Although there are multiple genes available for some comparisons, it would violate the assumptions of statistical independence to include more than one data point per comparison in a single statistical test.
Two separate analyses were conducted, one where differences in rate of molecular evolution were calculated for the single longest available gene for each radiation, and one where differences in rate were calculated for concatenated alignment of all available genes. Different genes can have different average substitution rates, just as sites within a gene can have different substitution rates, so we allowed for variation in rates across all sites in the alignment by using a gamma parameter in the substitution model the gamma shape parameter was estimated for each alignment using maximum likelihood.
Because the hypothesis being tested concerns genome-wide rates of substitution, concatenation of sequences is an appropriate analysis of multigene data. In addition, we assessed the power of our comparative study by testing for an association between island age and rate difference, and between number of variable sites per comparison and rate difference, using Spearman rank correlations.
Of the 19 comparisons of substitution rates between island and mainland lineages, 6 showed a significant difference in estimated substitution rate for the single gene alignments 4 significant differences for the concatenated alignments: Table 2.
However, the island clade had the faster substitution rate in only half of these significant comparisons, so the Rhino results do not support the hypothesis that explosive island radiations should have a faster rate of molecular evolution. Estimated relative substitution rates and ratios of nonsynonymous to synonymous substitutions, for the comparisons between island and mainland lineages listed in Table 1. Substitutions per site estimated by maximum likelihood in Rhino see Methods.
The ratio of island to mainland substitution rates is given for the single longest gene and the concatenated alignment of all sequences. Significant differences between island and mainland lineages P are marked by asterisks:. There was no apparent influence of genome nuclear, mitochondrial, chloroplast , sequence type protein-coding or non—protein-coding , or taxon vertebrate, invertebrate, or plant on direction of the rate difference for these comparisons.
There was no significant association between island age and rate difference, nor between number of variable sites per comparison and rate difference. Comparisons with more lineages did not appear more likely to give a significant result, as the mean number of island taxa 14 and mainland taxa 5 for the significant comparisons was similar to the mean for all comparisons 13 island, 5 mainland.
The discrepancy between molecular and palaeontological dates for three explosive radiations—metazoans, birds, and mammals—has engendered a widely held reluctance to trust the molecular clock. The molecular dates have been defended by highlighting potential deficiencies in the palaeontological data that could result in a systematic gap in the fossil record.
Is it also possible for molecular dates to be systematically biased? If the molecular clock runs fast in lineages undergoing rapid evolutionary diversification, then dates for explosive radiations could be consistently overestimated. This study has aimed to test this hypothesis using multiple independent incidences of explosive radiations on islands: if rapid radiation speeds the molecular clock, these island endemic lineages should have faster rates of molecular evolution than their nonexploding mainland relatives.
We found no evidence that a lineage undergoing rapid adaptive radiation has a faster rate of molecular evolution, for the taxa and genes examined here. How confident can we be that these data represent an appropriate model for the adaptive radiations of the past? Have we looked at the right sequences? Positive selection during an adaptive radiation might generate a burst of substitutions in specific genes associated with diversification e.
The genes included in this study were originally sequenced for their phylogenetic utility, typically genes for basic metabolic functions that would be unlikely to be under direct selection in an adaptive radiation see Table 1. Although these housekeeping genes are not ideal for examining the more general question of whether rapid adaptation can influence molecular evolution, they are the correct focus for a study of the reliability of molecular clocks.
Molecular date estimates for the metazoan, avian, and mammalian radiations have been based primarily on housekeeping genes, precisely because they are considered less likely to be influenced by variable evolutionary pressure, and are therefore deemed the most reliable keepers of evolutionary time.
Moreover, although there is wide variation in published date estimates for these explosive radiations, studies based on a broad range of sequences all point to divergences long before the first fossils e. So only a process that affected genome-wide rates of molecular evolution—not just the substitution rate in genes under direct selection—could account for a systematic bias in published molecular date estimates for the metazoan, avian, and mammalian radiations.
We would expect to detect such a genome-wide bias in the genes used in this study. Do we have sufficient data to detect an effect of explosive radiations on molecular evolution? The statistical power of each Rhino test i. However, there is no apparent association between the number of variable sites per comparison and the difference in rate between island and mainland lineages, suggesting that statistical power of each comparison is not preventing this study from revealing an effect of adaptive radiation on molecular evolution rates.
The number of comparisons may also limit the statistical power of this study. However, the 19 comparisons were evenly divided between positive and negative rate differences, so there is no hint of a weak trend that could become significant if more data was added.
Are the comparisons too recent to have allowed for the accumulation of sufficient differences in number of substitutions? The gene sequences used in this study will have been specifically selected to have a substitution rate appropriate to the estimation of phylogenies of the island endemic radiations, and so should provide sufficient genetic differences.
The older island radiations do not show a greater tendency to have faster rates, suggesting that the analysis is not limited by insufficient time for the island taxa to accumulate a greater number of substitutions. For example, although the Cambrian explosion occurred over half a billion years ago, the period of hypothesized increased rates may have been as little as 10 million years Bowring et al.
Unless, of course, the number of substitutions added is influenced by the magnitude of the phenotypic change.
Some researchers may be unconvinced that recent island radiations provide an appropriate model for the more dramatic radiations of the past, when a high degree of morphological disparity was generated in an apparently short period of time. The Cambrian explosion has fascinated biologists because of the apparent evolution of phylum-level differences in body plan—such as change in the organization of body segments—in as little as 10 million years.
Modern mammals and birds apparently radiated into a great variety of forms and niches from a more generalized ancestor in a similarly short period. Although some of the island radiations included here do show dramatic differences in morphology, such as the Hawaiian silverswords, most show more modest variation from their mainland relatives. Can the modifications of beak shape and size in Darwin's finches compare to variation in number of limbs or body segments of the metazoan phyla? It has been argued that the radiations of animal phyla and other higher taxa involve fundamentally different kinds of evolutionary changes: in particular that new phyla originated through modifications of Hox gene expression patterns that generated new body plans, rather than by the gradual accumulation of incremental changes to morphology Gellon and McGinnis, This argument is macroevolutionary—that the evolution of higher taxa is qualitatively different from the microevolutionary changes that drive evolution of species-level differences—and contrary to the widely accepted neo-Darwinian model of evolution.
Whether or not this model of evolution can be proven, we can ask: if this were true, would it influence the speed of the molecular clock? If a higher-order explosive radiation involved few genetic changes, each of large phenotypic effect, then the few genetic changes that occurred would be unlikely to have occurred in the housekeeping genes, and therefore these genes would be expected to accumulate changes at the normal rate.
This would apply whether the significant changes were in the form of substitutions within key genes, gene duplication events, or changes to the developmental architecture. None of these changes would be expected to directly influence the molecular clock as it is currently applied, based on nucleotide substitutions in housekeeping genes, such as those coding for ribosomal RNAs or metabolic enzymes.
Explosive radiation could only bias molecular clock estimates if it influenced genome-wide rates of molecular evolution. This study was designed to detect such an influence by looking at a range of genes and taxa in many independent adaptive radiations, all characterized by high rates of morphological change. These island radiations provide an informative test case because they combine a number of factors that could influence rate of molecular evolution in adaptive radiations, such as reduced population size, adaptation to new niches, and release from previous constraints.
The relatively young age of the island radiations should not bias the results, as they are comparable to the projected duration of explosive radiations of the past, and gene sequences were selected that would provide a sufficient sample of substitutions.
Even small increases in the substitution rate should be sufficient to be detected across such a wide range of comparisons, and yet there is no sign of an influence of adaptive radiation on molecular evolution rates in these sequences. Archibald J.
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How enzymes adapt: lessons from directed evolution. Trends Biochem Sci 26 , — Indeed, after the exclusion of mutations from the branches that carried only 1 or 2 individuals from the oldest clades, the rate differences extended to 2 and 2. After the correction, the substitution ratios of the oldest clades were comparable with the ratios seen in the distance between human and Neandertal mtDNA or between two chimpanzees Figure 1.
At the same time these values were still 2—3 times higher than the rates derived from a human-chimpanzee comparison. The latter ratio was considered to be stable because negative selection would have wiped out all deleterious substitutions since the split of the lineages of the two species. Notably, about a half of the observed rise from the stable rate in RNA gene and a third of the rise in non-synonymous substitutions took place only after the end of the Last Ice Age, Both empirical curves significantly differed from a simulated dataset with uniform distribution of substitutions over the entire tree Figure 1 , Table 1.
The empirical mutation ratios exceeded the average simulated values in the younger end of the time-scale but were lower in the older end. This pattern is compatible with a scenario of purifying selection gradually removing the slightly deleterious non-synonymous and RNA mutations from the population. Black dots represent the estimates based on the empirical dataset.
Grey dots show the estimates from the reference dataset with a uniform distribution of mutations. Open circles show the values for the 4 oldest clades. The lower open circle shows the substitution rate without counting the mutations from the youngest branches, those that carried only 1 or 2 individuals. Open triangle shows the substitution rate between two chimpanzee sequences X [58] and EU [56].
The plus sign and cross represent data points for the comparisons of human rCRS -Neandertal [60] and human rCRS [40] -chimpanzee [56] , respectively. The upper axis rhoS gives the ages as average amount of accumulated synonymous substitutions. The lower axis shows the ages in calendar years, with 1 rhoS corresponding to years.
The right axis gives relative changes in respect of the substitution rate inferred from the human-chimpanzee comparison. See the details of the sliding window analysis and estimates for the Neandertal and chimpanzee sequences in the text and Methods. Although there is no evidence of strong directional selection on human mtDNA [23] , [25] , [32] , [36] , it is possible that weak positive selection acts on many branches of mtDNA tree [25] , [33].
Indeed, if positive selection favored many younger branches, it could result in a substitution pattern similar to that produced by incomplete purifying selection. To test whether the influence of positive selection could have caused the apparent rate acceleration, we excluded all sub-trees defined by mutations at evolutionarily conserved sites that have shown evidence of back-mutation to a more favorable state [25] , [33].
Altogether, individual sequences were removed from the tree and the number of clades in the sliding window analysis reduced to Nevertheless, the patterns of substitution ratios changed only marginally Figure 2. We conclude that the overall effect of positive selection on the substitution pattern of mtDNA is weak. Black dots represent the results after the removal of the mutations and grey dots show the results of the full dataset Figure 1.
The upper axis rhoS gives the ages as average amount of accumulated synonymous transitions. The sliding window analysis was performed as with full dataset, except that windows were analyzed.
See the additional details in the text, Figure 1 legend and Methods. Synonymous changes are assumed to be free of strong natural selection. Despite of that, the effect of apparent rate change has also been observed for synonymous mutations.
Both undetected saturation and variation of substitution rates in between synonymous sites have been proposed as the main factors behind the discrepancy between the intra- and interspecies apparent substitution rates [9] , [14] , [25]. Rate variation among sites can be caused by basic molecular biases like strand asymmetry or by natural selection.
We studied the rate variation of synonymous sites by analyzing the distribution of the number of synonymous sites by mutation hits they had received Table 2. The number of mutation hits at each synonymous site was counted on the Mitomap tree of sequences. A table was created showing the number of sites and the respective number of mutation hits the sites had received Table 2. We then fitted the summary distribution of several Poisson distributions, generated with variable number of different rates, with the observed values.
Fitting the expected distributions with the observed distribution for all sites, variable and invariable, revealed that the overall average rate of 1. The finding that just 3 discrete classes of substitution rates can explain rate variation at synonymous sites stands somewhat in contrast to earlier assumptions of a continuous gamma distribution of substitution rates over all coding sites [15] , [37] , [38].
The observed human-chimpanzee distance of synonymous transitions was used to test the applicability of the 3-rates model of synonymous sites for the correction of the genetic distance for multiple mutation hits. Assuming equal probability for all transitions, the correction would stretch the distance to mutations Table 4.
This, however, would yield a divergence date of 4. We took the commonly referred date of 6. The obtained distance, synonymous transitions, was only insignificantly different from the empirical value of , showing that it is sufficient to assume only 3 different substitution rates for the correction Table 4. Notably, the common calculation of corrected distances from the observed differences was problematic for several reasons. First of all, the sites with the highest rate were expected to be fully saturated.
To exclude the possible effect of recent rapid growth of human populations, the reference sequence rCRS [40] that represents extant variation was replaced by the reconstructed root sequence of the human mtDNA tree. The resulting difference was still significant for the sites that had mutated at least 6 times in the human mtDNA tree Table 5.
The overall apparent substitution rate of mtDNA coding region shows no notable change for hundreds of thousands of years implying that the decrease of non-neutral mutations with time must be very slow Figure 3. This observation is based on the human-Neandertal comparison and is supported by a comparison between two chimpanzees. We have dated the coalescence ages of these lineage splits according to our newly revised rate of synonymous transitions and by the application of the 3-rates model.
This date estimate makes it unlikely that the direct ancestors of Neandertals would have been around in Europe ky ago, assuming that the human-neanderthal split occurred in Africa.
However, the commonly referred divergence dates for the two species, a — kya, assuming the continuity between H. The latter hypothesis seems though more consistent with the mtDNA date estimate as it allows for a reasonable convergence time of DNA lineages within the ancestral species: if the species split occurred — kya then the additional — ky could be reserved for the coalescence of mtDNA lineages within the ancestral population, which would be comparable to the observed value in modern humans — about ky.
The last open circle shows the value for the 4 oldest clades. The grey circle shows the apparent substitution rate for the 4 oldest clades without counting the mutations from the youngest branches, those that carried only 1 or 2 individuals.
Open triangle shows the substitution rate derived from the comparison of two chimpanzee sequences. The plus sign and cross represent data points for the comparisons of human-Neandertal and human-chimpanzee, respectively.
The lower axis shows the ages in calendar years. See the details in Figure 1 legend and in the Methods. Our results imply that the change in the apparent substitution rate of mtDNA coding region involves fluctuations Figure 3.
In addition, the growth trend in the proportion of synonymous substitutions along with growing clade ages is wavy Figure 4A. Furthermore, this growth trend is lost after sorting the data according to the accumulated total variation, which includes substitutions that are under selection Figure 4B.
The fluctuations in the apparent substitution rate imply interrelated influences of variable strength in natural selection and population size changes. Indeed, the earliest turning point in the apparent substitution rate coincides with the population expansion following the out-of Africa migration of modern humans, associated with the first diversification of the two mtDNA superclades M and N.
Notably, a recently proposed correction for the molecular clock of mtDNA was based on a growth function derived from a dataset of proportions of synonymous substitutions that was sorted, like in Figure 4B , according to the accumulated total variation [15]. It is possible that the distortion of the growth trend is more evident in our dataset compared to theirs [15] because of the larger proportion of non-neutral variation in our data due to the exclusion of the non-coding control region.
Detailed understanding of the behavior of mtDNA substitution rate would certainly increase our knowledge on the factors that have conditioned the development of existing genetic variation in human populations.
The dataset of coalescence ages was sorted according to the accumulated amount of synonymous variation A or total variation B and the sliding window analysis was performed as with the other mutation ratios see Methods.
Open circle shows the values for the 4 oldest clades. Note that the time axis derived from total variation on panel B is in fact not linear, contrary to what is shown in panel A. The values on panel B have been stretched out along the younger end of the dataset because of the generally higher fraction of non-synonymous and RNA gene mutations there. Our intrahuman data show systematically lower fraction of synonymous substitutions compared to the results of Soares et al [15]. For instance, the proportion of synonymous substitutions 0.
Unaccounted saturation cannot be the explanation for the discrepancy as the extent of saturation in the long branches of human mtDNA tree is very low [15]. The difference in the results of the two studies, however, has implications for the strength of purifying selection. According to Soares et al [15] the proportion of synonymous changes would be virtually stable by the time of the divergence of human and Neandertal mtDNA lineages, indicating only marginal role of purifying selection on mutations that have preserved from this time depth.
An indirect support for the weakness of purifying selection comes from the comparison of the two chimp sequences that diverged about ky ago, which show a similar fraction of synonymous substitutions to the human-Neandertal pair. Notably, the estimates for the fraction of synonymous substitutions for the human-chimp distance were similar in our study and Soares et al. Our substitution rate of years per synonymous mutation is slightly slower than the average result of Soares et al. We chose to retain the commonly used 6.
Nevertheless, our rate estimate is in the range of the four estimates derived by four different methods applied by Soares et al. Notably, there is a difference in the results of the two studies that were both derived from the accumulated amount of synonymous transversions. Soares et al. According to the substitution rate of years per synonymous substitution, geographically pooled sequences show that the coalescence times for the two non-African superclades, M and N, are in reasonable agreement with the earliest archaeological evidence for the presence of anatomically modern humans outside Africa Table 6.
The results of the present study confirm the higher non-synonymous and RNA variation in human mtDNA genealogy as compared to the level of interspecies variation [12] , [14] , [15] , [21] , [43] and reveal that the rate of accumulation of substitutions is not gradual but has accelerated since the beginning of the Holocene There are three possible explanations for the recent excessive accumulation of functional substitutions in our species: 1 adaptive shifts and positive selection, 2 insufficient time for purifying selection or 3 relaxation of selective constraints.
These are not mutually exclusive and probably all of them have shaped the distribution of substitutions in human mtDNA tree. The contribution of adaptive mutations is small [25] , [33]. The abundance of infrequent non-synonymous variants in human populations has been difficult to explain in terms of adaptive shift. Instead, a transient perpetuation of slightly deleterious variants [21] , [22] , [25] or relaxation of selection [44] have been proposed as explanations. The distinction between these two scenarios is difficult to make [23].
Comparisons to other species would enable to disentangle the effect of relaxation of natural selection due to technological, cultural and anatomical innovations of modern humans from other factors slowing down the purification of DNA from slightly deleterious mutations, such as post-glacial population growth.
Indeed, various other species have been found to have higher intra-species non-synonymous variation than anticipated from interspecies differences [19] , [21] , [43].
There is also evidence of a delayed purifying selection after the rapid population expansion following a severe bottleneck at the LGM for the North American migratory bird species [45]. Nevertheless, because of scarcity of data, such interspecies comparisons of variation in mitochondrial genomes are so far very limited. The low efficiency of purifying selection may be a more general pattern as there is no sign of a rapid population growth in the genetic variation of chimpanzees [46] , [47]. It is known that published human mtDNA sequences contain sequencing errors [51] , [52] , [53] , which may bias the results of neutrality tests [54].
Sequencing errors mostly occur on terminal branches, where no phylogenetic check or independent confirmation from another study is available. As the share of terminal branches is the largest in the youngest clades, sequencing errors can generate the accumulation of non-synonymous and RNA mutations in the youngest clades. We assessed the effect of putative sequencing errors on the results of this study by three indirect means.
First, provided that the sequencing errors in the dataset are random in respect to mutation class, one can presume that the number of errors in each mutation class is proportional to the number of nucleotide positions that allow such mutations. As the number of non-synonymous positions is nearly two times higher than the number of synonymous positions, most of the randomly generated errors should appear as non-synonymous mutations.
The number of positions in genes coding for RNAs is comparable to that of synonymous positions and sequencing errors are expected to occur equally likely at the two classes of nucleotide sites. However, the ratio of non-synonymous to synonymous substitutions was not elevated compared to the ratio of RNA to synonymous changes Figure 1 , suggesting no major impact of sequencing errors on our results.
Secondly, we assessed the effect of sequencing mistakes by removing all non-synonymous and RNA mutation counts from terminal branches and also from branches that carried 2 individuals and repeated the sliding window analysis. This reduced the number of non-synonymous mutations in the analysis from to or and the number of RNA mutations from to or The observed decrease in mutation ratios was expected for all time-windows as the high ratios of external branches also contributed to the ratios of the older clades Figure 5.
After the removal of the external branches the mutation ratios were still higher for the younger clades. Black dots represent the results after the removal of terminal branches, open circles represent the outcome after further removal of the branches that carried 2 individuals, and grey dots show the results of the full dataset Figure 1. The clade ages were taken from the analysis of full dataset. The sliding window analysis was performed as with full dataset.
See the details in the text and Methods. Third, as phantom transversions are a common type of sequencing errors, the ratio of transversions to transitions was compared for the internal and terminal branches.
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