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Thermal habit, metabolic rate and the evolution of mitochondrial DNA
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Thermalhabit, metabolicrate and the evolutionof mitochondrialDNA David M. Rand
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f one could design a customThe hallmarks of animal mitochondrial subsequent estimates of the rate ized genetic marker for use in DNA (mtDNA) are a rapid rate of sequence of mtDNA evolution in other verpopulation and evolutionary evolution, a small genome carrying the tebrates also supported a figure biology, it is quite likely that same set of homologousgenes, maternal of about 2% divergence per million one would reinvent mtDNA.This is inheritance and lack of recombination. years6,7. This led to a widespread essentially the conclusion reached Over the past few years, a variety of acceptance of mtDNAas a reliable by Avise and colleagues1 in drawdifferent observations has challenged molecular clock for divergenceing up a wish list of the attributes these accepted notions of mitochondrlal times which are less than 10 of a molecular marker for use in biology. Notable examples include million years (Refs 6-8). phylogenetic analysis. It is no coevidence for variable rates of mtDNA Most of these estimates of the incidence that the unique genetic sequence evolution among taxa, evidence rate of mtDNA evolution were obcharacteristics of animal mtDNA for large and variable mitochondrlal tained from restriction-enzyme (rapid rate of sequence evolution; genome sizes in certain groups, and a surveys that sampled the entire compact genome carrying a unigrowing number of cases in metazoans of mitochondrial genome. Despite form set of homologous genes; ‘paternal leakage’ in the inheritance of the evidence for different molecumaternal inheritance; and lack of mtDNA. Several recent studies have lar clocks among genes within recombination, to name a few) are uncovered different lines of evidence mtDNAs,it came as a bit of a surthe very characteristics that prosuggesting that an organism’s thermal prise when the average rates of vide evolutionary biologists with a habit, or metabolic rate, can influence the mtDNA and single-copy nuclear unique picture of population difevolution of mtDNA. DNA (scnDNA) evolution were ferentiation and species relationshown to be very similar in sea ships*J. urchins9 and Dmsophilu~~.These David Rand is at the Dept of Ecology and Evolutionaty But what if our customized studies provided the first robust Biology, Box G-W, Brown University, Providence, marker was (1) shown to evolve rejection of the notion of a general RI 02912, USA. slowly, (2) was observed to be molecular clock of DNA. Rate variable in genome size due to variation among genes (either mitochondrial or nuclear) within a lineage could be due sequence duplications, and (3) was capable of being transmitted by both parents? Some evolutionists might react to different functional constraints 6J1,differences in the likelihood that a nucleotide will be mutated or correctedl*, or by casting off the evidence as rare exceptions to the rules that will not compromise continued use of the marker; some combination of these factors. The obvious ramifiothers might abandon the marker for fear of spurious cation of these findings is that both the rate and the asresults; while others might focus in on the exceptions as sociated error of any molecular clock needs to be calibrated being important patterns in nature that could shed light for the specific gene and taxonomic group in question on the processes of molecular evolution. One general messbefore sequence divergence can be used to estimate age of this review is that each of the three reactions times of divergence or other evolutionary events. Where offered above (rejecting the evidence, abandoning the calibrations of absolute rates were possible, variation in marker, and researching the cause) can be appropriate the nuclear rate of evolution seemed to account for the responses to the continued use of mtDNA in light of such extensive variation among taxa in the relative rates of nuclear and mtDNAevolutiongJ*-14.These studies provided exceptions. However, it is the latter response (clarifying the mechanistic bases of these exceptions) that promises little or no evidence to suggest that the mitochondrial to break new ground in our understanding of the natural molecular clock was variablerqa. history of mtDNA and, indeed, the organisms that carry it. Here 1 call attention to some recent work that suggests a Thermal habit and the rate of mitochondrial DNA relationship between metabolic rates and the tempo and evolution: what is the evidence? mode of mtDNA evolution. In the past several years, comparisons within and among insectslsJ6 and vertebrateslr-23 have provided evidence Mitochondrlal DNA and the molecular clock for variation in the absolute rate of mtDNA evolution. One of the distinguishing characteristics of mtDNA, While it seems unlikely that this rate variation can be and one of the primary justifications for its use in evolaccounted for by a single mechanistic basis (compare utionary studies, is its rapid rate of sequence evolution. Refs 15 and 20) an intriguing correlation is emerging Mitochondrial DNAwas first estimated to evolve at a rate between the thermal habit of an organism and the rate of of l-2 x 10-8substitutions per nucleotide site per year, or mtDNA evolution. about 2% sequence divergence between species per million The first indication that thermal habit might affect the years (Refs 4 and 5). This suggested a rate S-10 times rates and patterns of mtDNA evolution came from studies faster than the evolution of nuclear DNA.Although these of mtDNA in fisheslrJ8. In comparing the sequences of initial rate calibrations were based on comparisons between four protein-coding genes (ATPase subunit 6, cytochrome mtDNA of sheep and goats4 and among primate speciess, oxidase subunit 3, NADH dehydrogenase subunit 3 and TREE uol. 9, no
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Divergence time (millions of years) Fig. 1. Different rates of nucleotide-sequence evolution in mtDNA of mammals and sharks20. The number of transversion differences (Pu -_) Py or Py + Pu change) per fourfold degenerate site (a site at which the code is unaffected by the nucleotide present at that site) in pairwise species comparisons is plotted against divergence time for each species pair. The number of transversion differences are from sequences of the cytochrome b, cytochrome oxidase I, ND4 and ND5 genes in primates (triangles), ungulates (circles) and sharks (squares), and are corrected for multiple substitutions. Transversions at fourfold degenerate sites were used since these changes accumulate linearly with time. The times of divergence can be found in Ref. 20 and references therein; the 60 million year point for ungulates is for Suidae versus Ruminantia*O. The regressions are through the origin and yield average evolutionary rates of 70, 60 and 10 x lo-lo transversions per fourfold degenerate site per million years in primates, ungulates and sharks, respectively. The slopes for primates and sharks are significantly different. Redrawn, with permission, from Ref. 20.
NADHdehydrogenase subunit 4L) in the mitochondria of salmon with the homologous sequences in Xenopus, mouse and humans, Thomas and Bechenbach found an unexpectedly high level of sequence similarity between the cold-blooded speciesIr. For these four genes, the amount of amino acid divergence between rainbow trout (Oncorhyncus mykiss) and Xenopus (diverged over 400 million years agoz4) is very similar to the divergence between mouse and human (diverged 65-80 million years ago24). Averaging across these four genes, there is an approximately fivefold reduction in rate of mtDNA evolution in cold-blooded, relative to warm-blooded, vertebrates. A similar result was observed independently for the cytochrome b gene of cold- and warm-blooded specie@. Several additional recent papers have provided further support for slow rates of mtDNA sequence evolution in cold-blooded vertebrates. Martin, Naylor and Palumbi*O showed that the rate of transversion substitutions at fourfold degenerate sites in two mitochondrial protein-coding genes is six to seven times slower in sharks than that in primates and ungulates (Fig. 1). The findings of Martin et al. are compelling since an effort was made to rule out differences between sharks and mammals in nucleotide composition, codon usage, natural selection and the mitochondrial loci used in the rate estimation. Moreover, there is little reason to challenge the rate calibration in sharks since this group is well represented in the fossil record at a range of divergence dates. A similar pattern has emerged in turtles, where mtDNA seems to be evolving at a ‘turtles pace’zl. As indicated by sequence analyses of the cytochrome b genez2 and restriction-enzyme surveys of the entire mtDNA molecule*], the rate of mtDNA evolution in turtles is reduced 126
between three- and eightfold relative to the conventional rate of 2% per million years in ungulates. This is comparable in magnitude to the difference in mtDNA evolutionary rate observed between sharks and mammals20. Two recent and more-extensive analyses have elaborated on the observations described above*3,*s.Adachi, Cao and Hasegawa23examined the rates of amino acid evolution in vertebrate mtDNAs based on 2711 amino acid positions in the complete mitochondrial genomes of carp, Xenopus, chicken, mouse, rat, whale, cow and human. Their analysis has shown convincingly that the mtDNA of cold-blooded vertebrates evolves at least five times slower than that of mammals. At the positions examined, there are about as many differences between a fish (carp) and Xenopus (452 residues) as between humans and the other mammals (between 433 and 497 residues). As noted above, the divergence times of these two comparisons differ by about a factor of five. Moreover, the qualitative distinction between organisms of the two thermal habits glosses over quantitative differences between the various species examined. The rate of amino acid sequence evolution in mitochondrial genes increases in a graded manner from slow sequence evolution in fishes, to slightly faster evolution in amphibians and birds, to the most rapid evolution in mammals*s. This is evident in the branch lengths of a phylogenetic tree derived from the data-set of mitochondrially encoded proteins (Fig. 2). These results extend an earlier study documenting variation in the rate of mtDNA evolution among orders of mammal@. Martin and Palumbi*s provide further evidence for both a qualitative and quantitative relationship between thermal habit and rate of mtDNAevolution. In a plot of the relationship between the rate of mtDNA evolution (as estimated from restriction enzyme surveys of the entire molecule) and an organism’s body size, endotherms and ectotherms fall out on two very different lines (see Fig. 3). Moreover, within each thermal habit, there appears to be a negative relationship between the mtDNA rate and body size. Body size is correlated with a number of different physiological traits, of which generation time and metabolic rate are potentially important with respect to the evolution of DNA25. Using estimates of rates of silent substitution in mtDNA from species where metabolic rates as well as generation times are published, Martin and Palumbi found strong correlations between the rate of evolution and each of these two physiological traits in simian primates. In a multiple regression with these data, only metabolic rate had a
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Fig. 2. Variable rates of amino acid evolution in vertebrates. A tree of mitochondrially encoded proteins was constructed23 using a maximumlikelihood model based on an empirically determined amino acid transition matrix. The horizontal length of each branch is proportional to the estimated number of substitutions by this model. There is a trend of increasing branch length indicating progressively faster rates of evolution in these lineages. Redrawn, with permission, from Ref. 23.
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REVIEWS significant contribution to this relationship. However, in a similar analysis using nucleotide-sequence data from the mitochondrial cytochrome b gene of mammals, neither metabolic rate nor generation time showed a significant correlation. A multiple regression using both metabolic rate and generation time did reveal a significant relationship. While the negative relationship between body size and the rate of mtDNA evolution is apparent in Fig. 3, the slope of this line is very sensitive to errors in the estimates of divergence dates for mice and whales, the two extremes of body size. Martin and Palumbi qualify their conclusions by suggesting that metabolic rate and generation time may interact in complex ways to determine the specific rate of molecular evolution. Thermal habit and mitochondrial genome size The traditional view of animal mitochondrial genomes is one of a small compact molecule, with no repetitive DNA, pseudogenes or introns, that is identical throughout the tissues of an individuallJJ. This view has changed considerably over the years with evidence for tandemly repeated and duplicated sequences that result in mtDNA length variation among and even within individuals (e.g. Ref. 26 and references therein). While mtDNA size variation was described some time ago in Drosophila (e.g. Ref. 27 and references therein), evidence in vertebrates for mtDNAsize variation and heteroplasmy (the presence of more than one mtDNA type within a cell) was lacking until coldblooded species were studied (e.g. Ref. 28 and references therein). A recent review has uncovered over 50 species that have been reported to exhibit intraspecific variation in mitochondrial genome size, ranging from nematodes to human.9. Among these species, endotherms tend to have smaller mitochondrial genomes than ectotherms while only the latter exhibit the extremely large (e.g. up to 40 kb; Ref. 30) genomes. There is no difference in the frequency of heteroplasmy between the two thermal habits. The most striking difference between endotherms and ectotherms is that in the former, the extent of mtDNA length variation is a much smaller percent of the standard genome size [i.e. (largest genome size-smallest genome size)/(standard genome size) is smaller in endotherms]. These correlations between thermal habit and mitochondrial genome size are also evident when only cold- and warm-blooded
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Fig. 3. Relationship between rate of mtDNA sequence divergence (percent divergence per million years) and body size in kg for various vertebrates. The percent sequence divergence estimates are from restriction fragment length polymorphisms (RFLPs) of mtDNA (tabulated in Ref. 25). The boxes represent the range of sequence divergence estimates and body sizes for a grven taxon: (1) mice; (2) dogs; (3) human-chimpanzee: (4) horses; (5) bears: (6) geese; (7) whales; (8) newts; (9) frogs; (10) tortoise: (11) salmon; (12) sea turtles: (13) sharks. The solid lines are not regressions but are drawn to pass through the boxes.29. The dashed line is the hypothesis of a constant rate of divergence of about 2% per million years. From Ref. 25, with permission.
vertebrates are compared29 (see Fig. 4). A trend towards smaller mtDNAs in endotherms is further suggested by the length of overlap of coding sequences in the ATPase 6 gene. In human, cow, mouse and whale, gene overlaps of 46, 40, 43, 31 base pairs are observed, respectively; in Xenopus and several fish species the overlap is only 10 base pair+. It has been suggested that these patterns are the result of differences between endotherms and ectotherms in the rates of mutation generating mtDNA length variation**, and in the strength of selection on mitochondrial genome size26Js. While the patterns do suggest that selection on genome size is different between endotherms and ectotherms, the relative importance of selection versus mutation needs to be addressed through comparative and functional studies (see below).
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Fig. 4. Patterns of mitochondrial genomesize variation among endotherms, and invertebrate and vertebrate ectotherms. The data are based on a total of 51 species reported to show intraspecific mtDNA length variation2Q. The means (horizontal arrows) and ranges (vertical bars) are shown for (a) the largest mtDNA and (b) the percent variation in genome srze [(largest mtDNA-smallest mtDNA)/modal mtDNA size]. The means and ranges of all three categories [includmg (c)] are based on 11 endotherms (triangles), 23 vertebrate ectotherms (circles) and 17 invertebrates (squares). The largest mtDNA and percent variation in mtDNA size is significantly smaller in endotherms when compared to the invertebrate and vertebrate ectotherms combined, and when compared to vertebrate ectotherms alone. (c) Relationship between the percent variation in mitochondrial genome size and the largest genome size reported within a species. Regression analyses revealed that this relationship differs significantly between each of the three categories (represented by triangles, circles and squares, as mentroned above). Redrawn, with permission. from Ref. 29.
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REVIEWS The work summarized above provides different lines of evidence indicating a relationship between the thermal habit of an organism and both the tempo and mode of mitochondrial genome evolution. An obvious biological difference between endotherms and ectotherms is metabolic rate. As the mitochondrial organelle and the genome it carries are fundamental to the metabolic flux in eukaryotic cells, it is intuitively appealing to focus on metabolic rate itself as a causal factor in these patterns of mtDNA evolution. However, metabolic rate is a complex biological trait that has intricate causal and correlative relationships with other important aspects of organisms such as body size, generation time and rates of cell division2sJ2. Generation time and the evolution of mitochondrial DNA Differences among taxa in generation time are commonly invoked to account for variation in the rate of a molecular clock (e.g. Ref. 33 and references therein). The assumption of this hypothesis is that species with shorter generation times have a greater number of DNAreplication events per unit time, which should provide a greater opportunity for mutations to occur. Turtles do have generation times which are much longer than those of mammaW, consistent with their slow rate of mtDNA evolution. In some cases, however, the generation-time hypothesis is not consistent with the available data. For example, sharks have generation times similar to those of primates and ungulates despite having a six- to sevenfold slower rate of mtDNA evolution, Among orders of mammals where rates of mtDNAevolution vary, Hasegawa and Kishino found no effect of generation time lg. Also, rates of evolution in the COI-CO11region of honeybee mtDNA are faster than that in Drosophila, despite a shorter generation time in flie+. These observations suggest that factors other than organismal generation time could play a role in determining the pace of mtDNA evolution. Little is known about the relative number of germ-cell generations per animal generation in most animals, and even less is known about the number of mtDNA generations within germ cells. These factors are important since they could serve to decouple the rates of mtDNA replication from rates of cellular35 and organismal reproduction, making the latter a poor predictor of the opportunities for mutation in mtDNA that occur during replication. In addition to differences in rates of mutation or efficiency of repairi2’13,these effects could be responsible for the variation between mtDNA and scnDNA in rates of evolution. Metabolic rates, oxidatlve damage and the evolution of mitochondrlal DNA In many of the cases where the generation-time hypothesis fails, variation in the rate of mtDNA evolution can be explained by differences in specific metabolic rates25. The metabolic-rate hypothesis is supported by biochemical studies showing that species with higher metabolic rates experience higher rates of oxidative DNA damage (Ref. 36 and references therein). Thus, species with lower metabolic rates may experience lower mutation rates due to reduced oxidative damage. Moreover, the amount of oxidative damage to mtDNA is greater than that to nuclear DNA,presumably owing to the central role of mitochondria in oxidative metabolism and the high rate at which oxygen radicals are produced in mitochondriasr. These differences could further explain the variation among taxa in the relative rates of nuclear and mtDNA evolution. 128
A recent study of the relationship between body mass and metabolic rate38 lends additional support to the metabolic-rate hypothesis. In animal cells, there is a constant leak of protons across the inner mitochondrial membrane. Pumping of these protons back across the inner membrane, which consumes a substantial portion of an animal’s oxygen budget, is a major source of heat production39 and is thought to be a significant component of metabolic ratedo. The new evidence shows that the rate of proton leak de creases with increasing body massaa. Since larger animals have fewer mitochondria per kg of body mass, the oxygen consumption needed to drive the proton pump will be further reduced in larger animal@. Hence, the DNA damage from oxygen radicals should be reduced in animals with large body mass, which could account for the negative relationship between body mass and rates of mtDNA evolution observed in both endotherms and ectotherms2” (Fig. 3). The distinct differences in rates of mtDNAevolution between species of the two thermal habits may also stem from lower rates of proton leak in ectotherms, but this relationship needs to be examined. Relaxed constraints and the evolution of mitochondrial DNA Thomas and Bechenbachi7 originally suggested that differences in functional constraints might account for the faster rate of amino acid evolution among mammals relative to salmonid fish. Since proteins must function at a wider range of temperatures in cold-blooded vertebrates, these proteins may be more constrained in terms of sequence evolution, The homogenous cellular environment of warmblooded vertebrates may have allowed for the relaxation of constraints on protein function with respect to physiologically acceptable amino acid substitutions. The relaxedconstraint hypothesis differs from the metabolic-rate hypothesis in that the former focuses on changes in the number of amino acid positions that are free to vary in different cellular environments, whereas the latter focuses on the underlying mutation pressure generating potential variants. Adachi et al.23 also invoke the suggestion17 that variation in the rate of amino acid evolution in mtDNA is the result of relaxed constraint in warm-blooded vertebrates. As a test of this hypothesis, Adachi et al. compare the ratio of the amino acid substitution rate, KA,to the synonymous substitution rate, Ks, between warm-blooded (primate) and cold-blooded (salmonid) species. Since the rate of silent substitution will be more closely proportional to the mutation rate, a higher K,:Ks ratio in warm-blooded vertebrates is taken as support for the view that constraints on amino acid evolution have been relaxed in these species. The data for 489 nucleotides (163 amino acids) of the ATPase 6 gene available for primate&l and salmonids17 do indicate a higher ratio in primates, consistent with the relaxed-constraint hypothesis (Fig. 5). However, Adachi et al. conclude that the elevated rate of protein evolution in primate mtDNA is a combination of relaxed constraints and higher mutation rates, relative to salmonids. It is likely that rate variation in mtDNA evolution is attributable to a more complex combination of factors than to changes in functional constraints17 and metabolic rate25. Small selection coefficients and variable population sizes15, and the population biology of the germ line cytoplasm26Z2g, may alter rates of mtDNA evolution. A useful concept that incorporates many of these factors is the ‘nucleotide generation time’, that is, the average length of time before a nucleotide is copied by replication or repair25. TREE vol.
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Fig. 5. Relationship between the number of amino acid differences per site (K,) and the number of synonymous differences per site (K,) of the ATPase 6 gene of mtDNA. The primates (squares) are rep resented by human (Homo sapiens), chimpanzee (Pan troglodytes), bonobo (Pan paniscus), gorilla (Gorilla gorilla] and orangutan (Pongo pygmaeus), and the salmonid fishes (circles) are represented by rainbow trout (Oncorhynchus mykiss), pink salmon (0. gorbuscha), coho salmon (0. kisutch), sockeye salmon (0. nerka), chinook salmon (0. tschawytscha) and cutthroat trout (0. clarki). The estimates of K, and K, are derived from 489 nucleotides (163 codons) that were sequenced for each of the two groups. The ratio of K, to K, is clearly higher in the primates and is interpreted as support for the hypothesis that the faster rate of amino acid evolution in mammals is due to re laxation of functional constraints on amino acid sequence, relative to that in salmonids23. Redrawn, with permission, from Ref. 23.
Metabolically active species with small body sizes, short generation times and a small effective number of mitochondria passed through the germ line may have higher rates of mtDNA evolution due to a shorter mean residence time of nucleotides. Further studies should focus on organisms whose biology allows one to separate the significant variables affecting the nucleotide generation time (see below). In particular, how such combinations of factors alter the residence times of nucleotides in germ line cells is the fundamental question regarding the evolution of DNA. Genome size variation, mutations and a race for replication Why might endotherms have smaller and less-variable mitochondrial genome sizes than ectotherms? Elevated mutation rates for mtDNA insertions and deletions in ectotherms28, or less-efficient repair, might account for the observed patterns. If the rate at which mtDNA length variants were generated was higher in ectotherms, but the rate at which this variation was lost (due to the effects of genetic drift during the mitotic events associated with the transmission of mitochondria2Qg) was similar among organisms of the two thermal habits, one might expect more mtDNA length variation in ectotherms. This balance of mutation and drift affecting mtDNA size variation has been examined in only a few organisms2@z343. A simple mutation hypothesis might lead to the prediction of differences in the incidence of mtDNA size variation or heteroplasmy in organisms with different rates of length mutation. The available data show no difference between endotherms and ectotherms in the frequency of heteroplasmy29. It is not clear that longer genomes, or a higher proportion of genome length exhibited as variation, would be an explicit prediction of a simple elevated mutation rate. These expectations depend critically on the models TREE uol
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of length mutation, and little is known about the mechanisms that generate mtDNA length variation beyond the general observations that sequence motifs that appear capable of forming secondary structures are often implicated in large-scale length mutations44 and tandem repeat variation26.42,43,45. To the extent that thermal habit or metabolic rate affects the models of mutation generating patterns of mtDNAlength variation, it is important to recognize that these predictions are in the opposite direction from the predictions regarding primary sequence evolution. In the latter, the metabolic-rate hypothesis invokes lower relative rates of mutation (oxidative damage) in low-metabolicrate species. Stronger directional and purifying selection for small genome size in the cytoplasm of endotherms, perhaps mediated by a stronger ‘race for replication’, is also compatible with the data263’9. Both within and between species, there is a correlation between the capacity of a tissue for oxidative respiration and the proportion of mtDNA molecules primed for replication46. Tissues with higher metabolic rates also appear to have higher turnover rates of mtDNA47.The additional nucleotides present in a mtDNA length variant could be a significant burden in terms of the time required to complete replication. Since replication of an entire mtDNA takes about one hour@, mtDNA size may play a more important role in a ‘race for replication’ than, say, the initiation of replication. It may be that, in species with higher metabolic rates, where there can be aggressive replication of the cytoplasmic population of their mtDNAs, a long mtDNA variant has a lower relative ‘fitness’ than a comparable variant in a cytoplasm of a low-metabolic-rate species, where replication is less frequent. Alternatively, the oxidative damage associated with metabolic processes37 could result in the introduction of more lesions in longer mtDNAs than in a shorter molecule. Since lesions in DNA can result in the stalling of polymerases, molecules with the fewest damaged sites will tend to be replicated preferentially which would select against longer mtDNA molecules in the cytoplasm. Higher rates of replication coupled with higher rates of DNA damage in high-metabolic-rate species could result in a mechanism that is both directional (favoring smaller genomes) and purifying (removing variation from cytoplasms), which might generate the observed patternP. Future studies: molecular biology and the comparative method Variation in the tempo and mode of mtDNA evolution, and the competing hypotheses that succeed to varying degrees to account for this variation, can and should be addressed from two very different perspectives. On the one hand, additional data are needed on fundamental aspects of mitochondrial genetics, such as mutational properties, replication, repair, transmission genetics and dynamics in the germ line. These types of studies are needed in a variety of different taxa that vary in biological properties which are relevant to the competing hypotheses (for example, metabolic rates, generation times, germ-line sequestration and development). These studies are tasks for molecular and cellular biologists, who are best equipped technically and intellectually to attack these problems. Greater knowledge of the molecular genetics of mtDNA in diverse taxa remains as an important hurdle to a more thorough understanding of how mtDNA evolve+. As these sorts of data accumulate, evolutionists lessinclined to engage in studies of molecular genetics and cell biology have a wealth of experimental material in the
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REVIEWS diversity of organisms where sequence comparisons alone will continue to uncover important patterns. This endeavor is indeed a collaborative effort, and interested biologists of all persuasions should take the time to stay up to date
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on the advances of one another. Molecular cell biologists need to be aware of the patterns of evolutionary diversity as a potential source of mutations for functional analysis, and molecular evolutionists have a responsibility to understand how mitochondria function, so the process can be placed in a phylogenetic context. The comparative method offers a fruitful means by which these individual and collaborative efforts should proceed. The aim here is to examine the evolution of characters within the context of phylogenetic relationships. In the light of the discussion above, the relevant characters are rates of sequence and genome-size evolution in mitochondria, and metabolic rates, body sizes, generation times and other important causal variables that need to be teased apart from one another (see Fig. 6). A number of examples come to mind that might be suitable for these types of tests, such as hummingbirds and a lower metabolic rate sister taxon, or tuna fish49and sharks20 that have evolved endothermy in specific tissues. Thermal habit appears to affect patterns of nucleotide composition and evolution in nuclear genes in diverse specie@, and these studies should provide interesting material for mitochondrial comparisons. In the light of the emerging relationship between physiology and molecular evolution, a casual glance at the tables and graphs showing the classic relationships between physiological traits, taxonomic rank and body size (e.g. Ref. 32) should stimulate more than a handful of thesis projects, postdoctoral fellowships, sabbatical leaves and lifetime research programs. Indeed, tests of the relationships between thermal habit, metabolic rates and the evolution of DNA offers fertile ground for synthetic advances among diverse disciplines of biology. Note added in proof A recent paper by M. Lynch and P.E. JarreW
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confirms the heterogeneity of rates of mtDNA evolution among animal lineages. Notably, primates and honeybees exhibit fast rates of mtDNA evolution while two sea urchin lineages exhibit fast and average rates, respectively. The data and methods of Lynch and Jarrell offer interesting material for further tests of the competing hypothesis discussed in this review.
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1 would like to thank Andrew Martin, Steve Palumbi and John Avise for providing manuscripts before publication. 1 would also like to thank David Clayton and Ken Miller for discussions about mitochondria. Chris Simon and an anonymous reviewer provided helpful comments on an earlier version. My work on mtDNA is supported by the US National Science Foundation.
References 1 Avise, J.C. et al. (1987)Annu. Rev. Ecol. Syst. 18,489-522 Moritz, C., Dowling, T.E. and Brown, W.M. (1987) Arm. Rev. Ecol.
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Syst. 18,269~292 3 4 5 Fig. 6. Hypothetical phylogenies of organisms that demonstrate tests of competing hypotheses to account for variation in the rate of mtDNA evolution. Species that have evolved (a) long versus short generation times, or (b) high versus low metabolic rates, independently, should exhibit accelerated or reduced rates of mtDNA evolution, respectively. Species exhibiting different combinations, (c), of short or long generation times with high or low metabolic rates could be used further to test for interactions among these factors in setting the pace of mtDNA evolution. By assigning taxa to real (versus random) grades and clades significantly longer branch lengths could be tested for where accelerated evolution is predicted.
Avise, J.C. (1991)Annu. Rev. &net. 25,45-69 Upholt, W.I. and Dawid, LB. (1977) Cell 11,571-583 Brown, W.M., George, M. and Wilson, A.C. (1979) Proc. Natl Acad. SC;. USA 76,1967-1971
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Brown, W.M. (1985) in Molecular Evolutionary Genetics (Maclntyre, R.J., ed.), pp. 95-130, Plenum Wilson, A.C. et al. (1985) Biol. J. Linn. Sot. 26, 375-400 Wilson,AC., Ochman, H. and Prager, E.M.(1987)Trends Genet 3,241-247 Vawter, L. and Brown, W.M. (1986) Science 234,194-196 Powell, J.R., Caccone, A., Amato, G.D.and Yoon, C. (1986) Proc. NatI
Acad. Sci. USA 83,9090-9093 11 Kimura, M. (1983) The Neutral Theory ofMolecular
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Britten, R.J. (1986) Science 231, 1393-1398 Caccone, A., Amato, G.D.and Powell, J.R. (1988) Generics 118,671-683 Caccone, A. and Powell, J.R. (1990) J. Mol. Euol. 30,273-280 DeSalle, R. and Templeton, A.R. (1988) Eoolution 42, 1076-1084 Crozier, R.H., Crozier, Y.C. and Mackinlay, A.G. (1989) Mol. Biol. Euol. 6.399-411
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Thomas, W.K. and Beckenbach, A.T. (1989) J. Mol. Euol. 29,233-245 Kocher, T.D. el al. (1989) Proc. Nat1Acad. Sci. USA 86,6196-6200 Hasegawa, M. and Kishino, H. (1989) Jap. J. Genet. 64,243-258 Martin, A.P., Naylor, C.J.P. and Palumbi, S.R.(1992)Nafare 357, 153-155 Avise, J.C., Bowen, B.W., Lamb, T.A., Meylan, B. and Bermingham, E. (1992) Mol. Biol. Euol. 9,457-473 22 Bowen, B.W., Nelson, W.S. and Avise, J.C. (1993) Proc. Nat/ Acad. Sci. USA 90,5574-5577
23 Adachi, J., Cao, Y. and Hasegawa, M. (1993) J. Mol. Euol. 36,270-281 24 Benton, M.J. (1990) Vertebrate Paleontology, Unwin Hyman 25 Martin, A.P. and Palumbi. S.R. (1993) Proc. Nat/ Acad. Sci. USA 90, 4087-4091 26 Rand, D.M. and Harrison, R.C. (1989) Genetics 121,551-569 27 Solignac, M., Monnerot, M. and Mounolou, J.C. (1983) froc. Nat/ Acad. Sci. USA 80,6942-6946 28 Wallis, G.P. (1987) Heredity 58,229-238 29 Rand, D.M. (1993) J. Mol. Evol. 37,281-295 30 Snyder, M., Fraser, A.R., LaRoche, J., Gartner-Kepkay, K.E.and Zouros, E. (1987) Proc. Nad Acad. Sci. USA 84,7595-7599 31 Meyer, A. (1993) in Biochemistry and Molecular Biology of Fishes, Vol. 2 (Hochachka, P.W. and Mommsen, T.P., eds), pp. l-38, Elsevier
32 Calder, W.A., Ill (1984) Size, Function and Life History, Harvard 33 34 35 36
University Press Sharp, P.M. and Li, W-H.(1989)J. Mol. Euol. 28,398-402 Gibbons, J.W. (1987) Bioscience 37,262-269 Bogenhagen, D. and Clayton, D.A. (1977) Cell 11, 719-727 Shigenaga, M.K.,Gimeno, C.J. and Ames, B.N. (1989) froc. Nat/Acad.
Sci. USA 86,9697-9701 37 Richter, C., Park, J-W. and Ames, B.N. (1988) Proc. NaUAcad. Su. USA 8526465-6467 38 Porter, R.K.and Brand, M.D. (1993) Nature 362,628-630 39 Nobes, CD., Brown, G.C., Olive, P.N. and Brand, M.D. (l990)5 Biol. Chem. 265,12903-12909 40 Brand, M.D. (1990) J. Theor. Bio/. 145,267-286 41 Horai, S. eta/. (1992) J. Mol. Euol. 35,32-43 42 Wilkinson, G.S. and Chapman, A.M. (1991) Genetics 128.607-617 43 Arnason, E. and Rand, D.M. (1992) Genetics 132,211-220 44 Moritz, CM. and Brown, W.M. (1987) froc. Nat1 Acad. Sci. USA 84, 7183-7187 45 Buroker, N.E. etal. (1990) Genetics 124, 157-163 46 Annex, B.H. and Williams, R.S. (1990) Mol. Ce/[ Biol. 10,5671-5678 47 Gross, N.J., Getz, ES. and Rabinowitz, M. (1969)J Biol. Chem. 244.
1552-1562 48 Clayton, D.A. (1982) Cell 28,693-705 49 Block, B.A., Finnerty, J.R., Stewart, A.F.R.and Kidd. J. (1993) Science
260,210-214 50 Bernardi, G. and Bernardi, G. (1991) J. Mol. Euol. 31,282-293 51 Lynch, M. and Jarrell, P.E. (1993) Genetics 135, 1197-1208
Patch-occupancydynamicsin landscapes llkka Hanski
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generalizations in ecology. The Recent work on the dynamics of species cologists are often faced species-area relationship, and the living in fragmented landscapes has with the dilemma of holdproduced much information on patterns of analogous species-isolation reing only observational data lationship (decreasing species habitat patch occupancy In a wide range on patterns, while being number with increasing isolation) of organisms. Building on an elementary hard pressed to say something were two critical building blocks Markov chain model of patch occupancy, about the processes that have supposedly produced these pat- a family of Incidence-function models can on which the equilibrium theory of island biogeographp was esbe constructed for particular kinds of terns. A popular example is the pattablished. These multi-species metapopulatlons. These models can be tern of habitat patch occupancy patterns tend to emerge from reguparameterlzed with field data on patch of species living in fragmented larities in the occurrence of indioccupancy, and the models can be used landscapes, and the processes of vidual species on true or habitat to make quantitative predictions about stochastic population extinction islands varying in area and isospecific metapopulations. This approach and patch recolonization. Patchprovides a potentially powerful tool for the lation. Below, I use the shorthand occupancy dynamics is a fashionterm ‘(habitat) patch’ for islands management of reserve networks and able’-3, though still controversia14,5, and also discrete fragments of a species living in fragmented landscapes. issue in conservation biology, particular kind of habitat. where there is the additional difPatterson7J has drawn attenficulty that experimentation would llkka Hanski is at the Dept of Zoology, University of tion to significant ‘nestedness’ of often be unethical as well as Helsinki, PO Box 17 (P. Rautatiekatu 13), species occurrences in habitat impractical. The purpose of this FIN-00014 Helsinki, Finland. patches. Theoretically, species review is to summarize the kind might occur as if randomly placed of pattern data that ecologists in patches, with the constraint that smaller patches have interested in ‘patch-occupancy dynamics’ have accumulated, and to outline a recent approach to modelling of a smaller probability of having any one of the species than larger patches. But this is not the pattern typically occupancy data for the purpose of answering questions observed. In reality, each species tends to occur in patches about the dynamics. exceeding an apparent threshold value in size. This threshold area varies among the species, which produces Patterns of patch occupancy the more-or-less regular increase in species number with It has been said that the increase in species number increasing area. That is, species that are present in a with increasing (island) area is one of the few valid TREE uol.
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Thermalhabit, metabolicrate and the evolutionof mitochondrialDNA David M. Rand
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f one could design a customThe hallmarks of animal mitochondrial subsequent estimates of the rate ized genetic marker for use in DNA (mtDNA) are a rapid rate of sequence of mtDNA evolution in other verpopulation and evolutionary evolution, a small genome carrying the tebrates also supported a figure biology, it is quite likely that same set of homologousgenes, maternal of about 2% divergence per million one would reinvent mtDNA.This is inheritance and lack of recombination. years6,7. This led to a widespread essentially the conclusion reached Over the past few years, a variety of acceptance of mtDNAas a reliable by Avise and colleagues1 in drawdifferent observations has challenged molecular clock for divergenceing up a wish list of the attributes these accepted notions of mitochondrlal times which are less than 10 of a molecular marker for use in biology. Notable examples include million years (Refs 6-8). phylogenetic analysis. It is no coevidence for variable rates of mtDNA Most of these estimates of the incidence that the unique genetic sequence evolution among taxa, evidence rate of mtDNA evolution were obcharacteristics of animal mtDNA for large and variable mitochondrlal tained from restriction-enzyme (rapid rate of sequence evolution; genome sizes in certain groups, and a surveys that sampled the entire compact genome carrying a unigrowing number of cases in metazoans of mitochondrial genome. Despite form set of homologous genes; ‘paternal leakage’ in the inheritance of the evidence for different molecumaternal inheritance; and lack of mtDNA. Several recent studies have lar clocks among genes within recombination, to name a few) are uncovered different lines of evidence mtDNAs,it came as a bit of a surthe very characteristics that prosuggesting that an organism’s thermal prise when the average rates of vide evolutionary biologists with a habit, or metabolic rate, can influence the mtDNA and single-copy nuclear unique picture of population difevolution of mtDNA. DNA (scnDNA) evolution were ferentiation and species relationshown to be very similar in sea ships*J. urchins9 and Dmsophilu~~.These David Rand is at the Dept of Ecology and Evolutionaty But what if our customized studies provided the first robust Biology, Box G-W, Brown University, Providence, marker was (1) shown to evolve rejection of the notion of a general RI 02912, USA. slowly, (2) was observed to be molecular clock of DNA. Rate variable in genome size due to variation among genes (either mitochondrial or nuclear) within a lineage could be due sequence duplications, and (3) was capable of being transmitted by both parents? Some evolutionists might react to different functional constraints 6J1,differences in the likelihood that a nucleotide will be mutated or correctedl*, or by casting off the evidence as rare exceptions to the rules that will not compromise continued use of the marker; some combination of these factors. The obvious ramifiothers might abandon the marker for fear of spurious cation of these findings is that both the rate and the asresults; while others might focus in on the exceptions as sociated error of any molecular clock needs to be calibrated being important patterns in nature that could shed light for the specific gene and taxonomic group in question on the processes of molecular evolution. One general messbefore sequence divergence can be used to estimate age of this review is that each of the three reactions times of divergence or other evolutionary events. Where offered above (rejecting the evidence, abandoning the calibrations of absolute rates were possible, variation in marker, and researching the cause) can be appropriate the nuclear rate of evolution seemed to account for the responses to the continued use of mtDNA in light of such extensive variation among taxa in the relative rates of nuclear and mtDNAevolutiongJ*-14.These studies provided exceptions. However, it is the latter response (clarifying the mechanistic bases of these exceptions) that promises little or no evidence to suggest that the mitochondrial to break new ground in our understanding of the natural molecular clock was variablerqa. history of mtDNA and, indeed, the organisms that carry it. Here 1 call attention to some recent work that suggests a Thermal habit and the rate of mitochondrial DNA relationship between metabolic rates and the tempo and evolution: what is the evidence? mode of mtDNA evolution. In the past several years, comparisons within and among insectslsJ6 and vertebrateslr-23 have provided evidence Mitochondrlal DNA and the molecular clock for variation in the absolute rate of mtDNA evolution. One of the distinguishing characteristics of mtDNA, While it seems unlikely that this rate variation can be and one of the primary justifications for its use in evolaccounted for by a single mechanistic basis (compare utionary studies, is its rapid rate of sequence evolution. Refs 15 and 20) an intriguing correlation is emerging Mitochondrial DNAwas first estimated to evolve at a rate between the thermal habit of an organism and the rate of of l-2 x 10-8substitutions per nucleotide site per year, or mtDNA evolution. about 2% sequence divergence between species per million The first indication that thermal habit might affect the years (Refs 4 and 5). This suggested a rate S-10 times rates and patterns of mtDNA evolution came from studies faster than the evolution of nuclear DNA.Although these of mtDNA in fisheslrJ8. In comparing the sequences of initial rate calibrations were based on comparisons between four protein-coding genes (ATPase subunit 6, cytochrome mtDNA of sheep and goats4 and among primate speciess, oxidase subunit 3, NADH dehydrogenase subunit 3 and TREE uol. 9, no
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Divergence time (millions of years) Fig. 1. Different rates of nucleotide-sequence evolution in mtDNA of mammals and sharks20. The number of transversion differences (Pu -_) Py or Py + Pu change) per fourfold degenerate site (a site at which the code is unaffected by the nucleotide present at that site) in pairwise species comparisons is plotted against divergence time for each species pair. The number of transversion differences are from sequences of the cytochrome b, cytochrome oxidase I, ND4 and ND5 genes in primates (triangles), ungulates (circles) and sharks (squares), and are corrected for multiple substitutions. Transversions at fourfold degenerate sites were used since these changes accumulate linearly with time. The times of divergence can be found in Ref. 20 and references therein; the 60 million year point for ungulates is for Suidae versus Ruminantia*O. The regressions are through the origin and yield average evolutionary rates of 70, 60 and 10 x lo-lo transversions per fourfold degenerate site per million years in primates, ungulates and sharks, respectively. The slopes for primates and sharks are significantly different. Redrawn, with permission, from Ref. 20.
NADHdehydrogenase subunit 4L) in the mitochondria of salmon with the homologous sequences in Xenopus, mouse and humans, Thomas and Bechenbach found an unexpectedly high level of sequence similarity between the cold-blooded speciesIr. For these four genes, the amount of amino acid divergence between rainbow trout (Oncorhyncus mykiss) and Xenopus (diverged over 400 million years agoz4) is very similar to the divergence between mouse and human (diverged 65-80 million years ago24). Averaging across these four genes, there is an approximately fivefold reduction in rate of mtDNA evolution in cold-blooded, relative to warm-blooded, vertebrates. A similar result was observed independently for the cytochrome b gene of cold- and warm-blooded specie@. Several additional recent papers have provided further support for slow rates of mtDNA sequence evolution in cold-blooded vertebrates. Martin, Naylor and Palumbi*O showed that the rate of transversion substitutions at fourfold degenerate sites in two mitochondrial protein-coding genes is six to seven times slower in sharks than that in primates and ungulates (Fig. 1). The findings of Martin et al. are compelling since an effort was made to rule out differences between sharks and mammals in nucleotide composition, codon usage, natural selection and the mitochondrial loci used in the rate estimation. Moreover, there is little reason to challenge the rate calibration in sharks since this group is well represented in the fossil record at a range of divergence dates. A similar pattern has emerged in turtles, where mtDNA seems to be evolving at a ‘turtles pace’zl. As indicated by sequence analyses of the cytochrome b genez2 and restriction-enzyme surveys of the entire mtDNA molecule*], the rate of mtDNA evolution in turtles is reduced 126
between three- and eightfold relative to the conventional rate of 2% per million years in ungulates. This is comparable in magnitude to the difference in mtDNA evolutionary rate observed between sharks and mammals20. Two recent and more-extensive analyses have elaborated on the observations described above*3,*s.Adachi, Cao and Hasegawa23examined the rates of amino acid evolution in vertebrate mtDNAs based on 2711 amino acid positions in the complete mitochondrial genomes of carp, Xenopus, chicken, mouse, rat, whale, cow and human. Their analysis has shown convincingly that the mtDNA of cold-blooded vertebrates evolves at least five times slower than that of mammals. At the positions examined, there are about as many differences between a fish (carp) and Xenopus (452 residues) as between humans and the other mammals (between 433 and 497 residues). As noted above, the divergence times of these two comparisons differ by about a factor of five. Moreover, the qualitative distinction between organisms of the two thermal habits glosses over quantitative differences between the various species examined. The rate of amino acid sequence evolution in mitochondrial genes increases in a graded manner from slow sequence evolution in fishes, to slightly faster evolution in amphibians and birds, to the most rapid evolution in mammals*s. This is evident in the branch lengths of a phylogenetic tree derived from the data-set of mitochondrially encoded proteins (Fig. 2). These results extend an earlier study documenting variation in the rate of mtDNA evolution among orders of mammal@. Martin and Palumbi*s provide further evidence for both a qualitative and quantitative relationship between thermal habit and rate of mtDNAevolution. In a plot of the relationship between the rate of mtDNA evolution (as estimated from restriction enzyme surveys of the entire molecule) and an organism’s body size, endotherms and ectotherms fall out on two very different lines (see Fig. 3). Moreover, within each thermal habit, there appears to be a negative relationship between the mtDNA rate and body size. Body size is correlated with a number of different physiological traits, of which generation time and metabolic rate are potentially important with respect to the evolution of DNA25. Using estimates of rates of silent substitution in mtDNA from species where metabolic rates as well as generation times are published, Martin and Palumbi found strong correlations between the rate of evolution and each of these two physiological traits in simian primates. In a multiple regression with these data, only metabolic rate had a
1
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Fig. 2. Variable rates of amino acid evolution in vertebrates. A tree of mitochondrially encoded proteins was constructed23 using a maximumlikelihood model based on an empirically determined amino acid transition matrix. The horizontal length of each branch is proportional to the estimated number of substitutions by this model. There is a trend of increasing branch length indicating progressively faster rates of evolution in these lineages. Redrawn, with permission, from Ref. 23.
I TREE vol.
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REVIEWS significant contribution to this relationship. However, in a similar analysis using nucleotide-sequence data from the mitochondrial cytochrome b gene of mammals, neither metabolic rate nor generation time showed a significant correlation. A multiple regression using both metabolic rate and generation time did reveal a significant relationship. While the negative relationship between body size and the rate of mtDNA evolution is apparent in Fig. 3, the slope of this line is very sensitive to errors in the estimates of divergence dates for mice and whales, the two extremes of body size. Martin and Palumbi qualify their conclusions by suggesting that metabolic rate and generation time may interact in complex ways to determine the specific rate of molecular evolution. Thermal habit and mitochondrial genome size The traditional view of animal mitochondrial genomes is one of a small compact molecule, with no repetitive DNA, pseudogenes or introns, that is identical throughout the tissues of an individuallJJ. This view has changed considerably over the years with evidence for tandemly repeated and duplicated sequences that result in mtDNA length variation among and even within individuals (e.g. Ref. 26 and references therein). While mtDNA size variation was described some time ago in Drosophila (e.g. Ref. 27 and references therein), evidence in vertebrates for mtDNAsize variation and heteroplasmy (the presence of more than one mtDNA type within a cell) was lacking until coldblooded species were studied (e.g. Ref. 28 and references therein). A recent review has uncovered over 50 species that have been reported to exhibit intraspecific variation in mitochondrial genome size, ranging from nematodes to human.9. Among these species, endotherms tend to have smaller mitochondrial genomes than ectotherms while only the latter exhibit the extremely large (e.g. up to 40 kb; Ref. 30) genomes. There is no difference in the frequency of heteroplasmy between the two thermal habits. The most striking difference between endotherms and ectotherms is that in the former, the extent of mtDNA length variation is a much smaller percent of the standard genome size [i.e. (largest genome size-smallest genome size)/(standard genome size) is smaller in endotherms]. These correlations between thermal habit and mitochondrial genome size are also evident when only cold- and warm-blooded
Mass
(kg)
Fig. 3. Relationship between rate of mtDNA sequence divergence (percent divergence per million years) and body size in kg for various vertebrates. The percent sequence divergence estimates are from restriction fragment length polymorphisms (RFLPs) of mtDNA (tabulated in Ref. 25). The boxes represent the range of sequence divergence estimates and body sizes for a grven taxon: (1) mice; (2) dogs; (3) human-chimpanzee: (4) horses; (5) bears: (6) geese; (7) whales; (8) newts; (9) frogs; (10) tortoise: (11) salmon; (12) sea turtles: (13) sharks. The solid lines are not regressions but are drawn to pass through the boxes.29. The dashed line is the hypothesis of a constant rate of divergence of about 2% per million years. From Ref. 25, with permission.
vertebrates are compared29 (see Fig. 4). A trend towards smaller mtDNAs in endotherms is further suggested by the length of overlap of coding sequences in the ATPase 6 gene. In human, cow, mouse and whale, gene overlaps of 46, 40, 43, 31 base pairs are observed, respectively; in Xenopus and several fish species the overlap is only 10 base pair+. It has been suggested that these patterns are the result of differences between endotherms and ectotherms in the rates of mutation generating mtDNA length variation**, and in the strength of selection on mitochondrial genome size26Js. While the patterns do suggest that selection on genome size is different between endotherms and ectotherms, the relative importance of selection versus mutation needs to be addressed through comparative and functional studies (see below).
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35
40
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Fig. 4. Patterns of mitochondrial genomesize variation among endotherms, and invertebrate and vertebrate ectotherms. The data are based on a total of 51 species reported to show intraspecific mtDNA length variation2Q. The means (horizontal arrows) and ranges (vertical bars) are shown for (a) the largest mtDNA and (b) the percent variation in genome srze [(largest mtDNA-smallest mtDNA)/modal mtDNA size]. The means and ranges of all three categories [includmg (c)] are based on 11 endotherms (triangles), 23 vertebrate ectotherms (circles) and 17 invertebrates (squares). The largest mtDNA and percent variation in mtDNA size is significantly smaller in endotherms when compared to the invertebrate and vertebrate ectotherms combined, and when compared to vertebrate ectotherms alone. (c) Relationship between the percent variation in mitochondrial genome size and the largest genome size reported within a species. Regression analyses revealed that this relationship differs significantly between each of the three categories (represented by triangles, circles and squares, as mentroned above). Redrawn, with permission. from Ref. 29.
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REVIEWS The work summarized above provides different lines of evidence indicating a relationship between the thermal habit of an organism and both the tempo and mode of mitochondrial genome evolution. An obvious biological difference between endotherms and ectotherms is metabolic rate. As the mitochondrial organelle and the genome it carries are fundamental to the metabolic flux in eukaryotic cells, it is intuitively appealing to focus on metabolic rate itself as a causal factor in these patterns of mtDNA evolution. However, metabolic rate is a complex biological trait that has intricate causal and correlative relationships with other important aspects of organisms such as body size, generation time and rates of cell division2sJ2. Generation time and the evolution of mitochondrial DNA Differences among taxa in generation time are commonly invoked to account for variation in the rate of a molecular clock (e.g. Ref. 33 and references therein). The assumption of this hypothesis is that species with shorter generation times have a greater number of DNAreplication events per unit time, which should provide a greater opportunity for mutations to occur. Turtles do have generation times which are much longer than those of mammaW, consistent with their slow rate of mtDNA evolution. In some cases, however, the generation-time hypothesis is not consistent with the available data. For example, sharks have generation times similar to those of primates and ungulates despite having a six- to sevenfold slower rate of mtDNA evolution, Among orders of mammals where rates of mtDNAevolution vary, Hasegawa and Kishino found no effect of generation time lg. Also, rates of evolution in the COI-CO11region of honeybee mtDNA are faster than that in Drosophila, despite a shorter generation time in flie+. These observations suggest that factors other than organismal generation time could play a role in determining the pace of mtDNA evolution. Little is known about the relative number of germ-cell generations per animal generation in most animals, and even less is known about the number of mtDNA generations within germ cells. These factors are important since they could serve to decouple the rates of mtDNA replication from rates of cellular35 and organismal reproduction, making the latter a poor predictor of the opportunities for mutation in mtDNA that occur during replication. In addition to differences in rates of mutation or efficiency of repairi2’13,these effects could be responsible for the variation between mtDNA and scnDNA in rates of evolution. Metabolic rates, oxidatlve damage and the evolution of mitochondrlal DNA In many of the cases where the generation-time hypothesis fails, variation in the rate of mtDNA evolution can be explained by differences in specific metabolic rates25. The metabolic-rate hypothesis is supported by biochemical studies showing that species with higher metabolic rates experience higher rates of oxidative DNA damage (Ref. 36 and references therein). Thus, species with lower metabolic rates may experience lower mutation rates due to reduced oxidative damage. Moreover, the amount of oxidative damage to mtDNA is greater than that to nuclear DNA,presumably owing to the central role of mitochondria in oxidative metabolism and the high rate at which oxygen radicals are produced in mitochondriasr. These differences could further explain the variation among taxa in the relative rates of nuclear and mtDNA evolution. 128
A recent study of the relationship between body mass and metabolic rate38 lends additional support to the metabolic-rate hypothesis. In animal cells, there is a constant leak of protons across the inner mitochondrial membrane. Pumping of these protons back across the inner membrane, which consumes a substantial portion of an animal’s oxygen budget, is a major source of heat production39 and is thought to be a significant component of metabolic ratedo. The new evidence shows that the rate of proton leak de creases with increasing body massaa. Since larger animals have fewer mitochondria per kg of body mass, the oxygen consumption needed to drive the proton pump will be further reduced in larger animal@. Hence, the DNA damage from oxygen radicals should be reduced in animals with large body mass, which could account for the negative relationship between body mass and rates of mtDNA evolution observed in both endotherms and ectotherms2” (Fig. 3). The distinct differences in rates of mtDNAevolution between species of the two thermal habits may also stem from lower rates of proton leak in ectotherms, but this relationship needs to be examined. Relaxed constraints and the evolution of mitochondrial DNA Thomas and Bechenbachi7 originally suggested that differences in functional constraints might account for the faster rate of amino acid evolution among mammals relative to salmonid fish. Since proteins must function at a wider range of temperatures in cold-blooded vertebrates, these proteins may be more constrained in terms of sequence evolution, The homogenous cellular environment of warmblooded vertebrates may have allowed for the relaxation of constraints on protein function with respect to physiologically acceptable amino acid substitutions. The relaxedconstraint hypothesis differs from the metabolic-rate hypothesis in that the former focuses on changes in the number of amino acid positions that are free to vary in different cellular environments, whereas the latter focuses on the underlying mutation pressure generating potential variants. Adachi et al.23 also invoke the suggestion17 that variation in the rate of amino acid evolution in mtDNA is the result of relaxed constraint in warm-blooded vertebrates. As a test of this hypothesis, Adachi et al. compare the ratio of the amino acid substitution rate, KA,to the synonymous substitution rate, Ks, between warm-blooded (primate) and cold-blooded (salmonid) species. Since the rate of silent substitution will be more closely proportional to the mutation rate, a higher K,:Ks ratio in warm-blooded vertebrates is taken as support for the view that constraints on amino acid evolution have been relaxed in these species. The data for 489 nucleotides (163 amino acids) of the ATPase 6 gene available for primate&l and salmonids17 do indicate a higher ratio in primates, consistent with the relaxed-constraint hypothesis (Fig. 5). However, Adachi et al. conclude that the elevated rate of protein evolution in primate mtDNA is a combination of relaxed constraints and higher mutation rates, relative to salmonids. It is likely that rate variation in mtDNA evolution is attributable to a more complex combination of factors than to changes in functional constraints17 and metabolic rate25. Small selection coefficients and variable population sizes15, and the population biology of the germ line cytoplasm26Z2g, may alter rates of mtDNA evolution. A useful concept that incorporates many of these factors is the ‘nucleotide generation time’, that is, the average length of time before a nucleotide is copied by replication or repair25. TREE vol.
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Fig. 5. Relationship between the number of amino acid differences per site (K,) and the number of synonymous differences per site (K,) of the ATPase 6 gene of mtDNA. The primates (squares) are rep resented by human (Homo sapiens), chimpanzee (Pan troglodytes), bonobo (Pan paniscus), gorilla (Gorilla gorilla] and orangutan (Pongo pygmaeus), and the salmonid fishes (circles) are represented by rainbow trout (Oncorhynchus mykiss), pink salmon (0. gorbuscha), coho salmon (0. kisutch), sockeye salmon (0. nerka), chinook salmon (0. tschawytscha) and cutthroat trout (0. clarki). The estimates of K, and K, are derived from 489 nucleotides (163 codons) that were sequenced for each of the two groups. The ratio of K, to K, is clearly higher in the primates and is interpreted as support for the hypothesis that the faster rate of amino acid evolution in mammals is due to re laxation of functional constraints on amino acid sequence, relative to that in salmonids23. Redrawn, with permission, from Ref. 23.
Metabolically active species with small body sizes, short generation times and a small effective number of mitochondria passed through the germ line may have higher rates of mtDNA evolution due to a shorter mean residence time of nucleotides. Further studies should focus on organisms whose biology allows one to separate the significant variables affecting the nucleotide generation time (see below). In particular, how such combinations of factors alter the residence times of nucleotides in germ line cells is the fundamental question regarding the evolution of DNA. Genome size variation, mutations and a race for replication Why might endotherms have smaller and less-variable mitochondrial genome sizes than ectotherms? Elevated mutation rates for mtDNA insertions and deletions in ectotherms28, or less-efficient repair, might account for the observed patterns. If the rate at which mtDNA length variants were generated was higher in ectotherms, but the rate at which this variation was lost (due to the effects of genetic drift during the mitotic events associated with the transmission of mitochondria2Qg) was similar among organisms of the two thermal habits, one might expect more mtDNA length variation in ectotherms. This balance of mutation and drift affecting mtDNA size variation has been examined in only a few organisms2@z343. A simple mutation hypothesis might lead to the prediction of differences in the incidence of mtDNA size variation or heteroplasmy in organisms with different rates of length mutation. The available data show no difference between endotherms and ectotherms in the frequency of heteroplasmy29. It is not clear that longer genomes, or a higher proportion of genome length exhibited as variation, would be an explicit prediction of a simple elevated mutation rate. These expectations depend critically on the models TREE uol
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of length mutation, and little is known about the mechanisms that generate mtDNA length variation beyond the general observations that sequence motifs that appear capable of forming secondary structures are often implicated in large-scale length mutations44 and tandem repeat variation26.42,43,45. To the extent that thermal habit or metabolic rate affects the models of mutation generating patterns of mtDNAlength variation, it is important to recognize that these predictions are in the opposite direction from the predictions regarding primary sequence evolution. In the latter, the metabolic-rate hypothesis invokes lower relative rates of mutation (oxidative damage) in low-metabolicrate species. Stronger directional and purifying selection for small genome size in the cytoplasm of endotherms, perhaps mediated by a stronger ‘race for replication’, is also compatible with the data263’9. Both within and between species, there is a correlation between the capacity of a tissue for oxidative respiration and the proportion of mtDNA molecules primed for replication46. Tissues with higher metabolic rates also appear to have higher turnover rates of mtDNA47.The additional nucleotides present in a mtDNA length variant could be a significant burden in terms of the time required to complete replication. Since replication of an entire mtDNA takes about one hour@, mtDNA size may play a more important role in a ‘race for replication’ than, say, the initiation of replication. It may be that, in species with higher metabolic rates, where there can be aggressive replication of the cytoplasmic population of their mtDNAs, a long mtDNA variant has a lower relative ‘fitness’ than a comparable variant in a cytoplasm of a low-metabolic-rate species, where replication is less frequent. Alternatively, the oxidative damage associated with metabolic processes37 could result in the introduction of more lesions in longer mtDNAs than in a shorter molecule. Since lesions in DNA can result in the stalling of polymerases, molecules with the fewest damaged sites will tend to be replicated preferentially which would select against longer mtDNA molecules in the cytoplasm. Higher rates of replication coupled with higher rates of DNA damage in high-metabolic-rate species could result in a mechanism that is both directional (favoring smaller genomes) and purifying (removing variation from cytoplasms), which might generate the observed patternP. Future studies: molecular biology and the comparative method Variation in the tempo and mode of mtDNA evolution, and the competing hypotheses that succeed to varying degrees to account for this variation, can and should be addressed from two very different perspectives. On the one hand, additional data are needed on fundamental aspects of mitochondrial genetics, such as mutational properties, replication, repair, transmission genetics and dynamics in the germ line. These types of studies are needed in a variety of different taxa that vary in biological properties which are relevant to the competing hypotheses (for example, metabolic rates, generation times, germ-line sequestration and development). These studies are tasks for molecular and cellular biologists, who are best equipped technically and intellectually to attack these problems. Greater knowledge of the molecular genetics of mtDNA in diverse taxa remains as an important hurdle to a more thorough understanding of how mtDNA evolve+. As these sorts of data accumulate, evolutionists lessinclined to engage in studies of molecular genetics and cell biology have a wealth of experimental material in the
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REVIEWS diversity of organisms where sequence comparisons alone will continue to uncover important patterns. This endeavor is indeed a collaborative effort, and interested biologists of all persuasions should take the time to stay up to date
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on the advances of one another. Molecular cell biologists need to be aware of the patterns of evolutionary diversity as a potential source of mutations for functional analysis, and molecular evolutionists have a responsibility to understand how mitochondria function, so the process can be placed in a phylogenetic context. The comparative method offers a fruitful means by which these individual and collaborative efforts should proceed. The aim here is to examine the evolution of characters within the context of phylogenetic relationships. In the light of the discussion above, the relevant characters are rates of sequence and genome-size evolution in mitochondria, and metabolic rates, body sizes, generation times and other important causal variables that need to be teased apart from one another (see Fig. 6). A number of examples come to mind that might be suitable for these types of tests, such as hummingbirds and a lower metabolic rate sister taxon, or tuna fish49and sharks20 that have evolved endothermy in specific tissues. Thermal habit appears to affect patterns of nucleotide composition and evolution in nuclear genes in diverse specie@, and these studies should provide interesting material for mitochondrial comparisons. In the light of the emerging relationship between physiology and molecular evolution, a casual glance at the tables and graphs showing the classic relationships between physiological traits, taxonomic rank and body size (e.g. Ref. 32) should stimulate more than a handful of thesis projects, postdoctoral fellowships, sabbatical leaves and lifetime research programs. Indeed, tests of the relationships between thermal habit, metabolic rates and the evolution of DNA offers fertile ground for synthetic advances among diverse disciplines of biology. Note added in proof A recent paper by M. Lynch and P.E. JarreW
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confirms the heterogeneity of rates of mtDNA evolution among animal lineages. Notably, primates and honeybees exhibit fast rates of mtDNA evolution while two sea urchin lineages exhibit fast and average rates, respectively. The data and methods of Lynch and Jarrell offer interesting material for further tests of the competing hypothesis discussed in this review.
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Acknowledgements
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1 would like to thank Andrew Martin, Steve Palumbi and John Avise for providing manuscripts before publication. 1 would also like to thank David Clayton and Ken Miller for discussions about mitochondria. Chris Simon and an anonymous reviewer provided helpful comments on an earlier version. My work on mtDNA is supported by the US National Science Foundation.
References 1 Avise, J.C. et al. (1987)Annu. Rev. Ecol. Syst. 18,489-522 Moritz, C., Dowling, T.E. and Brown, W.M. (1987) Arm. Rev. Ecol.
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Syst. 18,269~292 3 4 5 Fig. 6. Hypothetical phylogenies of organisms that demonstrate tests of competing hypotheses to account for variation in the rate of mtDNA evolution. Species that have evolved (a) long versus short generation times, or (b) high versus low metabolic rates, independently, should exhibit accelerated or reduced rates of mtDNA evolution, respectively. Species exhibiting different combinations, (c), of short or long generation times with high or low metabolic rates could be used further to test for interactions among these factors in setting the pace of mtDNA evolution. By assigning taxa to real (versus random) grades and clades significantly longer branch lengths could be tested for where accelerated evolution is predicted.
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Patch-occupancydynamicsin landscapes llkka Hanski
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generalizations in ecology. The Recent work on the dynamics of species cologists are often faced species-area relationship, and the living in fragmented landscapes has with the dilemma of holdproduced much information on patterns of analogous species-isolation reing only observational data lationship (decreasing species habitat patch occupancy In a wide range on patterns, while being number with increasing isolation) of organisms. Building on an elementary hard pressed to say something were two critical building blocks Markov chain model of patch occupancy, about the processes that have supposedly produced these pat- a family of Incidence-function models can on which the equilibrium theory of island biogeographp was esbe constructed for particular kinds of terns. A popular example is the pattablished. These multi-species metapopulatlons. These models can be tern of habitat patch occupancy patterns tend to emerge from reguparameterlzed with field data on patch of species living in fragmented larities in the occurrence of indioccupancy, and the models can be used landscapes, and the processes of vidual species on true or habitat to make quantitative predictions about stochastic population extinction islands varying in area and isospecific metapopulations. This approach and patch recolonization. Patchprovides a potentially powerful tool for the lation. Below, I use the shorthand occupancy dynamics is a fashionterm ‘(habitat) patch’ for islands management of reserve networks and able’-3, though still controversia14,5, and also discrete fragments of a species living in fragmented landscapes. issue in conservation biology, particular kind of habitat. where there is the additional difPatterson7J has drawn attenficulty that experimentation would llkka Hanski is at the Dept of Zoology, University of tion to significant ‘nestedness’ of often be unethical as well as Helsinki, PO Box 17 (P. Rautatiekatu 13), species occurrences in habitat impractical. The purpose of this FIN-00014 Helsinki, Finland. patches. Theoretically, species review is to summarize the kind might occur as if randomly placed of pattern data that ecologists in patches, with the constraint that smaller patches have interested in ‘patch-occupancy dynamics’ have accumulated, and to outline a recent approach to modelling of a smaller probability of having any one of the species than larger patches. But this is not the pattern typically occupancy data for the purpose of answering questions observed. In reality, each species tends to occur in patches about the dynamics. exceeding an apparent threshold value in size. This threshold area varies among the species, which produces Patterns of patch occupancy the more-or-less regular increase in species number with It has been said that the increase in species number increasing area. That is, species that are present in a with increasing (island) area is one of the few valid TREE uol.
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