The complete mitochondrial genome of the verongid sponge Aplysina cauliformis: implications for DNA barcoding in demosponges

DNA “barcoding,” the determination of taxon-specific genetic variation typically within a fragment of the mitochondrial cytochrome oxidase 1 (cox1) gene, has emerged as a useful complement to morphological studies, and is routinely used by expert taxonomists to identify cryptic species and by non-experts to better identify samples collected during field surveys. The rate of molecular evolution in the mitochondrial genomes (mtDNA) of nonbilaterian animals (sponges, cnidarians, and placozoans) is much slower than in bilaterian animals for which DNA barcoding strategies were developed. If sequence divergence among nonbilaterian mtDNA and specifically cox1 is too slow to generate diagnostic variation, alternative genes for DNA barcoding and species-level phylogenies should be considered. Previous study across the Aplysinidae (Demospongiae, Verongida) family of sponges demonstrated no nucleotide substitutions in the traditional cox1 barcoding fragment among the Caribbean species of Aplysina. As the mitochondrial genome of Aplysina fulva has previously been sequenced, we are now able to make the first comparisons between complete mtDNA of congeneric demosponges to assess whether potentially informative variation exists in genes other than cox1. In this article, we present the complete mitochondrial genome of Aplysina cauliformis, a circular molecule 19620 bp in size. The mitochondrial genome of A. cauliformis is the same length as is A. fulva and shows six confirmed nucleotide differences and an additional 11 potential SNPs. Of the six confirmed SNPs, NADH dehydrogenase subunit 5 (nad5) and nad2 each contain two, and in nad2 both yield amino acid substitutions, suggesting balancing selection may act on this gene. Thus, while the low nucleotide diversity in Caribbean aplysinid cox1 extends to the entire mitochondrial genome, some genes do display variation. If these represent interspecific differences, then they may be useful alternative markers for studies in recently diverged sponge clades.     

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

Background  

The 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).     

Deep sympatric mtDNA divergence in the autumnal moth (Epirrita autumnata)

Deep sympatric intraspecific divergence in mtDNA may reflect cryptic species or formerly distinct lineages in the process of remerging. Preliminary results from DNA barcoding of Scandinavian butterflies and moths showed high intraspecific sequence variation in the autumnal moth, Epirrita autumnata. In this study, specimens from different localities in Norway and some samples from Finland and Scotland, with two congeneric species as outgroups, were sequenced with mitochondrial and nuclear markers to resolve the discrepancy found between mtDNA divergence and present species‐level taxonomy. We found five COI sub‐clades within the E. autumnata complex, most of which were sympatric and with little geographic structure. Nuclear markers (ITS2 and Wingless) showed little variation and gave no indications that E. autumnata comprises more than one species. The samples were screened with primers for Wolbachia outer surface gene (wsp) and 12% of the samples tested positive. Two Wolbachia strains were associated with different mtDNA sub‐clades within E. autumnata, which may indicate indirect selection/selective sweeps on haplotypes. Our results demonstrate that deep mtDNA divergences are not synonymous with cryptic speciation and this has important implications for the use of mtDNA in species delimitation, like in DNA barcoding.     

Rampant polyphyly indicates cryptic diversity in a clade of Neotropical flycatchers (Aves: Tyrannidae)

Polyphyletic arrangements in DNA phylogenies are often indicators of cryptic species diversity masked by erroneous taxonomic treatments that are frequently based on morphological data. Although mitochondrial (mt)DNA polyphyly is detected relatively rarely in phylogenetic studies, it has recently been found in a variety of tyrant‐flycatcher (Tyrannidae) groups. In the present study, we provide a DNA phylogeny for a mitochondrial and a nuclear locus with a complete species sampling in Zimmerius flycatchers, showing that the genus is characterized by multiple mtDNA polyphyly. Based on phylogenetic and life‐history information, we suggest the elevation of a number of taxa to species status, leading to a doubling of Zimmerius species‐level diversity compared to taxonomic treatments conducted before 2001. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, ●●, ●●–●●.     

The mitochondrial genome of stygobitic sponge Eunapius subterraneus: mtDNA is highly conserved in freshwater sponges

The complete mitochondrial DNA (mtDNA) genome of the Eunapius subterraneus (Porifera, Demospongiae), a unique stygobitic sponge, was analyzed and compared with previously published mitochondrial genomes from this group. The 24,850 bp long mtDNA genome is circular with the same gene composition as found in other metazoans. Intergenic regions (IGRs) comprise 24.7% of mtDNA and are abundant with direct and inverted repeats and palindromic elements as well as with open reading fames (ORFs) whose distribution and homology was compared with other available mt genomes with a special focus on freshwater sponges. Phylogenetic analyses based on concatenated amino acid sequences from 12 mt protein genes placed E. subterraneus in a well-supported monophyletic clade with the freshwater sponges, Ephydatia muelleri and Lubomirskia baicalensis. Our study showed high homology of mtDNA genomes among freshwater sponges, implying their recent split.     

Contrasting intra versus interspecies DNA sequence variation for representatives of the Batrachospermales (Rhodophyta): Insights from a DNA barcoding approach

Sixty‐five accessions of the species‐rich freshwater red algal order Batrachospermales were characterized through DNA sequencing of two regions: the mitochondrial cox1 gene (664 bp), which is proposed as the DNA barcode for red algae, and the UPA (universal plastid amplicon) marker (370 bp), which has been recently identified as a universally amplifying region of the plastid genome. upgma phenograms of both markers were consistent in their species‐level relationships, although levels of sequence divergence were very different. Intraspecific variation of morphologically identified accessions for the cox1 gene ranged from 0 to 67 bp (divergences were highest for the two taxa with the greatest number of accessions; Batrachospermum helminthosum and Batrachospermum macrosporum); while in contrast, the more conserved universal plastid amplicon exhibited much lower intraspecific variation (generally 0–3 bp). Comparisons to previously published mitochondrial cox2–3 spacer sequences for B. helminthosum indicated that the cox1 gene and cox2–3 spacer were characterized by similar levels of sequence divergence, and phylogeographic patterns based on these two markers were consistent. The two taxa represented by the largest numbers of specimens (B. helminthosum and B. macrosporum) have cox1 intraspecific divergence values that are substantially higher than previously reported, but no morphological differences can be discerned at this time among the intraspecific groups revealed in the analyses. DNA barcode data, which are based on a short fragment of an organellar genome, need to be interpreted in conjunction with other taxonomic characters, and additional batrachospermalean taxa need to be analyzed in detail to be able to draw generalities regarding intraspecific variation in this order. Nevertheless, these analyses reveal a number of batrachospermalean taxa worthy of more detailed DNA barcode study, and it is predicted that such research will have a substantial effect on the taxonomy of species within the Batrachospermales in the future.     

Presence of nuclear copies of mitochondrial origin (NUMTs) in two related species of stingless bee genus Melipona (Hymenoptera: Meliponini)

Transferred copies of mitochondrial DNA (mtDNA) into the nuclear genome (numts) have been reported in several Hymenoptera species, even at a high density in the honey bee nuclear genome. The accidental amplification of numts in phylogenetic studies focused on mtDNA highlights the importance of a correct determination of numts and their related mtDNA sequences. We report here the presence of numts derived from a mitochondrial rDNA 16S gene in the genome of the stingless bee species Melipona colimana and M. fasciata (tribe Meliponini) from Western Mexico. PCR products were cloned in both species obtaining thirty paralogous numts. Numts were identified by the presence of insertions and deletions and the disruption of the 16S secondary structure. Further phylogenetic analyses including alternative mitochondrial cox1 and nuclear ITS1 genes have revealed the presence of another numt (cox1) in the nuclear genome of these two species, and place both as sister lineages within the subgenus Michmelia. This is one of the first studies reporting the presence of numts in Meliponini species, and supports previous studies suggesting frequent transfer of mtDNA to the nuclear genome in Hymenoptera.     

Molecular and morphological boundaries of the predatory mite Neoseiulus californicus (McGregor) (Acari: Phytoseiidae)

Neoseiulus californicus (McGregor) is a natural enemy of pest mites used worldwide in many crops. Its correct identification is thus essential to ensure biological control success. The present study aimed to characterize molecular and morphological intraspecific variations for assisting in the diagnosis of the species and to build baseline information about expected variations within a commercially important phytoseiid species. Morphological and molecular [12S rRNA, cytochrome b mitochondrial (mt)DNA, and internal transcribed spacer] analyses were carried out on fourteen populations collected worldwide and on one mass‐reared strain. The genetic distances between the specimens of N. californicus and another related species were high and no overlap was observed, sustaining the reliability of such molecular methods for assisting a specific diagnosis. Furthermore, the genetic distances between populations of N. californicus were very low and overlap between intra‐ and interpopulations distances was observed, except for two populations collected in France (Marsillargues and Midi‐Pyrénées). The high mitochondrial differentiation between these two latter populations and the others questions their specific status: do they belong to the species N. californicus or to another cryptic species? However, using nuclear DNA marker analyses, no distinct differences were observed. Furthermore, even if significant morphological differences were observed between the populations, these differences were very small and the standard errors within each population were very low. We thus concluded that all the populations studied belong to the species N. californicus, despite unexpected high mitochondrial variations. The present study thus displays the importance of an integrative taxonomic approach for avoiding misidentifications. A discussion on morphological and mtDNA variations in relation to diagnostic reliability is developped. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 393–406.     

Uncovering species boundaries in the Neotropical ant complex Ectatomma ruidum (Ectatomminae) under the presence of nuclear mitochondrial paralogues

Nuclear mitochondrial (mt) paralogues (numts) are non‐functional fragments of mtDNA integrated into the nuclear genome that can overestimate the number of species in analyses based on mtDNA sequences. As numts have relatively slow mutation rates, they can pass undetected by conventional procedures such as inspecting for internal stop codons, indels or apparent polymorphism in chromatograms. Species boundaries based on mtDNA markers therefore require a thorough assessment of numts, especially in insects, where this phenomenon appears to be relatively frequent. Ectatomma ruidum is a widely distributed Neotropical ant species that is distributed from northern Mexico to northern Brazil. Previous behavioural and molecular evidence suggests that this species actually represents a composite taxon. Here we assessed the species boundaries in E. ruidum based on two mt (COI, cyt b) and one nuclear (H3) marker, as well as on external morphology. Ancient and recent mt paralogues were detected in several specimens, although pre‐PCR dilution of DNA template helped to recover most of the mt orthologues. Based on the congruence found between our species delineation obtained from the mt genealogies and the discriminated morphospecies, we propose that E. ruidum is actually composed of at least three species. Two of these species have a wide geographical distribution in the Neotropics, whereas the remaining one was restricted to localities situated near the Pacific coast in south‐east Mexico. We also found extensive intra‐ and interspecific variation in the barcoding locus. Moreover, the nuclear evidence suggests the existence of hybrids between two of these species in Oaxaca, south‐east Mexico. This study agrees with previous studies of other closely related animal taxa, which have revealed a complex evolutionary history and overlooked species diversity in the latter region.     

GENE FLOW AND SPECIES DELIMITATION: A CASE STUDY OF TWO PINE SPECIES WITH OVERLAPPING DISTRIBUTIONS IN SOUTHEAST CHINA

Species delimitation detected by molecular markers is complicated by introgression and incomplete lineage sorting between species. Recent modeling suggests that fixed genetic differences between species are highly related to rates of intraspecific gene flow. However, it remains unclear whether such differences are due to high levels of intraspecific gene flow overriding the spread of introgressed alleles or favoring rapid lineage sorting between species. In pines, chloroplast (cp) and mitochondrial (mt) DNAs are normally paternally and maternally inherited, respectively, and thus their relative rates of intraspecific gene flow are expected to be high and low, respectively. In this study, we used two pine species with overlapping geographical distributions in southeast China, P. massoniana and P. hwangshanensis, as a model system to examine the association between organelle gene flow and variation within and between species. We found that cpDNA variation across these two pine species is more species specific than mtDNA variation and almost delimits taxonomic boundaries. The shared mt/cp DNA genetic variation between species shows no bias in regard to parapatric versus allopatric species’ distributions. Our results therefore support the hypothesis that high intraspecific gene flow has accelerated cpDNA lineage sorting between these two pine species.     

Molecular and morphological analyses revealed a cryptic species of dojo loach Misgurnus anguillicaudatus (Cypriniformes: Cobitidae) in Japan

Although it has been reported that populations of the Japanese dojo loach Misgurnus anguillicaudatus (Cypriniformes: Cobitidae) belong to two distinct mitochondrial (mt)DNA (Type I and Type II), the taxonomic status of the species remains unresolved. To address this question, nuclear DNA and morphological analyses were performed on M. anguillicaudatus population in the Nakaikemi Wetland, where Type I and Type II lineages are sympatric. Results suggest the existence of a cryptic species (Type I) within the Japanese dojo loach.     

A mirage of cryptic species: Genomics uncover striking mitonuclear discordance in the butterfly Thymelicus sylvestris

Mitochondrial DNA (mtDNA) sequencing has led to an unprecedented rise in the identification of cryptic species. However, it is widely acknowledged that nuclear DNA (nuDNA) sequence data are also necessary to properly define species boundaries. Next generation sequencing techniques provide a wealth of nuclear genomic data, which can be used to ascertain both the evolutionary history and taxonomic status of putative cryptic species. Here, we focus on the intriguing case of the butterfly Thymelicus sylvestris (Lepidoptera: Hesperiidae). We identified six deeply diverged mitochondrial lineages; three distributed all across Europe and found in sympatry, suggesting a potential case of cryptic species. We then sequenced these six lineages using double‐digest restriction‐site associated DNA sequencing (ddRADseq). Nuclear genomic loci contradicted mtDNA patterns and genotypes generally clustered according to geography, i.e., a pattern expected under the assumption of postglacial recolonization from different refugia. Further analyses indicated that this strong mtDNA/nuDNA discrepancy cannot be explained by incomplete lineage sorting, sex‐biased asymmetries, NUMTs, natural selection, introgression or Wolbachia‐mediated genetic sweeps. We suggest that this mitonuclear discordance was caused by long periods of geographic isolation followed by range expansions, homogenizing the nuclear but not the mitochondrial genome. These results highlight T. sylvestris as a potential case of multiple despeciation and/or lineage fusion events. We finally argue, since mtDNA and nuDNA do not necessarily follow the same mechanisms of evolution, their respective evolutionary history reflects complementary aspects of past demographic and biogeographic events.     

Comparative Analyses of Coding and Noncoding DNA Regions Indicate that Acropora (Anthozoa: Scleractina) Possesses a Similar Evolutionary Tempo of Nuclear vs. Mitochondrial Genomes as in Plants

Evidence suggests that the mitochondrial (mt)DNA of anthozoans is evolving at a slower tempo than their nuclear DNA; however, parallel surveys of nuclear and mitochondrial variations and calibrated rates of both synonymous and nonsynonymous substitutions across taxa are needed in order to support this scenario. We examined species of the scleractinian coral genus Acropora, including previously unstudied species, for molecular variations in protein-coding genes and noncoding regions of both nuclear and mt genomes. DNA sequences of a calmodulin (CaM)-encoding gene region containing three exons, two introns and a 411-bp mt intergenic spacer (IGS) spanning the cytochrome b (cytb) and NADH 2 genes, were obtained from 49 Acropora species. The molecular evolutionary rates of coding and noncoding regions in nuclear and mt genomes were compared in conjunction with published data, including mt cytochrome b, the control region, and nuclear Pax-C introns. Direct sequencing of the mtIGS revealed an average interspecific variation comparable to that seen in published data for mt cytb. The average interspecific variation of the nuclear genome was two to five times greater than that of the mt genome. Based on the calibration of the closure of Panama Isthmus (3.0 mya) and closure of the Tethy Seaway (12 mya), synonymous substitution rates ranged from 0.367% to 1.467% Ma⁻¹ for nuclear CaM, which is about 4.8 times faster than those of mt cytb (0.076–0.303% Ma⁻¹). This is similar to the findings in plant genomes that the nuclear genome is evolving at least five times faster than those of mitochondrial counterparts. I-Ping Chen and Chung-Yu Tang, co-first author (equal contribution)     

The Updated Phylogenies of the Phasianidae Based on Combined Data of Nuclear and Mitochondrial DNA

The phylogenetic relationships of species in the Phasianidae, Order Galliformes, are the object of intensive study. However, convergent morphological evolution and rapid species radiation result in much ambiguity in the group. Further, matrilineal (mtDNA) genealogies conflict with trees based on nuclear DNA retrotransposable elements. Herein, we analyze 39 nearly complete mitochondrial genomes (three new) and up to seven nuclear DNA segments. We combine these multiple unlinked, more informative genetic markers to infer historical relationships of the major groups of phasianids. The nuclear DNA tree is largely congruent with the tree derived from mt genomes. However, branching orders of mt/nuclear trees largely conflict with those based on retrotransposons. For example, Gallus/Bambusicola/Francolinus forms the sister-group of Coturnix/Alectoris in the nuclear/mtDNA trees, yet the tree based on retrotransposable elements roots the former at the base of the tree and not with the latter. Further, while peafowls cluster with Gallus/Coturnix in the mt tree, they root at the base of the phasianids following Gallus in the tree based on retrotransposable elements. The conflicting branch orders in nuclear/mtDNA and retrotransposons-based trees in our study reveal the complex topology of the Phasianidae.     

The evolutionary history of the order Antipatharia (Cnidaria: Anthozoa: Hexacorallia) as inferred from mitochondrial and nuclear DNA: implications for black coral taxonomy and systematics

Although black corals inhabit all the world's oceans, they have been relatively understudied as ～185 of 247 species occur at depths > 50 m. Antipatharians have been included in several phylogenetic studies; however, sample sizes are small and taxonomic coverage minimal. Low levels of mitochondrial (mt) sequence divergence within Scleractinia and Octocorallia are assumed to apply to all anthozoans, although no formal study has been conducted on the order Antipatharia. To quantify genetic variation in the black coral mitogenome, we analysed DNA sequences of the two longest intergenic regions (IGRs) and cox3‐cox1 for 26 of 41 genera, representing all families and subfamilies. We also quantified divergence at the intraspecific level using six mtIGRs and their flanking protein‐coding genes and rRNA for 100+ colonies of Antipathes griggi. Utilizing sequence data from the two mtIGRs, cox3‐cox1, as well as nuclear 18S and 28S, we constructed the first multi‐locus phylogenies of the Antipatharia. Reconstructions revealed that species in the genus Stichopathes are split across two families, Sibopathes macrospina groups among North Atlantic Parantipathes (suggesting the actinopharynx and mesenteries were secondarily lost), and three families are polyphyletic. These and other results provide novel, independent insights into the evolutionary history of antipatharians and support placement of species into higher‐level groupings based on microscopic skeletal features rather than gross colony morphology. An illustrated key to the seven currently recognized families is also provided. © 2013 The Linnean Society of London     

A striking lack of genetic diversity across the wide-ranging amphibian <Emphasis Type="Italic">Gastrophryne carolinensis</Emphasis> (Anura: Microhylidae)

We examine phylogeographic structure across a wide-ranging microhylid frog (Gastrophryne carolinensis) using both mitochondrial (mtDNA) and nuclear (AFLP) data. Species with similar ecological characteristics such as large range size, low vagility, or existence across known biogeographic barriers, often are comprised of multiple, cryptic lineages. Surprisingly, our analyses of both portions of the genome show very little phylogeographic or population genetic structure. The family Microhylidae is one of the largest families of anurans with over 60 genera and around 400 species distributed across much of the world (Americas, Asia, Africa, and Madagascar), but very few phylogeographic studies have assessed intraspecific genetic diversity across the mitochondrial and nuclear genomes. Our results suggest that G. carolinensis, one of only three species of microhylid native to the US, has experienced a severe population bottleneck with subsequent range expansion. Comparable molecular data from closely related microhylids, in addition to demographic and ecological analyses, will provide valuable insight into patterns of genetic diversity and the processes driving phylogeographic diversity in these wide-ranging frogs.     

Wolbachia Infections Mimic Cryptic Speciation in Two Parasitic Butterfly Species,Phengaris teleius and P. nausithous (Lepidoptera: Lycaenidae)

Deep mitochondrial divergence within species may result from cryptic speciation, from phylogeographic isolation or from endosymbiotic bacteria like Wolbachia that manipulate host reproduction. Phengaris butterflies are social parasites that spend most of their life in close relationship with ants. Previously, cryptic speciation has been hypothesised for two Phengaris species based on divergent mtDNA sequences. Since Phengaris species are highly endangered, the existence of cryptic species would have drastic consequences for conservation and management. We tested for cryptic speciation and alternative scenarios in P. teleius and P. nausithous based on a comprehensive sample across their Palaearctic ranges using COI gene sequences, nuclear microsatellites and tests for Wolbachia. In both species a deep mitochondrial split occurring 0.65–1.97 myrs ago was observed that did not correspond with microsatellite data but was concordant with Wolbachia infection. Haplotypes previously attributed to cryptic species were part of the Wolbachia-infected clades. In both species remaining phylogeographic structure was largely consistent between mitochondrial and nuclear genomes. In P. teleius several mitochondrial and nuclear groups were observed in East Asia while a single haplogroup and nuclear cluster prevailed across continental Eurasia. Neutrality tests suggested rapid demographic expansion into that area. In contrast, P. nausithous had several mitochondrial and nuclear groups in Europe, suggesting a complex phylogeographic history in the western part of the species range. We conclude that deep intraspecific divergences found in DNA barcode studies do not necessarily need to represent cryptic speciation but instead can be due to both infection by Wolbachia and phylogeographic structure.     

Evolutionary and demographic processes shaping geographic patterns of genetic diversity in a keystone species,the African forest elephant (Loxodonta cyclotis)

The past processes that have shaped geographic patterns of genetic diversity may be difficult to infer from current patterns. However, in species with sex differences in dispersal, differing phylogeographic patterns between mitochondrial (mt) and nuclear (nu) DNA may provide contrasting insights into past events. Forest elephants (Loxodonta cyclotis) were impacted by climate and habitat change during the Pleistocene, which likely shaped phylogeographic patterns in mitochondrial (mt) DNA that have persisted due to limited female dispersal. By contrast, the nuclear (nu) DNA phylogeography of forest elephants in Central Africa has not been determined. We therefore examined the population structure of Central African forest elephants by genotyping 94 individuals from six localities at 21 microsatellite loci. Between forest elephants in western and eastern Congolian forests, there was only modest genetic differentiation, a pattern highly discordant with that of mtDNA. Nuclear genetic patterns are consistent with isolation by distance. Alternatively, male‐mediated gene flow may have reduced the previous regional differentiation in Central Africa suggested by mtDNA patterns, which likely reflect forest fragmentation during the Pleistocene. In species like elephants, male‐mediated gene flow erases the nuclear genetic signatures of past climate and habitat changes, but these continue to persist as patterns in mtDNA because females do not disperse. Conservation implications of these results are discussed.     

Conservative and compensatory evolution in oxidative phosphorylation complexes of angiosperms with highly divergent rates of mitochondrial genome evolution

Interactions between nuclear and mitochondrial gene products are critical for eukaryotic cell function. Nuclear genes encoding mitochondrial‐targeted proteins (N‐mt genes) experience elevated rates of evolution, which has often been interpreted as evidence of nuclear compensation in response to elevated mitochondrial mutation rates. However, N‐mt genes may be under relaxed functional constraints, which could also explain observed increases in their evolutionary rate. To disentangle these hypotheses, we examined patterns of sequence and structural evolution in nuclear‐ and mitochondrial‐encoded oxidative phosphorylation proteins from species in the angiosperm genus Silene with vastly different mitochondrial mutation rates. We found correlated increases in N‐mt gene evolution in species with fast‐evolving mitochondrial DNA. Structural modeling revealed an overrepresentation of N‐mt substitutions at positions that directly contact mutated residues in mitochondrial‐encoded proteins, despite overall patterns of conservative structural evolution. These findings support the hypothesis that selection for compensatory changes in response to mitochondrial mutations contributes to the elevated rate of evolution in N‐mt genes. We discuss these results in light of theories implicating mitochondrial mutation rates and mitonuclear coevolution as drivers of speciation and suggest comparative and experimental approaches that could take advantage of heterogeneity in rates of mtDNA evolution across eukaryotes to evaluate such theories.