mtDNA Substitution Rates Research Articles

Genome copy number predicts extreme evolutionary rate variation in plant mitochondrial DNA

Nuclear and organellar genomes can evolve at vastly different rates despite occupying the same cell. In most bilaterian animals, mitochondrial DNA (mtDNA) evolves faster than nuclear DNA, whereas this trend is generally reversed in plants. However, in some exceptional angiosperm clades, mtDNA substitution rates have increased up to 5,000-fold compared with closely related lineages. The mechanisms responsible for this acceleration are generally unknown. Because plants rely on homologous recombination to repair mtDNA damage, we hypothesized that mtDNA copy numbers may predict evolutionary rates, as lower copy numbers may provide fewer templates for such repair mechanisms. In support of this hypothesis, we found that copy number explains 47% of the variation in synonymous substitution rates of mtDNA across 60 diverse seed plant species representing ~300 million years of evolution. Copy number was also negatively correlated with mitogenome size, which may be a cause or consequence of mutation rate variation. Both relationships were unique to mtDNA and not observed in plastid DNA. These results suggest that homologous recombinational repair plays a role in driving mtDNA substitution rates in plants and may explain variation in mtDNA evolution more broadly across eukaryotes. Our findings also contribute to broader questions about the relationships between mutation rates, genome size, selection efficiency, and the drift-barrier hypothesis.

On the conservation of white-clawed crayfish in the Iberian Peninsula: Unraveling its genetic diversity and structure, and origin.

European crayfish species are a clear example of the drastic decline that freshwater species are experiencing. In particular, the native species of the Iberian Peninsula, the white clawed-crayfish (WCC) Austropotamobius pallipes, is listed as "endangered" by the IUCN and included in Annex II of the EU Habitat Directive and requires especially attention. Currently, implemented conservation management strategies require a better understanding of the genetic diversity and phylogeographic patterns, as well as of its evolutionary history. For this purpose, we have generated the largest datasets of two informative ribosomal mitochondrial DNA regions, i.e., cytochrome oxidase subunit I and 16S, from selected populations of the WCC covering its geographical distribution. These datasets allowed us to analyze in detail the (i) genetic diversity and structure of WCC populations, and (ii) divergence times for Iberian populations by testing three evolutionary scenarios with different mtDNA substitution rates (low, intermediate, and high rates). The results indicate high levels of haplotype diversity and a complex geographical structure for WCC in the Iberian Peninsula. The diversity found includes new unique haplotypes from the Iberian Peninsula and reveals that most of the WCC genetic variability is concentrated in the northern and central-eastern regions. Despite the fact that molecular dating analyses provided divergence times that were not statistically supported, the proposed scenarios were congruent with previous studies, which related the origin of these populations with paleogeographic events during the Pleistocene, which suggests an Iberian origin for these WCC. All results generated in this study, indicate that the alternative hypothesis of an introduced origin of the Iberian WCC is highly improbable. The result of this study, therefore, has allowed us to better understand of the genetic diversity, structure patterns, and evolutionary history of the WCC in the Iberian Peninsula, which is crucial for the management and conservation needs of this endangered species.

The role of mitonuclear incompatibilities in allopatric speciation.

Aerobic metabolism in eukaryotic cells requires extensive interactions between products of the nuclear and mitochondrial genomes. Rapid evolution of the mitochondrial genome, including fixation of both adaptive and deleterious mutations, creates intrinsic selection pressures favoring nuclear gene mutations that maintain mitochondrial function. As this process occurs independently in allopatry, the resulting divergence between conspecific populations can subsequently be manifest in mitonuclear incompatibilities in inter-population hybrids. Such incompatibilities, mitonuclear versions of Bateson-Dobzhansky-Muller incompatibilities that form the standard model for allopatric speciation, can potentially restrict gene flow between populations, ultimately resulting in varying degrees of reproductive isolation. The potential role of mitonuclear incompatibilities in speciation is further enhanced where mtDNA substitution rates are elevated compared to the nuclear genome and where population structure maintains allopatry for adequate time to evolve multiple mitonuclear incompatibilities. However, the fact that mitochondrial introgression occurs across species boundaries has raised questions regarding the efficacy of mitonuclear incompatibilities in reducing gene flow. Several scenarios now appear to satisfactorily explain this phenomenon, including cases where differences in mtDNA genetic load may drive introgression or where co-introgression of coadapted nuclear genes may support the function of introgressed mtDNA. Although asymmetries in reproductive isolation between taxa are consistent with mitonuclear incompatibilities, interactions between autosomes and sex chromosomes yield similar predictions that are difficult to disentangle. With regard to establishing reproductive isolation while in allopatry, existing studies clearly suggest that mitonuclear incompatibilities can contribute to the evolution of barriers to gene flow. However, there is to date relatively little definitive evidence supporting a primary role for mitonuclear incompatibilities in the speciation process.

Mitochondrial substitution rates estimation for divergence time analyses in modern birds based on full mitochondrial genomes

Mitochondrial DNA (mtDNA) is a popular resource used for the study of evolutionary processes in birds, especially to infer divergence times between lineages. In many cases, due to the scarcity of fossils with which to constrain the age of nodes on a phylogenetic topology, dating relies on mtDNA substitution rates that, ideally, are specific to the taxa being studied. As such values are often unavailable, many studies use fixed‐rate values from other studies, such as the ‘standard molecular clock’. The validity of this universal rate across all bird lineages and for the different mtDNA loci has been questioned by multiple authors. We here performed the most comprehensive calibration of the mtDNA molecular clock in birds, with the inclusion of complete mitochondrial genomes for 622 bird species and 25 reliable fossil calibrations. The results show variation in the rates among lineages and among markers, confirming the lack of universality of the standard molecular clock. Moreover, we provide specific rates for every mtDNA marker (except D‐loop) in each of the sampled avian orders, a valuable tool for molecular clock analyses in the absence of fossil calibrations.

Long-Lived Species of Bivalves Exhibit Low MT-DNA Substitution Rates.

Bivalves represent valuable taxonomic group for aging studies given their wide variation in longevity (from 1–2 to >500 years). It is well known that aging is associated to the maintenance of Reactive Oxygen Species homeostasis and that mitochondria phenotype and genotype dysfunctions accumulation is a hallmark of these processes. Previous studies have shown that mitochondrial DNA mutation rates are linked to lifespan in vertebrate species, but no study has explored this in invertebrates. To this end, we performed a Bayesian Phylogenetic Covariance model of evolution analysis using 12 mitochondrial protein-coding genes of 76 bivalve species. Three life history traits (maximum longevity, generation time and mean temperature tolerance) were tested against 1) synonymous substitution rates (dS), 2) conservative amino acid replacement rates (Kc) and 3) ratios of radical over conservative amino acid replacement rates (Kr/Kc). Our results confirm the already known correlation between longevity and generation time and show, for the first time in an invertebrate class, a significant negative correlation between dS and longevity. This correlation was not as strong when generation time and mean temperature tolerance variations were also considered in our model (marginal correlation), suggesting a confounding effect of these traits on the relationship between longevity and mtDNA substitution rate. By confirming the negative correlation between dS and longevity previously documented in birds and mammals, our results provide support for a general pattern in substitution rates.

Relation between mitochondrial DNA hyperdiversity, mutation rate and mitochondrial genome evolution in Melarhaphe neritoides (Gastropoda: Littorinidae) and other Caenogastropoda

Mitochondrial DNA hyperdiversity is primarily caused by high mutation rates (µ) and has potential implications for mitogenome architecture and evolution. In the hyperdiverse mtDNA of Melarhaphe neritoides (Gastropoda: Littorinidae), high mutational pressure generates unusually large amounts of synonymous variation, which is expected to (1) promote changes in synonymous codon usage, (2) reflect selection at synonymous sites, (3) increase mtDNA recombination and gene rearrangement, and (4) be correlated with high mtDNA substitution rates. The mitogenome of M. neritoides was sequenced, compared to closely related littorinids and put in the phylogenetic context of Caenogastropoda, to assess the influence of mtDNA hyperdiversity and high µ on gene content and gene order. Most mitogenome features are in line with the trend in Mollusca, except for the atypical secondary structure of the methionine transfer RNA lacking the TΨC-loop. Therefore, mtDNA hyperdiversity and high µ in M. neritoides do not seem to affect its mitogenome architecture. Synonymous sites are under positive selection, which adds to the growing evidence of non-neutral evolution at synonymous sites. Under such non-neutrality, substitution rate involves neutral and non-neutral substitutions, and high µ is not necessarily associated with high substitution rate, thus explaining that, unlike high µ, a high substitution rate is associated with gene order rearrangement.

Disentangling a cryptic species complex and defining new species within the Eumerus minotaurus group (Diptera: Syrphidae), based on integrative taxonomy and Aegean palaeogeography

This study provides an overview of the Eumerus minotaurus taxon group, diagnosing a new species, E. anatolicus Grković, Vujić and Radenković sp. n. (Muğla, Turkey), and unraveling three cryptic species within E. minotaurus: E. karyates Chroni, Grković and Vujić sp. n. (Peloponnese, Greece), E. minotaurus Claussen and Lucas, 1988 (Crete and Karpathos, Greece) and E. phaeacus Chroni, Grković and Vujić sp. n. (Corfu and Mt Olympus, Greece; Mt Rumija, Montenegro). We applied an integrative taxonomic approach based on molecular, morphological and wing geometric morphometric data to corroborate and delimit cryptic species within the complex. In addition, we discuss the latent biogeographic patterns and speciation processes leading to configuration of the E. minotaurus group based on palaeogeographic evolution of the Aegean. Mitochondrial phylogeographic analysis suggested that speciation within the E. minotaurus group is attributable to formation of the mid-Aegean Trench and Messinian Salinity Crisis, and was integrated at the Pleistocene. We show that more accurate estimates of divergence times may be based on geological events rather than the standard arthropod mtDNA substitution rate.

Mode and Rate of Evolution of Haemosporidian Mitochondrial Genomes: Timing the Radiation of Avian Parasites.

Haemosporidians are a diverse group of vector-borne parasitic protozoa that includes the agents of human malaria; however, most of the described species are found in birds and reptiles. Although our understanding of these parasites’ diversity has expanded by analyses of their mitochondrial genes, there is limited information on these genes’ evolutionary rates. Here, 114 mitochondrial genomes (mtDNA) were studied from species belonging to four genera: Leucocytozoon, Haemoproteus, Hepatocystis, and Plasmodium. Contrary to previous assertions, the mtDNA is phylogenetically informative. The inferred phylogeny showed that, like the genus Plasmodium, the Leucocytozoon and Haemoproteus genera are not monophyletic groups. Although sensitive to the assumptions of the molecular dating method used, the estimated times indicate that the diversification of the avian haemosporidian subgenera/genera took place after the Cretaceous–Paleogene boundary following the radiation of modern birds. Furthermore, parasite clade differences in mtDNA substitution rates and strength of negative selection were detected. These differences may affect the biological interpretation of mtDNA gene lineages used as a proxy to species in ecological and parasitological investigations. Given that the mitochondria are critically important in the parasite life cycle stages that take place in the vector and that the transmission of parasites belonging to particular clades has been linked to specific insect families/subfamilies, this study suggests that differences in vectors have affected the mode of evolution of haemosporidian mtDNA genes. The observed patterns also suggest that the radiation of haemosporidian parasites may be the result of community-level evolutionary processes between their vertebrate and invertebrate hosts.

Peeking through the trapdoor: Historical biogeography of the Aegean endemic spider Cyrtocarenum Ausserer, 1871 with an estimation of mtDNA substitution rates for Mygalomorphae

The Aegean region, located in the Eastern Mediterranean, is an area of rich biodiversity and endemism. Its position, geographical configuration and complex geological history have shaped the diversification history of many animal taxa. Mygalomorph spiders have drawn the attention of researchers, as excellent model systems for phylogeographical investigations. However, phylogeographic studies of spiders in the Aegean region are scarce. In this study, we focused on the phylogeography of the endemic ctenizid trap-door spider Cyrtocarenum Ausserer, 1871. The genus includes two morphologically described species: C. grajum (C.L. Koch, 1836) and C. cunicularium (Olivier, 1811). We sampled 60 specimens from the distributions of both species and analyzed four mitochondrial and two nuclear markers. Cyrtocarenum served as an example to demonstrate the importance of natural history traits in the inference of phylogeographic scenarios. The mtDNA substitution rates inferred for the genus are profoundly higher compared to araneomorph spiders and other arthropods, which seems tightly associated with their biology. We evaluate published mtDNA substitution rates followed in the literature for mygalomorph spiders and discuss potential pitfalls. Following gene tree (maximum likelihood, Bayesian inference) and species tree approaches (*BEAST), we reconstructed a time-calibrated phylogeny of the genus. These results, combined with a biogeographical ancestral-area analysis, helped build a biogeographic scenario that describes how the major palaeogeographic and palaeoclimatic events of the Aegean may have affected the distribution of Cyrtocarenum lineages. The diversification of the genus seems to have begun in the Middle Miocene in the present west Aegean area, while major phylogenetic events occurred at the Miocene–Pliocene boundary for C. cunicularium, probably related to the Messinian Salinity Crisis. Our results also demonstrate the clear molecular distinction of the two morphologically described species, but possible cryptic lineages may exist within C. cunicularium.

Examining the role of effective population size on mitochondrial and multilocus divergence time discordance in a songbird.

Estimates of speciation times are subject to a number of potential errors. One source of bias is that effective population size (Ne) has been shown to influence substitution rates. This issue is of particular interest for phylogeographic studies because population sizes can vary dramatically among genetically structured populations across species’ ranges. In this study, we used multilocus data to examine temporal phylogeographic patterns in a widespread North American songbird, the Northern Cardinal (Cardinalis cardinalis). Species tree estimation indicated that the phylogeographic structure of C. cardinalis was comprised of four well-supported mainland lineages with large population sizes (large Ne) and two island lineages comprised of much smaller populations (small Ne). We inferred speciation times from mtDNA and multilocus data and found there was discordance between events that represented island-mainland divergences, whereas both estimates were similar for divergences among mainland lineages. We performed coalescent simulations and found that the difference in speciation times could be attributed to stochasticity for a recently diverged island lineage. However, the magnitude of the change between speciation times estimated from mtDNA and multilocus data of an older island lineage was substantially greater than predicted by coalescent simulations. For this divergence, we found the discordance in time estimates was due to a substantial increase in the mtDNA substitution rate in the small island population. These findings indicate that in phylogeographic studies the relative tempo of evolution between mtDNA and nuclear DNA can become highly discordant in small populations.

Quantifying the Elevation of Mitochondrial DNA Evolutionary Substitution Rates Over Nuclear Rates in the Intertidal Copepod Tigriopus californicus

Mitochondrial DNA (mtDNA) genomes generally evolve rapidly in animals, but considerable variation in the rates of evolution of mtDNA occurs among taxa. Higher levels of mutation will tend to increase the amount of polymorphism, which should also scale with population size, but there are mixed signals from previous studies on the evolutionary outcomes of the interactions of these processes. The copepod Tigriopus californicus provides an interesting model in which to study the evolution of mtDNA because it has high levels of divergence among populations and there is the suggestion that this divergence could be involved in reproductive isolation. This species also appears to have an elevated mtDNA substitution rate, but previous studies did not provide an accurate measurement. This article examines the rate of mtDNA substitution versus nuclear substitution in T. californicus and finds that the mtDNA rate for synonymous sites averages 55-fold higher, a level that exceeds the rates found in most other invertebrates. Levels of polymorphism are also examined in both mtDNA and nuclear genes, and it is shown that the effective population size of mtDNA genes is much lower than that of nuclear genes. In addition, no correlation between polymorphism in mtDNA and nuclear genes is found across populations, which suggest factors other than demography may shape polymorphism in this species. The results from this study suggest that mtDNA is evolving at a very rapid rate in this copepod species, and this could increase the likelihood that mtDNA evolution is involved in the generation of reproductive isolation.

Evolution ofCryptocephalusleaf beetles related toC. sericeus(Coleoptera: Chrysomelidae) and the role of hybridization in generating species mtDNA paraphyly

Gómez‐Zurita, J., Sassi, D., Cardoso, A. & Balke, M. (2011). Evolution ofCryptocephalusleaf beetles related toC. sericeus(Coleoptera: Chrysomelidae) and the role of hybridization in generating species mtDNA paraphyly. —Zoologica Scripta,41, 47–67.Autochthonous European insect diversity is the result of the very complex geological, geographic and climatic history of the Mediterranean area. The leaf beetle genusCryptocephalushas over 250 species in this area. Among them stands out a group nearly endemic from this region consisting of conspicuous metallic green or blue beetles which can be found visiting yellow Asteraceae flowers in most mid‐ to high‐altitude European grasslands: the Linnaean speciesC. hypochaeridis,C. sericeus, and all their relatives. In all, these are 32 species forming several taxonomically complex groups across Europe. We sampled all morphological diversity in this lineage and characterized it for two mitochondrial DNA genes. The mtDNA phylogeny of this assemblage was inferred, as well as the timing of its diversification using standard mtDNA substitution rates and a hypothetical Messinian vicariant split. The origin of the group can be traced back to western continental Eurasia in the Lower Miocene. Its subsequent taxonomic splits can be linked to specific periods in the formation of Europe, with a marked trend to east–west phylogenetic divides throughout time and space, and a nearly constant rate of diversification. Only during the Pleistocene, a significant increase in diversification rate can be associated with species formation in theC. hypochaeridisandC. sericeusspecies complexes. Within these latter groups, most taxa show some degree of mtDNA paraphyly as a result of their recent separation and remarkably by episodes of gene flow in areas of secondary contact among recently diverged species, possibly driven by climatic change.

Mitochondrial phylogeny and biogeographic history of the Greek endemic land-snail genus Codringtonia Kobelt 1898 (Gastropoda, Pulmonata, Helicidae)

The aim of this work was to infer the phylogeny of the Greek endemic land-snail genus Codringtonia Kobelt 1898, estimate the time frame of the radiation of the genus, and propose a biogeographic scenario that could explain the contemporary distribution of Codringtonia lineages. The study took place in the districts of Peloponnese, Central Greece and Epirus of mainland Greece. Sequence data originating from three mtDNA genes (COI, COII, and 16S rDNA) were used to infer the phylogeny of the eight nominal Codringtonia species. Furthermore, the radiation time-frame of extant Codringtonia species was estimated using a relaxed molecular clock analysis and mtDNA substitution rates of land snails. The phylogenetic analysis supported the existence of six Codringtonia lineages in Greece and indicated that one nominal species (Codringtonia neocrassa) might belong to a separate genus distantly related to Codringtonia. The time frame of differentiation of Codringtonia species was placed in the Late Miocene–Pleistocene epoch. The dispersal–vicariance analysis performed indicated that most probably Codringtonia exhibited a north-to-south spread with the ancestral area being that of central Greek mainland, accompanied with duplication (speciation) and vicariance events.

Revisiting the Insect Mitochondrial Molecular Clock: The Mid-Aegean Trench Calibration

Phylogenetic trees in insects are frequently dated by applying a "standard" mitochondrial DNA (mtDNA) clock estimated at 2.3% My(-1), but despite its wide use reliable calibration points have been lacking. Here, we used a well-established biogeographic barrier, the mid-Aegean trench separating the western and eastern Aegean archipelago, to estimate substitution rates in tenebrionid beetles. Cytochrome oxidase I (cox1) for six codistributed genera across 28 islands (444 individuals) on both sides of the mid-Aegean trench revealed 60 independently coalescing entities delimited with a mixed Yule-coalescent model. One representative per entity was used for phylogenetic analysis of mitochondrial (cox1, 16S rRNA) and nuclear (Mp20, 28S rRNA) genes. Six nodes marked geographically congruent east-west splits whose separation was largely contemporaneous and likely to reflect the formation of the mid-Aegean trench at 9-12 Mya. Based on these "known" dates, a divergence rate of 3.54% My(-1) for the cox1 gene (2.69% when combined with the 16S rRNA gene) was obtained under the preferred partitioning scheme and substitution model selected using Bayes factors. An extensive survey suggests that discrepancies in mtDNA substitution rates in the entomological literature can be attributed to the use of different substitution models, the use of different mitochondrial gene regions, mixing of intraspecific with interspecific data, and not accounting for variance in coalescent times or postseparation gene flow. Different treatments of these factors in the literature confound estimates of mtDNA substitution rates in opposing directions and obscure lineage-specific differences in rates when comparing data from various sources.

No variation and low synonymous substitution rates in coral mtDNA despite high nuclear variation

BackgroundThe mitochondrial DNA (mtDNA) of most animals evolves more rapidly than nuclear DNA, and often shows higher levels of intraspecific polymorphism and population subdivision. The mtDNA of anthozoans (corals, sea fans, and their kin), by contrast, appears to evolve slowly. Slow mtDNA evolution has been reported for several anthozoans, however this slow pace has been difficult to put in phylogenetic context without parallel surveys of nuclear variation or calibrated rates of synonymous substitution that could permit quantitative rate comparisons across taxa. Here, I survey variation in the coding region of a mitochondrial gene from a coral species (Balanophyllia elegans) known to possess high levels of nuclear gene variation, and estimate synonymous rates of mtDNA substitution by comparison to another coral (Tubastrea coccinea).ResultsThe mtDNA surveyed (630 bp of cytochrome oxidase subunit I) was invariant among individuals sampled from 18 populations spanning 3000 km of the range of B. elegans, despite high levels of variation and population subdivision for allozymes over these same populations. The synonymous substitution rate between B. elegans and T. coccinea (0.05%/site/106 years) is similar to that in most plants, but 50–100 times lower than rates typical for most animals. In addition, while substitutions to mtDNA in most animals exhibit a strong bias toward transitions, mtDNA from these corals does not.ConclusionSlow rates of mitochondrial nucleotide substitution result in low levels of intraspecific mtDNA variation in corals, even when nuclear loci vary. Slow mtDNA evolution appears to be the basal condition among eukaryotes. mtDNA substitution rates switch from slow to fast abruptly and unidirectionally. This switch may stem from the loss of just one or a few mitochondrion-specific DNA repair or replication genes.

MtDNA substitution rate and segregation of heteroplasmy in coding and noncoding regions

MtDNA substitution rate and segregation of heteroplasmy in coding and noncoding regions

MtDNA substitution rate and segregation of heteroplasmy in coding and noncoding regions

The mitochondrial DNA (mtDNA) substitution rate and segregation of heteroplasmy were studied for the non-coding control region (D-loop) and 500 bp of the coding region between nucleotide positions 5550 and 6050, by sequence analysis of blood samples from 194 individuals, representing 33 maternal lineages. No homoplasmic nucleotide substitutions were detected in a total of 292 transmissions. The estimated substitution rate per nucleotide per million years for the control region (micro>0.21, 95% CI 0-0.6) was not significantly different from that for the coding region (micro>0.54, 95% CI 0-1.0). Variation in the length of homopolymeric C streches was observed at three sites in the control region (positions 65, 309 and 16,189), all of which were in the heteroplasmic state. Segregation of heteroplasmic genotypes between generations was observed in several maternal pedigrees. At position 309, a longer poly C tract length was strongly associated with a higher probability for heteroplasmy and rapid segregation between generations. The length heteroplasmy at positions 65 and 16,189 was found at low frequency and was confined to a few families.

Characterisation of Four Species of the Genus Kluyveromyces by Mitochondrial DNA Restriction Analysis

Summary In the present work, we determine the relationships at the within-species level among strains of Kluyveromyces dobzhanskii, K. lactis, K. marxianus, and K. thermotolerans, through the restriction analysis of their mtDNAs. The three first species showed a high level of intraspecific mtDNA divergence, this polymorphism is correlated to the varieties or species defined according to the original taxonomy of the genus, which is in concordance with that shown by other phenotypic or genotypic markers codified for by the nuclear genome. In these species, the analysis of the relationships among strains based on mtDNA restriction data agrees with previous classifications based on morphological traits and physiological abilities. On the contrary, K. thermotolerans showed a reduced level of intraspecific divergence compared to the other three, which indicates that this species either is a more recent species, has suffered a reduction of polymorphism due to the action of evolutionary forces, or presents a lower mtDNA substitution rate.

Body size, metabolic rate, generation time, and the molecular clock.

There is increasing evidence for variation in rates of nucleotide substitution among divergent taxonomic groups. Here, we summarize published rate data and show a strong relationship between substitution rate and body size. For instance, rates of nuclear and mtDNA evolution are slow in whales, intermediate in primates, and fast in rodents. A similar relationship exists for poikilothermic vertebrates. However, these taxa have slower mtDNA substitution rates overall than do homeotherms of similar size. A number of physiological and life history variables are highly correlated with body size. Of these, generation time and metabolic rate explain some patterns of rate heterogeneity equally well. In many cases, however, differences in metabolic rate explain important exceptions to the generation time model. Correlation between metabolic rate and nucleotide substitution may be mediated by (i) the mutagenic effects of oxygen radicals that are abundant by-products of aerobic respiration, and (ii) increased rates of DNA synthesis and nucleotide replacement in organisms with higher metabolic rates. Both of these factors increase mutation rate by decreasing the "nucleotide generation time," the average length of time before a nucleotide is copied either through replication or repair. Reconsideration of the generation time hypothesis to include physiological effects such as metabolic rate improves the theoretical underpinnings of molecular evolution.