). In Drosophila, the estimated proportion of functional sequence is less contentious, though also less precise; a recent study estimated that 4070% of the Drosophila melanogaster genome is conserved (Andolfatto 2005). Other studies support a value at the upper end of this range (Halligan and Keightley 2006; Keith et al. 2008). Because the Drosophila genome is about one-tenth the size of the human genome, this corresponds to approximately the same quantity of conserved sequence as currently thought to occur in the human genome. The mouse and dog genome papers (Waterston et al. 2002; Lindblad-Toh et al. 2005) estimated the proportion of conserved genomic sequence in the human genome by calculating percent identity in nonoverlapping windows of 50 bp or more, using pairwise alignments. The method involves comparing the statistical distribution of evolutionary rates in the total genome h(r) (dark blue in g. 1) to the distribution of evolutionary rates f(r) in some subset of genomic sequence assumed to be evolving neutrally (red). These two distributions are related to the distribution of rates g(r) in nonneutrally-evolving sequence (light blue) as follows:

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Mol. Biol. Evol. 27(4):942953. 2010 doi:10.1093/molbev/msp299

Advance Access publication December 2, 2009

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Third, the method is potentially sensitive to inaccuracies in estimates of f(r) and h(r), particularly if there are large relative errors for any r. Fourth, the use of large nonoverlapping windows has a smoothing effect on estimates of f(r) and h(r); we show in this paper that this smoothing dramatically alters both distributions, so much so that the estimates based on them should be regarded as invalid. Fifth, no justication has yet been offered for assuming p % pmax. We suggest that the justication is primarily visual. The overall distribution h(r) in gure 1 looks like a mixture of two symmetric, unimodal components, and the neutral distribution f(r) looks like it could be one of these components. Setting p % pmax results in an almost symmetrical, unimodal estimate for g(r) (if we ignore a small additional mode on the left). However, it is equally possible that the curve results from a mixture of components with different distributions with p representing one subset. Sixth, there are considerable differences between the observed versus expected pattern of sequence divergence using random substitutions (even when mono and dinucleotide content are maintainedsee Materials and Methods section for details) (g. 2). There are two possible explanations for this: either there are variable selection forces and histories operating on these sequences or there are regionally variable underlying rates of mutation. We consider the latter explanation unlikely, as it would require neutral evolutionary rates to vary over very ne scales. We show below that the average segment length for all rate classes is less than 100 nt, but ne-scale variation is already apparent in the more primitive analysis shown in gure 2. However, the former explanation is often rejected because it conicts with the long-standing expectation that most of the genome is comprised of evolutionary debris. Recent data show that the vast majority of the genomes of humans and other complex organisms is transcribed, apparently in a developmentally regulated manner (Mattick 2007), suggesting that the number and extent of functional sequences may be much greater than anticipated, and that these sequences may comprise a complex population evolving at different rates under different structurefunction constraints, selection strengths, and selection histories. In this paper, we show that more sensitive estimates of h(r) and f(r) reveal complex, multimodal distributions, rendering a multimodal g(r) more plausible and invalidating the visual justication for assuming p % pmax. We compute such estimates for an alignment of four mammals (human, chimpanzee, rhesus monkey, and mouse) and for an alignment of four Drosophilid species (D. melanogaster, Drosophila simulans, Drosophila yakuba, and Drosophila erecta). Alternative methods have been used to estimate the proportion of conserved sequence in the human genome (e.g., Siepel et al. 2005; Lunter et al. 2006). These tend to support a gure of 38% of the genome being conserved. However, we contend that such studies either explicitly assume a two-state model or else lack the sensitivity to detect more than two rate classes. It is not surprising that if the genome is separated into two evolutionary rate classes by
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FIG. 1. The distribution of a conservation score calculated for 50bp windows in the human versus mouse alignment. The conservation score may be taken as a proxy for evolutionary rate. The distribution of the similarity measure is shown for total genomic sequence (dark blue) and for ancestral repeats (red)a model of neutrally evolving sequence. The latter distribution is scaled so that the area under the curve is pmax. The light blue distribution is the difference between the two curves and is taken as an estimate of the distribution of evolutionary rates in conserved sequences. Figure reproduced from Waterston et al. (2002), with permission.

hr 5 p f r 1 p gr; where p is the proportion of neutrally evolving sequence. The method is simple: Because g(r) cannot be negative, p must be less than or equal to the ratio h(r)/f(r) for all rates r. Thus, the maximum amount of neutrally evolving sequence (pmax) must be less than or equal to the minimum value of this ratio. Technically, the method only provides an upper bound on p, but it is usually interpreted as an estimate of it (p % pmax). Although the original mousehuman genome comparison paper stated that the ;5% gure only pertained to sequences under selection for functions common to these species, it also noted that some functionally important sequence cannot be separated cleanly from the tail of the distribution of neutral conservation (Waterston et al. 2002; Lindblad-Toh et al. 2005), clearly implying an expectation that such sequence would not occupy a signicant fraction of the remaining ;95%. There are at least six problems with estimating the proportion of conserved sequence in this way. First, the estimate is affected by window size and ranges from 3% to 8% (Stone et al. 2005). Second, there is currently no undisputed model of neutrally evolving sequence. Whole-genome sequenceconservation comparisons have generally chosen ancient transposon-derived sequences (ancient repeats or ARs) as the index of the neutral evolutionary rate, on the questionable assumptions that such sequences (which have resided in mammalian genomes for .100 My) are largely nonfunctional and that the discernable extant sequences (many of which are at the border of recognizability) do not represent the more conserved end of a broader spectrum. If either of these assumptions is wrong, and there is considerable emerging evidence that many transposonderived sequences are dynamically expressed and have genetic functions (Faulkner et al. 2009), there will be an underestimate of the true neutral evolutionary rate (pmax) and hence underestimates of the amount of constrained sequence (Pheasant and Mattick 2007).

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FIG. 2. A 1-Mb binary sequence (where 1 represents match, 0 represents mismatch and indels are ignored) was selected from Chromosome 21 of the humanmouse alignment. The solid line shows the fraction of matches in this sequence using sliding windows of size (A) 1 kb, (B) 10 kb, and (C) 100 kb. For comparison, binary sequences were simulated through a second-order Markov process, with transition probabilities estimated from the original (alignment derived) binary sequence. The red dashed lines are the 2.5% and 97.5% quantiles for the fraction identity in sliding windows using the simulated data. A large proportion of the solid line lies outside the red dashed lines, indicating that the data are poorly modeled by a second-order Markov model.

multiple methods, the proportions in each class are roughly consistent. However, we demonstrate here that a two-class model does not adequately capture the complexity of the
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evolutionary rate distribution. In light of the multimodal distribution that we nd here, one can no longer condently separate conserved from nonconserved sequence.

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ond transformation, each column was replaced by its parsimony score: the smallest number of mutations that must have occurred (0, 1, 2, or 3). This was computed using Fitchs algorithm (Fitch 1971) and depends on the assumed phylogeny. Boundaries between blocks were considered xed change points, represented using a # character. This was done because there were some substantial gaps between alignment blocks and it would not be sensible to assume that the sequence either side of such gaps is evolving at the same rate.

A potential complication in quantifying nonneutrally evolving sequence is that neutral rates are thought to vary regionally. In humans, the rate of synonymous mutations varies between chromosomes (Castresana 2002), between genes (Williams and Hurst 2002) and with GC content (Wolfe et al. 1989; Hurst and Williams 2000). Mutation rate and relative frequencies of various transitions vary on a scale as small as 1 Mbp (Smith et al. 2002; Arndt et al. 2005), and other mutational events also very regionally (Hardison et al. 2003). The rate of CpG substitutions varies with local GC content (Elango et al. 2008), which itself shows regional variability (Nekrutenko and Li 2000). However, to attribute these observations to regional variation in rates of neutral evolution involves assuming that the bulk of the sequence studied, whether that be synonymous sites, ancestral repeats, or nonprotein-coding sequence, is neutrally evolving. Such assumptions have been criticized elsewhere (Pheasant and Mattick 2007); here, we present evidence that at least 30% of ancestral repeat sequences in the human genome are evolving nonneutrally. Furthermore, we show that evolutionary rate variation occurs on a much ner scale than previously observedas low as 100 bp. Such ne-scale variation is difcult to explain if the bulk of the sequence studied is evolving neutrally. These ndings call into question whether the supposed regional variation in neutral rates is actually due to variation in the evolutionary rates of unrecognized nonneutral sequence.

Random Substitution Model

The motivating results shown in gure 2 and presented in the Introduction were obtained as follows: We transformed a 1-Mb section of chromosome 21 from the humanmouse alignment into a binary sequence (1 represented a match, 0 a mismatch and indels were deleted) and calculated the fraction identity along this sequence using sliding windows of various scales (1, 10, and 100 kb). We simulated 1,000 binary sequences under a random substitution model, where mutations occur independently of position, accounting for mono and dinucleotide frequencies. This process was modeled by a second-order Markov chain with transition probabilities estimated from the alignment. The fraction identity for each of the simulated sequences was also computed using a sliding window over the same range of scales, giving a distribution of fraction identity values expected under the random substitution model.

Materials and Methods

Alignment Data and Transformations
Alignments of the four mammal species (March 2006) and four Drosophila species (April 2006) were downloaded from http://genome.ucsc.edu (Karolchik et al. 2008). Our algorithms can readily be generalized for alignments of more than four species. However, we deliberately chose to work with small, closely related phylogenies, as is appropriate if there is a rapid turnover (gain and loss) of functional elements in genomes (see Conclusion). For Drosophila, we used alignment blocks in which D. melanogaster sequences were on chromosome arm 2L; for mammals, we used those in which human sequences were on chromosome 21. Only blocks containing all four species were included. (Note that this does bias the data toward more conserved sequences, and thus, we may have failed to detect some rapidly evolving rate classes. However, the inclusion of such data could only strengthen our conclusions.) Extraneous species were removed. Blocks containing sequences appearing in more than one block were removed, as were columns containing indels. The total postltered sequence amounted to 83% of chromosome arm 2L and 27% of chromosome 21. Each four-way alignment was transformed into two distinct sequences: The rst we refer to as the maximum frequency transformation, and it ascribes scores of 14 to each alignment column depending on the greatest number of nucleotides that are identical; for example, a column containing three A#s and one G became a 3. In the sec-

Estimating Distributions of Evolutionary Rate

We developed a Bayesian model of the generation of the four-character sequences that involves nding the positions in the sequence that delineate homogenous segments, the number of which is unknown. Each segment is classied based on the probability of observing each of the four sequence characters; the number of classes is also unknown. We used an efcient varying-dimensional technique for simulating from the posterior distribution for the number of segment boundaries and segment parameters for different numbers of classes (noting that segments are dened by their characteristics rather than a xed length).

Modeling
Our model generalizes that of Keith (2006) and Keith et al. (2008) to cater for sequences containing more than two characters. The Bayesian change-point model is described in detail for binary sequences in previous papers by Keith (2006) and Keith et al. (2008). The generalized model is presented in Supplementary Material online. Note that we have not included uncertainty about the alignment in the model; we take the UCSC alignments as given. This is not ideal, as it is known that variations in alignments can affect downstream comparative analyses (Wong et al. 2008). However, uncertainty in alignment is greatest for rapidly evolving sequences. As we show below (see g. 5 and Discussion), the distribution of evolutionary rate separates into multiple modes across the full range,
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with even the most slowly evolving half of the sequences exhibiting multiple rate classes. Thus, our conclusions will hold even with extensive ltering of poor quality alignments. Quantifying the effects of alignment uncertainty in the present context is a major undertaking and may form the subject of a future paper.

Optimal Number of Classes

An important feature of the model is that it involves classifying segments into T classes. To discriminate between models with different numbers of classes, we developed an approximation to the Bayesian information criterion (BIC) (Schwarz 1978)a common model selection criterion. The BIC is a compromise between model t and complexity, with smaller values indicating the preferred model. The BIC is thought to be conservative in that it strongly favors models with fewer parameters. The standard form of the BIC relies on maximum likelihood solutions. Our approximation instead uses posterior samples, a practice that has precedent (Carlin  and Louis 2000). We dene B IC5k Ta 1 lnn 2lnL ; where k is the posterior average number of change points, T the number of classes, a the size of the alphabet, n the length of the input sequence, and lnL the posterior average log-likelihood.
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Results
We simulated between 1,000 and 5,000 segmentationclassication pairs from the resulting posterior distribution 1) and repeated for models with the number of segment classes varying from 2 to 30. Values of adjusted BIC are shown in gure 3. For both the Drosophilid and mammalian alignments, the parsimony transformation produces lower adjusted BIC values than the maximum frequency transformation. The BIC values for the two transformations are not strictly comparable, because they are calculated for different data. However, the underlying data are the same, and the two transformations are so similar that the comparison is at least suggestive that the parsimony transformation should be preferred. That is, in any case, what we would expect, because the parsimony transformation makes use of additional information: the phylogeny. All subsequent results are based on the parsimony transformation. For the Drosophilid alignment, the addition of classes caused large reductions in BIC up until 9 or 10 model classes. We thus conservatively selected the nine-class model. Similarly, we conservatively selected the seven-class model as best for the mammalian alignment. Figure 3 indicates the clear inadequacy of a two-class model of evolutionary rates. A difference of 101 in BIC is generally considered to be very strong evidence in favor of the lower scoring model; here, we are dealing with differences of the order of 105. The twoclass model can thus be rejected with absolute condence, and thus, it is already clear that the conservation patterns of the genome are complex and reciprocally unclear which modes, if any, reect the neutral rate. To assess convergence of model parameters, and to determine how well resolved the classes are, we plotted, for
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1.601e+07 1.596e+07 1.591e+07 1.586e+07 1.581e+07 2 4 6 8 10 12 14 16 18

Number of classes

FIG. 3. Use of approximate BIC value to determine optimum number of classes for the (A) Drosophilid alignment and (B) mammalian alignment. The two lines show the BIC values resulting from two methods of generating the input sequence; the solid line is minimum parsimony and the dotted line maximum frequency.

each class, the mean proportion of columns in which no mutations occur versus the estimated proportion of segments in that class. For example,P the parsimony data for t t transformation, we plotted a0 = aj versus pt for each class t (see Supplementary Material online for denitions of terms). Examples of such plots are shown in gure 4. There was no evidence of systematic movement, which is evidence that convergence has occurred. Moreover, the classes are well resolved, and there is little occurrence of label switchinga common problem for Bayesian classication algorithms where the posterior distribution is invariant to permutations of the component indices. This indicates that the observed granularity of conservation levels is not merely an artifact of a lack of t between the model and the true distribution. The average number of change points detected in Drosophila was 457,079 (of which 187,227 were xed

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Figure 5 shows tted densities of a proxy for conservation level for the Drosophilid and mammalian alignments. The proxy is the proportion of columns in which no mutations occur, computed as above, parameterized by a0. The distributions of conservation levels are mixtures of beta distributions, with the mixture proportions estimated by taking posterior sample means and beta parameters estimated using the posterior medians (the medians were used in this case because a small number of large values had large effects on the means). The gures show clearly the multimodality of these distributions of evolutionary rate. These ndings have important implications for estimation of the proportion of functional sequence in the human and mammalian genomes. First, our results indicate that the estimated distribution of evolutionary rates shown in gure 1 differs substantially from the true distribution: It is smoother and less complex. Smoothing has occurred for two reasons: because of the local averaging effect inherent in window analyses and because only two species (human and mouse) were used. We do not claim that our estimate cannot be further rened, but we have dealt with the issue of local averaging by using a segmentation model instead of randomly located windows, and we have used more than two species. Our estimate is thus indicative of the complexity and multimodality of the true distribution, and of how far the estimate shown in gure 1 departs from reality. This issue alone is sufcient to invalidate the estimates of the proportion of functional sequence obtained in the mouse and dog genome papers. Nevertheless, gure 5 also raises a second problem: It removes the essentially visual justication for assuming that p % pmax. We argued above that this approximation seems compelling because it results in a unimodal estimate of the distribution of evolutionary rates in conserved sequences, and thus provides a pleasingly simple picture of the overall distribution as a mixture of two componentsconserved and nonconservedallowing the mixture decomposition that resulted in the earlier ;5% estimate. However, with our more sensitive estimates, this is no longer true and the visual justication evaporates. In previous work (Keith et al. 2008), we found that Drosophila sequences can be classied into many distinct classes on the basis of sequence similarity and base composition considered jointly, using a whole-genome alignment of only two species. However, the ability to identify a large number of classes depended on the inclusion of base composition in the analysis; all our previous work based on sequence similarity alone found at most three distinct classes. At the time, these could reasonably be attributed to purifying selection, neutral evolution, and positive selection, respectively, in the order of increasing degree of divergence and thus did not challenge the existing simple picture of the distribution of evolutionary rates. However, our new results based on multiple species alignments indicate that all of these classes can be subdivided into multiple modes, and thus, none of them can at this stage be condently identied as neutrally evolving.
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FIG. 4. Resolution of classes. To assess convergence of model parameters, conservation is plotted against proportion for each of the nine classes from the (A) Drosophilid alignment and for the seven classes from the (B) mammalian alignment. It is clear the classes are well resolved with no systematic drift. These parameters are plotted again for the model where indels are represented as a fth character in the input sequence in (C) mammals and (D) Drosophila. There is a distinct negative linear relationship between the proportion of indels and conservation score for the estimated classes in (E) drosophila and (F) mammals.

change points corresponding to alignment block boundaries). The total sequence length was 19,127,001 bp (compare with total D. melanogaster-genome length ;169 Mbp), giving an average segment length of 42 bp. The average number of change points detected in mammals was 166,189 (of which 82,575 were xed). The total sequence length was 9,162,237 bp (compare with total human genome length ; 3.1 Bbp), giving an average segment length of 55 bp. These average segment lengths are considerably smaller than we would have predicted and suggest extremely ne-scale variation in evolutionary rate across both alignments. The presence of xed change points was found to have little inuence on the number of detected change points or number of detected modes of the posterior distribution for simulated data (see supplementary section Inuence of xed change points, Supplementary Materials online, for details).

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FIG. 5. Fitted density distribution for conservation levels in (A) Drosophilid alignment (B) DINE-1 repeat sequence, (C) mammalian alignment and (D) ancient repeat sequence relative to human mouse divergence in the mammalian alignment. The proportion of columns with no mutations is used as a proxy for conservation. It is unclear which of these classes if any represent the true neutral rate of evolution.

Moreover, the distribution of evolutionary rates seen in gure 5 is not what would be expected if genomes consisted of a small amount of functional sequence hidden in a vast sea of neutrally evolving sequence. One would expect to see one or more small classes of slowly evolving sequence, and a large, featureless class of relatively rapidly evolving sequence. Instead, no class stands out as a clear candidate for neutrally evolving sequence, and the pattern of evolutionary rates that we observe suggests that the entire alignments are subject to segmentally varying degrees of constraint. It may well be that there are multiple subclasses of neutrally evolving sequence, which differ in rate of evolution due to differing physical and chemical constraints or sequence compositions that are subject to different mutational biases (see below), rather than differing degrees of selection. However, if that is the case, the problem now is to identify them, which must be done before any conclusions about the conservation of other sequences can be made. As noted already, the mousehuman and doghuman genome comparisons estimated rates of neutral evolution based on analysis of alignable AR sequences, which were assumed to be under no evolutionary constraint (Waterston et al. 2002; Lindblad-Toh et al. 2005). This assumption has subsequently been questioned (Pang et al. 2006; Pheasant and Mattick 2007). We extracted AR regions from the
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four-way mammal alignment using previously published AR sequences relative to the humanmouse divergence (Lindblad-Toh et al. 2005) and analyzed the distribution of conservation in these sequences using our segmentation method. We identied at least three clearly dened evolutionary rate classes in these ARs, with proportions 22%, 70%, and 8% in order of decreasing divergence (g. 5D). It is tempting to infer that the three classes correspond to sequences under positive, neutral and negative selection. However, even if the largest peak is attributed to neutral evolution, this still leaves 30% of AR sequences evolving nonneutrally. In addition, this peak does not correspond precisely to any of the major peaks seen in the whole chromosome analysis (g. 5C) and a mixture decomposition analysis such as that carried out in previous estimations (Waterston et al. 2002; Lindblad-Toh et al. 2005) would indicate that .90% of the sequences are evolving at a rate different from the majority of ARs. We acknowledge the discrepancy could be due to biases toward detecting more conserved sections of repeating sequence, as these are easier to detect. Nevertheless, this illustrates the sensitivity of the mixture decomposition approach to errors. A very slight shift of the central AR peak to the left or right would have a big effect on the estimate of the proportion of negatively selected sequence. We performed a similar analysis for ARs in Drosophila. DINE-1 (Drosophila interspersed element, also named DNAREP1_DM) is the most abundant transposable element in Drosophila and is believed to be the relic of a family of ancient retroelements that lost mobility approximately 3 Mya (Kapitonov and Jurka 2003), after the split of the melanogaster lineage from the yakuba lineage. We analyzed the distribution of conservation for known DINE-1 locations in D. melanogaster that occur in alignment blocks containing all four species. We acknowledge that some of these DINE1 locations may be incorrectly aligned to nonorthologous regions; however, we believe the majority are likely to be ancestral because they are aligned. We found four classes of evolutionary rate (g. 5B). Again, the four modes seen in gure 5B do not correspond precisely to any of the modes seen in gure 5A. The locations correspond roughly to classes 1, 2/3, 4/5, and 6 in gure 5A, but the relative proportions are different, and again the mixture decomposition approach is inappropriate.

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Tolerance of Classes to Indels

Figure 5 indicates that what was formerly taken to be a single mode corresponding to neutrally evolving sequence (between about 0.4 and 0.8 in g. 5A and B) can now be resolved into several distinct subclasses. We consider this to be evidence of multiple types and degrees of constraint acting on sequences evolving at these relatively rapid rates, that is, evidence that these sequences are not in fact evolving neutrally. It is also known there exist regions under selection against indels arising in functional sequences (Lunter et al. 2006). If that is so, the sequences in question should also exhibit varying degrees of tolerance to insertions and deletions. Specically, there should be an

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approximately linear relationship between substitution rate and indel frequency. To test this hypothesis, we added a fth character 4 to the parsimony transformation of each alignment, wherever an indel or run of indels occurred in the alignment. The Bayesian segmentation and classication analysis was then repeated for this ve-character sequence. Essentially the same classes were identied (compare g. 4A and B with g. 4C and D). Moreover, when we plotted, for each class, the mean proportion of columns P containing no substitutions a0t = ait versus the mean P proportion of indels a4t = ait , we observed a linear relationship as predicted (g. 4E and F).

Mean GC content

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Variations in the Neutral Rate

In this section, we consider the possibility that neutrally evolving sequences may have a multimodal distribution of evolutionary rates and hence that several of the modes seen in gure 5A and C may correspond to neutrally evolving sequence. The most plausible mechanism by which this could occur is that the distinct classes differ in their G C content and that G C content inuences the neutral evolutionary rate, noting that Hurst and Williams (2000) argue that G C content affects neutral evolutionary rate at 4fold degenerate sites and claim a positive relationship between GC content and degree of divergence. The proportion of G C content in each of the proled classes varied from 31% to 46% in D. melanogaster and from 37% to 44% in human (supplementary tables S1 and S2, Supplementary Material online, and gs. 6A and B). In D. melanogaster, there is a weak correlation between G C content and evolutionary rate among the nine classes but in the opposite direction to that observed by Hurst and Williams. In humans, there does not appear to be any correlation. However, if one assumes that the four classes with the greatest divergence (classes 1, 2, 3, and 4 in supplementary table S2, Supplementary Material online) are neutrally evolving, whereas the remaining classes are under selective constraint, then the neutrally evolving classes exhibit the relationship observed by Hurst and Williams. However, we regard this division of the classes to be arbitrary and the arguments that could be advanced for it to be unsound. We are aware of only three such arguments: 1) Previous studies (Waterston et al. 2002; Lindblad-Toh et al. 2005) identied only two classes of evolutionary rate, corresponding roughly to the proposed division; 2) The four peaks in question correspond to evolutionary rates observed in neutral sequences; and 3) Such a division reproduces the positive relationship between neutral divergence and G C content observed by Hurst and Williams. With regard to the rst argument, we contend that the binary classication made in earlier studies no longer appears plausible in light of our more sensitive determination of the distribution of evolutionary rates. There is nothing in gure 5C to suggest that the proposed division is more appropriate than any other arbitrary division. With regard to the second argument, we note that with our more sensitive method the distribution of evolutionary rates in AR sequences (g. 5D) does not resemble the left-hand part
B
Mean GC content
0.48 0.46 0.44 0.42 0.4 0.38 0.36 0.34 0.32 0.3 0.0 0.2

Mean Conservation

0.4

0.6

0.8

1.0

Mean Conservation

FIG. 6. Mean G C versus conservation for the (A) seven proled mammalian classes and (B) nine proled Drosophilid classes. No relationship can be seen for the mammal classes and only a weak relationship for the Drosophilid classes.

of the overall distribution of evolutionary rates (g. 5C), and in fact, there is only one peak that seems clearly to correspond (class 3). We thus submit that only for class 3 is there any suggestion of neutral evolution. We estimated the percentage of segment length for this class (calculated as the weighted average total length of this class divided by the total sequence length) to be 55% and conclude that 45% (with 2.5% and 97.5% posterior distribution quantiles being 42% and 51%) of the alignable sequences in the genome are evolving at a rate that in no way resembles neutrality. Similarly, we estimate 30% of AR sequence evolving at a nonneutral rate (2.5% and 97.5% posterior distribution quantiles of 20% and 40% respectively). With regard to the third argument, we note that numerous functional roles have been identied for synonymous sites (reviewed in Pheasant and Mattick 2007). Thus, the positive relationship observed by Hurst and Williams may result from functional 4-fold degenerate sites having atypical G C content. More generally, we question whether the observed regional variations in evolutionary rate discussed in the introduction may in fact be due to variations in the evolutionary rates of undetected nonneutrally evolving sequences. A further argument against classes 1, 2, 3, and 4 representing different classes of neutrally evolving sequence is
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the classes we have identied display any systematic correlation between the two. Our procedure is described in Supplementary Material online. We found no evidence for such correlation.

the extremely short median length of segments in these classes. The median lengths of all classes identied in the two alignments are shown in supplementary tables S1 and S2, Supplementary Material online. It is apparent that changes, not just in evolutionary rate, but also in evolutionary rate class, occur on a very ne scale, on average around 100 nt or less. (Note that these estimates of average segment length are slightly longer than those above based on the number of change points. This occurs because we delineate segments in a class by including only sequence positions with a posterior probability greater than 0.5 of belonging to that class. Some low probability change points are thus ignored.) Such ne-scaled variation could be readily explained in terms of spatially varying selective constraints, such as observed in protein-coding sequences, but we know of no mechanism that could produce such ne-scale variation in neutral rates.

Genomic Location of Most Conserved Classes

It is not our intention in this paper to attribute specic functional roles to the classes of evolutionary rate we have identied. Indeed, it is unlikely that any type of functional genomic element can be characterized solely by its evolutionary rate. However, we do expect the classes we have identied will be enriched in characteristic types of functional element, and we thus briey investigate where the classes are situated in the genome. For each class and each sequence position, we calculated the posterior probability of that position belonging to that class. We compared the locations where this probability exceeded a threshold of 0.5 with the locations of known protein-coding genes (Pruitt et al. 2007). The number of sequence positions exceeding the threshold in each of the following genomic regions was counted: 3# untranslated region (UTR), 5# UTR, exon, upstream (1,000 bp), downstream (1,000 bp), intron, and intergenic regions. For comparison, the expected counts were calculated through simulation of a null model, where we assume the genomic region is independent of segment class. To simulate the null model, the lengths of sequences for each class were rst recorded. Segments of these lengths were then positioned randomly in the sequence with all possible starting positions being equally likely. Expected counts for a given class and genic region were then computed as the average number of intersecting bases for that class and genic region over all simulations. Summaries of these counts are given for the four most conserved classes in the Drosophilid (table 1) and mammalian (table 2) alignments. In the Drosophilid alignment, the most conserved class (labeled D9) is severely depleted in exons and introns but highly enriched in intergenic and 3# regions. In contrast, class D7 is enriched in exons and depleted in intergenic regions. Classes D8 and D6 also show signicant and characteristic patterns of enrichment and depletion, as do the four most conserved classes in the mammal alignment shown in table 2. The fact that these classes show different patterns of enrichment conrms that these classes are shaped by different constraints. This is also apparent by plotting the tted conservation density for each genic region (supplementary gs. S5 and S6, discussion also in Supplementary Material online). A striking feature of the most conserved Drosophila class was that, of 431 uniquely 3# UTR segments, roughly half (191) were within 100 bp from the tail end of the UTR. Supplementary gure S3A, Supplementary Material online, shows an example of a typical occurrence. Intriguingly, the most conserved class in the mammal alignment also has a tendency to occur in the tail end of 3#UTRs, often in tandem with a segment at the coding end of the 3# UTR (supplementary g. S3B, Supplementary Material online). It is plausible that these highly conserved classes are enriched

Tree Topology and Recombination

The summary statistics that we use to represent alignment columns (i.e., parsimony and maximum frequency) are intended to indicate an amount of evolution. These amounts are products of rate and time. If all sites share a common topology and if the amounts of time represented for this shared topology are invariant among sites, then differences among regions in amounts of evolution can be attributed to differences in evolutionary rates. But, due to recombination, different regions can have different evolutionary histories. For example, parts of the human genome are more recently diverged from gorilla than chimpanzee, though the majority is more recently diverged from chimpanzee (Patterson et al. 2006; Hobolth et al. 2007). Our key conclusion (that the complexity of evolutionary forces shaping genomes invalidates simplistic attempts to estimate the proportion of functional sequence) would still stand even if the multimodality observed in gure 5 is due to variation in both phylogenetic history and evolutionary rate. However, it will simplify both our argument and the interpretation of these gures if it can be shown that the classes are due primarily to variation in rate. We therefore performed phylogenetic analyses for each of the classes, as described in the Supplementary section, Supplementary Material online. As noted there, we found no evidence for variation in phylogenetic history between classes, and indeed, the estimates of relative branch lengths displayed in supplementary gures S1A and S1B, Supplementary Material online, are remarkably stable for all but the most divergent classes. Moreover, these highly divergent classes represent only a tiny proportion of the total sequence, and thus, their removal would not greatly reduce the multimodality observed in gure 5. Another factor that could conceivably explain the multimodal distribution of evolutionary rates in terms of neutral forces (i.e., forces unrelated to the presence of functional elements) is ne-scale variation in recombination rates. Although we know of no mechanism by which variation in recombination rates can affect rates of divergence, we nevertheless thought it prudent to investigate whether
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Table 1. Observed (Obs) and Expected (Exp) Counts for the Four Most Conserved Drosophila Classes in Each of the RefSeq Genic Regions of Known Protein-Coding DNA.
D9 3# UTR 5# UTR Coding exon Intron Upstream Downstream Intergenic Obs 73,131 46,901 163,722 629,104 125,893 138,423 760,050 Exp 56,550 44,267 336,577 666,794 202,404 192,094 552,814 Obs 134,623 77,625 753,166 1,049,083 265,546 284,014 991,802 D8 Exp 99,372 77,761 590,308 1,172,386 354,895 337,631 965,476 Obs 173,914 145,941 1,908,332 1,740,649 618,565 664,993 1,234,719 D7 Exp 169,404 132,499 1,007,379 2,000,907 607,011 576,345 1,651,386 Obs 73,618 78,008 421,322 951,206 327,295 306,117 604,589 D6 Exp 72,578 56,647 432,198 856,322 259,332 246,662 709,458

Expected counts computed by simulation.

in a signal that participates in the process of polyadenylation site choice or polyadenylation per se.

Substitution Rate Variation

We estimated the average substitution rate per million years for each of the proled classes. Using a total tree length of 41.2 My for Drosophila (Tamura et al. 2004), we found distinct departures (supplementary table S1, Supplementary Material online) from the previously published estimate of 0.01 substitutions per site per million years (Tamura et al. 2004). The most rapidly evolving class corresponded to a substitution rate of 0.02 substitutions per site per million years and the most slowly evolving class corresponded to 0.0002 per site per million years. Similarly in mammals (supplementary table S2, Supplementary Material online), with a total tree length of 180.5 My (Murphy et al. 2005), our most rapidly evolving class had a substitution rate of 0.004 per site per million years, and the most slowly evolving class had a rate of 0.0002. This compares with the average substitution rate in mammals of 0.0022 (Kumar and Subramanian 2002). The range of values for both Drosophila and mammals is quite similar, although this is probably coincidental given that the mouse lineage is believed to be evolving at a much faster rate than humans (Wu and Li 1985; Li et al. 1996). Both lineages display a broad spread of rates, Drosophila contains more highly conserved sequence, and similarly, mammals contain more rapidly evolving sequence; but these results are merely artefacts of the specic choice of species and their phylogenies. The evidence of enrichment and depletion of various genic fractions (coding sequence, UTRs, etc.) in these classes suggests that the differences in substitution rate detected are indeed biologically relevant. Supplementary

gures S5 and S6, Supplementary Material online, are rough estimates of these rates, and the degree of variation in these rates is apparent. The distribution of rates for introns is similar to that of intergenic regions in both mammals and Drosophila.

Conclusion
It could be argued that there is a natural dichotomy between conserved and nonconserved sequencethe distinction being based on whether a sequence is or is not subject to purifying selection. This is certainly true, but immaterial to our point. We do not have direct access to which parts of genomes are under purifying selection; as proxy, we have a degree of relative divergence, and our results indicate that alignable sequences do not separate into two natural classes on the basis of this criterion. In light of these results, we propose a new framework for the estimation of the proportion of functional sequence in genomes and for the de novo bioinformatic discovery of new classes of functional elements. First and foremost, we propose temporarily abandoning attempts to dichotomously classify genomic sequence into conserved and nonconserved segments; we suggest that this cannot be done correctly until the catalog of identied functional elements is more complete and/or more is known about the physical, chemical and selective constraints shaping the evolution of genomes. In place of such attempts, we recommend the tting of multiclass models of evolutionary rates to multiple sequence alignments and the investigation of the content of individual classes and the evolutionary forces shaping them. In addition, we suggest that such attempts will be much more fruitful if they incorporate other

Table 2. Observed (Obs) and Expected (Exp) Counts (Computed by Simulation) for the Four Most Conserved Mammalian Classes in Each of the RefSeq Genic Regions of Known Protein-Coding DNA.
M7 3# UTR 5# UTR Coding exon Intron Upstream Downstream Intergenic Obs 6,036 1,191 14,490 15,863 1,403 920 13,831 Exp 865 132 1,278 18,864 552 510 24,395 Obs 13,663 1,669 93,209 56,844 2,853 1,858 97,320 M6 Exp 4,710 720 6,801 100,315 2,851 2,522 129,781 Obs 16,533 4,202 80,569 136,024 6,648 3,192 251,433 M5 Exp 8,715 1,388 12,707 189,772 5,324 4,719 243,202 Obs 20,332 2,687 20,309 353,414 8,916 8,374 587,795 M4 Exp 17,916 2,827 25,785 385,410 10,906 9,732 494,945

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potential indicators of function, perhaps including G C content, single nucleotide polymorphism frequency, epigenetic modications, transcription levels, and potential to form secondary structures. Current approaches to estimating the proportion of functional sequence in genomes are predicated on two key assumptions: First, they assume that turnover of functional elements is low, and thus, most functional sequences are preserved over large evolutionary distances. Second, they assume that the majority of functional elements are subject to strong selective pressures that are discernibly different from the bulk of genome. The results of the ENCODE pilot project strongly suggest that both of these assumptions are false: There is a large pool of biochemically active (i.e., functional) sequence that is not evolving at a rate which is signicantly different from the genome average (Birney et al. 2007) and that may represent regulatory elements that are evolving rapidly (Pheasant and Mattick 2007) by drift and/or lineage-specic adaptive radiation. We therefore propose that bioinformatic attempts at de novo detection of functional elements should consider closer range phylogenies, pairwise comparisons and even analyses of individual genomes. (This does not, of course, mean that there is no value in making comparisons among longer range phylogenies to identify sequences that are conserved and evolving slowly over longer evolutionary distances.) The failure of these two assumptions also emphasizes the need to classify genomic sequence into multiple classes, because it suggests a much more complex distribution of evolutionary rates than a dichotomous classication can represent and the need to integrate multiple indicators of function into such classications, because conservation alone will not be sufcient to delineate many functional elements.

Supplementary Material
Supplementary tables S1 and S2 and supplementary gures S1S6 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).

Acknowledgments
The authors would like to thank Peter Visscher for helpful comments on the manuscript. We would also like to thank the Associate Editor Jeffrey Thorne and two anonymous reviewers for helpful comments and suggestions that improved the manuscript. This work was supported by the Australian Research Council (grants DP0556631, DP0879308 and FF0561986), the National Health and Medical Research Council (grant ID 389892) and the Queensland University of Technology. Availability: Source code, data sets and results available upon request to the authors.

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