Evidence of a secondary interspecific mitochondrial DNA introgression in the pond loach Misgurnus dabryanus (Teleostei: Cobitidae) population introduced in Japan

The pond loach Misgurnus dabryanus is a freshwater fish with a distribution range spanning the eastern part of the Asian continent, the Korean Peninsula, and Taiwan. The pond loach was transplanted to the Japanese archipelago through the co-inclusion with dojo loach (Misgurnus anguillicaudatus species complex) populations, which were imported live from China for food materials, and it is currently distributed widely across Japan. A previous mitochondrial DNA (mtDNA) analysis revealed that a pond loach population in Ehime Prefecture (Shikoku Island, Japan) included two highly diverged mtDNA groups (Groups I and II). To examine the origin of these two distinct forms of mtDNA within the Japanese pond loach population, we performed phylogenetic analyses using sequences based on the mtDNA of cytochrome oxidase b (cyt b) and the nuclear DNA recombination activating gene 1 (RAG-1). We also conducted a random amplified polymorphic DNA (RAPD) analysis to examine the establishment of reproductive isolation between sympatric pond loaches with two different mtDNA groups. Our mtDNA phylogenetic results indicated that the two diverged pond loach mtDNA sequences showed polyphyletic relationships among Misgurnus species and its related genus Cobitis. In contrast, there were no clear divergence in nuclear DNA among the pond loaches irrespective of their mtDNA groups, and they all formed monomorphic clades in the phylogenetic relationships among the species. The discrepancy between the mtDNA and nuclear DNA genes support that the existence of two diverged forms of DNA within the pond loach population could be attributed to past mtDNA introgressions from other species rather than convergent evolution. Previous mtDNA phylogenetic studies among Cobitidae revealed that the dojo loach also consisted of two genetically diverged polyphyletic clades: an original Misgurnus mtDNA and an introgressed mtDNA from Cobitis species. In our mtDNA result, the Group II haplotype of the pond loach was included in the mtDNA from the introgressed dojo loach. This suggested that the Group II haplotype was derived from introgressed dojo loach mtDNA. The close relationships between the introgressed dojo loach and the pond loach mtDNA indicated that this secondary introgression had recently occurred via hybridization in a recent artificial aquaculture or transportation process. Common RAG-1 alleles and RAPD bands were shared between the sympatric pond loaches with original and introgressed mtDNAs. This indicates that the introgressed mtDNA haplotype is included as one of the polymorphic genotypes within the pond loach populations, and does not represent existence of different cryptic species.     

False-positive diagnosis of a single, large-scale mitochondrial DNA deletion by Southern blot analysis: the role of neutral polymorphisms

Single, large-scale deletions of mitochondrial DNA (mtDNA) are a common finding in the molecular investigation of patients with suspected mitochondrial disorders and are typically detected by Southern blot analysis of muscle DNA that has been linearized by a single cutter enzyme (BamHI or PvuII). We describe our investigations of a 47-year-old woman with exercise intolerance, myalgia, and ptosis who underwent a muscle biopsy for a suspected mitochondrial genetic abnormality. Southern blot analysis after digestion of muscle DNA with BamHI revealed the apparent presence of two mtDNA species, indicative of a heteroplasmic deletion of 2.0-2.5 kb in length involving approximately 50% of all molecules. Contrary to this observation, longrange polymerase chain reaction (PCR) amplified only wild-type mtDNA. Sequence analysis revealed that the patient harbored two previously recognized control region polymorphisms, a homoplasmic 16390G>A variant that introduces a new BamHI site and a heteroplasmic 16390G>A change that abolishes this site, thus explaining the initial false-positive testing for a heteroplasmic mtDNA deletion. Our findings highlight the potential problems associated with the diagnosis of mitochondrial genetic disease and emphasize the need to confirm positive cases of mtDNA deletions using more than one enzyme or an independent method such as long-range PCR amplification.     

Historic hybridization and introgression between two iconic Australian anemonefish and contemporary patterns of population connectivity

Endemic species on islands are considered at risk of extinction for several reasons, including limited dispersal abilities, small population sizes, and low genetic diversity. We used mitochondrial DNA (D-Loop) and 17 microsatellite loci to explore the evolutionary relationship between an endemic anemonefish, Amphiprion mccullochi (restricted to isolated locations in subtropical eastern Australia) and its more widespread sister species, A. akindynos. A mitochondrial DNA (mtDNA) phylogram showed reciprocal monophyly was lacking for the two species, with two supported groups, each containing representatives of both species, but no shared haplotypes and up to 12 species, but not location-specific management units (MUs). Population genetic analyses suggested evolutionary connectivity among samples of each species (mtDNA), while ecological connectivity was only evident among populations of the endemic, A. mccullochi. This suggests higher dispersal between endemic anemonefish populations at both evolutionary and ecological timeframes, despite separation by hundreds of kilometers. The complex mtDNA structure results from historical hybridization and introgression in the evolutionary past of these species, validated by msat analyses (NEWHYBRIDS, STRUCTURE, and DAPC). Both species had high genetic diversities (mtDNA h > 0.90, π = 4.0%; msat genetic diversity, gd > 0.670). While high gd and connectivity reduce extinction risk, identifying and protecting populations implicated in generating reticulate structure among these species should be a conservation priority.     

Biogeography of the southeastern United States: a comparison of salamander phylogeographic studies

Most phylogeographic studies of species from the southeastern United States have shown a simple east-west division of mtDNA variation. However, a study of the salamander Ambystoma maculatum resulted in a more complex pattern that includes a close affinity between populations from the Central Highlands of Missouri and Arkansas and the Coastal Plain separated by a genetically distinct central group of populations. We test the generality of this observation by surveying mitochondrial DNA (mtDNA) variation in the closely related species A. talpoideum. An Ambystoma-specific intergenic spacer was amplified and sequenced. The 26 resulting haplotypes varied from 380 to 800 base pairs, and alignments, including the outgroup, required 101 insertions/deletions. Sequence divergence among haplotypes ranged from 0.001 to 0.758. Population subdivision was extensive (theta = 0.64). Phylogenetic analysis of A. talpoideum mtDNA sequence reveals a close relationship between the populations from the Central Highlands and the Coastal Plain. This result is similar to that obtained for A. maculatum, although the A. talpoideum clade is not as well differentiated from its sister clades. We discuss the differences and similarities between the two Ambystoma species and previous studies and call for increased focus on multiple species with similar ecologies as a way to detect subtle biogeographic events.     

Transmission of human mitochondrial DNA along the paternal lineage in transmitochondrial mice

Previously we obtained heteroplasmic mice carrying murine and human mitochondrial DNA (mtDNA). Even the fourth generation of such mice had human mtDNA in their organs, hence, they were used to study the possibility of paternal mtDNA transmission. A lineage was obtained in which human mtDNA was transmitted by males to the progeny in four successive generations. This is the first observation of such a continuous paternal transmission of mtDNA. Persistence of paternal mtDNA in several successive generations of animals suggests that mechanisms aimed at elimination of paternally inherited mtDNA species are not as strict as has been postulated.     

Loss of CpG dinucleotides from DNA. VI. Methylation of mitochondrial and chloroplast genes

From nucleotide sequences of mitochondrial and chloroplast genes the probable frequency of the CpG----TpG + CpA substitutions was determined. These substitutions may indicate the level of prior DNA methylation. It was found that the level of this methylation is significantly lower in mitochondrial DNA (mtDNA) and chloroplast DNA (chDNA) than in nuclear DNA (nDNA) of the same species. The species (taxon) specificity of mtDNA and chDNA methylation was revealed. A correlation was found between the level of CpG methylation in nDNA, and mtDNA and chDNA in different organisms. It is shown that cytosine residues in CpG were not subjected to significant methylation in the fungi and invertebrate mtDNA and also in the algae chDNA. In contrast, the vertebrate mtDNA bears the impress of CpG-supression, which is confirmed by direct data on methylation of these DNA. Here the first data on the possible enzymatic methylation of the plant mtDNA and chDNA were obtained. It was shown that the degree of CpG-suppression in the 5S rRNA nuclear genes of lower and higher plants is significantly higher in the chloroplast genes of 4,5S and 5S rRNA. From data on pea chDNA hydrolysis with MspI and HpaII it was established that in CCGG sequences this DNA is not methylated. The role of DNA methylation in increasing the mutation rate and in accelerating the evolutionary rates of vertebrate mtDNA is discussed.     

Genetic population structure and introgression in Anopheles dirus mosquitoes in South-east Asia

Genetic structure and species relationships were studied in three closely related mosquito species, Anopheles dirus A, C and D in Thailand using 11 microsatellite loci and compared with previous mitochondrial DNA (mtDNA) data on the same populations. All three species were well differentiated from each other at the microsatellite loci. Given the almost complete absence of mtDNA differentiation between An. dirus A and D, this endorses the previous suggestion of mtDNA introgression between these species. The high degree of differentiation between the northern and southern population of An. dirus C (RST = 0.401), in agreement with mtDNA data, is suggestive of incipient species. The lack of genetic structure indicated by microsatellites in four populations of An. dirus A across northern Thailand also concurs with mtDNA data. However, in An. dirus D a limited but significant level of structure was detected by microsatellites over ~400 km in northern Thailand, whereas the mtDNA detected no population differentiation over a much larger area (>1200 km). There is prior evidence for population expansion in the mtDNA. If this is due to a selective sweep originating in An. dirus D, the microsatellite data may indicate greater barriers to gene flow within An. dirus D than in species A. Alternatively, there may have been historical introgression of mtDNA and subsequent demographic expansion which occurred first in An. dirus D so enabling it to accumulate some population differentiation. In the latter case the lack of migration-drift equilibrium precludes the inference of absolute or relative values of gene flow in An. dirus A and D.     

Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals.

DNA damage is considered of paramount importance in aging. Among causes of this damage, free radical attack, particularly from mitochondrial origin, is receiving special attention. If oxidative damage to DNA is involved in aging, long-lived animals (which age slowly) should show lower levels of markers of this kind of damage than short-lived ones. However, this possibility has not heretofore been investigated. In this study, steady-state levels of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) referred to deoxyguanosine (dG) were measured by high performance liquid chromatography (HPLC) in the mitochondrial (mtDNA) and nuclear (nDNA) DNA from the heart of eight and the brain of six mammalian species ranging in maximum life span (MLSP) from 3.5 to 46 years. Exactly the same digestion of DNA to deoxynucleosides and HPLC protocols was used for mtDNA and nDNA. Significantly higher (three- to ninefold) 8-oxodG/dG values were found in mtDNA than in nDNA in all the species studied in both tissues. 8-oxodG/dG in nDNA did not correlate with MLSP across species either in the heart (r=-0.68; P<0.06) or brain (r = 0.53; P<0.27). However, 8-oxodG/dG in mtDNA was inversely correlated with MLSP both in heart (r=-0.92; P<0.001) and brain (r=-0.88; P<0.016) tissues following the power function y = a(.)x(b), where y is 8-oxodG/dG and x is the MLSP. This agrees with the consistent observation that mitochondrial free radical generation is also lower in long-lived than in short-lived species. The results obtained agree with the notion that oxygen radicals of mitochondrial origin oxidatively damage mtDNA in a way related to the aging rate of each species.-Barja, G., Herrero, A. Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals.     

Rates and Patterns of Scndna and Mtdna Divergence within the Drosophila Melanogaster Subgroup

Levels of DNA divergence among the eight species of the Drosophila melanogaster subgroup and D. takahashii have been determined using the technique of DNA-DNA hybridization. Two types of DNA were used: single-copy nuclear DNA (scnDNA) and mitochondrial DNA (mtDNA). The major findings are: (1) A phylogeny has been derived for the group based on scnDNA which is congruent with chromosomal data, morphology, and behavior. The three homosequential species, simulans, sechellia, and mauritiana, are very closely related; the scnDNA divergence indicate the two island species are a monophyletic group. (2) The rates of change of scnDNA and mtDNA are not greatly different; if anything scnDNA evolves faster than mtDNA. (3) The rates of scnDNA evolution are not closely correlated to chromosomal (inversion) evolution. (4) The Drosophila genome appears to consist of two distinct classes of scnDNA with respect to rate of evolutionary change, a very rapidly evolving fraction and a relatively conservative fraction. (5) The absolute rate of change was estimated to be at least 1.7% nucleotide substitution per one million years. (6) DNA distance estimates based on restriction site variation are correlated with distances based on DNA-DNA hybridization, although the correlation is not very strong.     

Purification of mitochondrial DNA from total cellular DNA of small tissue samples

A method is presented for the isolation of highly purified mitochondrial (mt)DNA from a crude DNA extract, making use of the different mobilities of covalently closed circular mtDNA vs. endonuclease-digested nuclear DNA in agarose gels. The preparation is virtually free of any contaminating linear DNA, as judged from its electron microscopic appearance, and can be used for further procedures such as polymerase chain reaction (PCR). Since isolation of mitochondria is not a prerequisite for this method, it can be applied to tissue samples in the mg range. In principle, the method can be applied to every eukaryotic species, provided a molecular hybridization probe is available which permits the position of mtDNA to be located in an agarose gel. This probe can be a cDNA, a DNA fragment generated by PCR, or mtDNA itself, if only the approximate size of the genome is known.     

Phylogeographic genomics of mitochondrial DNA: Highly-resolved patterns of intraspecific evolution and a multi-species, microarray-based DNA sequencing strategy for biodiversity studies

Phylogeographic genomics, based on multiple complete mtDNA genome sequences from within individual vertebrate species, provides highly-resolved intraspecific trees for the detailed study of evolutionary biology. We describe new biogeographic and historical insights from our studies of the genomes of codfish, wolffish, and harp seal populations in the Northwest Atlantic, and from the descendants of the founding human population of Newfoundland. Population genomics by conventional sequencing methods remains laborious. A new biotechnology, iterative DNA “re-sequencing”, uses a DNA microarray to recover 30–300 kb of contiguous DNA sequence in a single experiment. Experiments with a single-species mtDNA microarray show that the method is accurate and efficient, and sufficiently species-specific to discriminate mtDNA genomes of moderately-divergent taxa. Experiments with a multi-species DNA microarray (the “ArkChip”) show that simultaneous sequencing of species in different orders and classes detects SNPs within each taxon with equal accuracy as single-species-specific experiments. Iterative DNA sequencing offers a practical method for high-throughput biodiversity genomics that will enable standardized, coordinated investigation of multiple species of interest to Species at Risk and conservation biologists.     

Decreased diversity but increased substitution rate in host mtDNA as a consequence of Wolbachia endosymbiont infection

A substantial fraction of insects and other terrestrial arthropods are infected with parasitic, maternally transmitted endosymbiotic bacteria that manipulate host reproduction. In addition to imposing direct selection on the host to resist these effects, endosymbionts may also have indirect effects on the evolution of the mtDNA with which they are cotransmitted. Patterns of mtDNA diversity and evolution were examined in Drosophila recens, which is infected with the endosymbiont Wolbachia, and its uninfected sister species D. subquinaria. The level of mitochondrial, but not nuclear, DNA diversity is much lower in D. recens than in D. subquinaria, consistent with the hypothesized diversity-purging effects of an evolutionarily recent Wolbachia sweep. The d(N)/d(S) ratio in mtDNA is significantly greater in D. recens, suggesting that Muller's ratchet has brought about an increased rate of substitution of slightly deleterious mutations. The data also reveal elevated rates of synonymous substitutions in D. recens, suggesting that these sites may experience weak selection. These findings show that maternally transmitted endosymbionts can severely depress levels of mtDNA diversity within an infected host species, while accelerating the rate of divergence among mtDNA lineages in different species.     

Low level of variability of mitochondrial DNA in sea otter populations from Kamchatka and Komandor Islands]

Variation in populations of sea otter Enhydra lutris lutris from Komandor islands and Kamchatka was studied in segment b1-b2 of cytochrome b gene and in the control region (main noncoding region) of mitochondrial DNA (mtDNA) by means of restriction analysis. The total sample size was 59 animals. Polymorphism was recorded only in the control mtDNA region for one restriction endonuclease (MboI). Frequencies of polymorphic variants were 64.7 and 35.3%, respectively. The low level of mtDNA variation in the sea otter populations examined is in good agreement with the published data on mtDNA polymorphism in populations of this species from north America. A discrepancy between the level of mtDNA variation and heterozygosity of protein-coding loci was demonstrated.     

New evidence from mitochondrial DNA of a progenitor-derivative species relationship between black spruce and red spruce (Pinaceae)

Mitochondrial DNA (mtDNA) markers were used to assess the genetic diversity in allopatric populations of black spruce (Picea mariana [Mill.] BSP) and red spruce (P. rubens Sarg.). Patterns of mitochondrial haplotypes (mitotypes) were strikingly different between the two species. All mtDNA markers surveyed were polymorphic in black spruce, revealing four different mitotypes and high levels of mtDNA diversity (P(p) = 100%, A = 2.0, H = 0.496). In contrast, populations of red spruce had only two mitotypes and harbored low levels of ggenetic diversity (P(p) = 13.2%, A = 1.1, H = 0.120). When the southernmost allopatric populations of red spruce were considered, only one mitotype was detected. As previously reported for nuclear gene loci, the diversity observed for mtDNA in red spruce was a subset of that found in black spruce. Comparison of present and previously published data supports the hypothesis of a recent progenitor-derivative relationship between these species, red spruce presumably being derived by allopatric speciation of an isolated population of black spruce during the Pleistocene.     

Multiple nuclear pseudogenes of mitochondrial DNA exist in the canine genome

Many copies of nuclear counterparts of mitochondrial DNA (mtDNA) were found in nuclear DNA from sperm heads of the domestic dog, Canis familiaris, by DNA-DNA hybridization and DNA sequencing. Nuclear counterparts homologous to the mtDNA D-loop region were cloned into lambda phage vectors (EMBL4 and lambda gt11), and nucleotide sequences of seven different mtDNA pseudogenes were then determined. The seven pseudogenes were E3 (474 bp; 82% homology with canine mtDNA), E13 (1867 bp; 67%), 8B (2375 bp; 78%), 12A (2650 bp; 79%), 33 (4131 bp; 86%), 47 (4251 bp; 86%), and E17 (5721 bp; 71%). These seven mtDNA pseudogenes corresponded to portions of cytoplasmic mtDNA containing the genes ile, ND1, leu, 16S rRNA, val, 12S rRNA, phe, D-loop, pro, thr, cytb, and glu. A neighbor-joining phylogenetic tree constructed from 12S rRNA sequences in mtDNA pseudogenes 8B, 33, 47, and E17 and in 10 mtDNA fragments from other species showed that these four pseudogenes form a monophyletic clade with canine mtDNA. A neighbor-joining phylogenetic tree based on the 318-bp cytb region showed that the canine pseudogenes existed before the divergence of 17 related canids, and their divergence dates were calculated at around 4.4 to 8.6 million years ago.     

Accumulation of adenine DNA glycosylase-sensitive sites in human mitochondrial DNA

The mitochondrial respiratory chain inevitably produces reactive oxygen species as byproducts of aerobic ATP synthesis. Mitochondrial DNA (mtDNA), which is located close to the respiratory chain, is reported to contain much more 8-oxoguanine (8-oxoG), an oxidatively modified guanine base, than nuclear DNA. Despite such a high amount of 8-oxoG in mtDNA (1-2 8-oxoG/10(4) G), mtDNA is barely cleaved by an 8-oxoG DNA glycosylase or MutM, which specifically excises 8-oxoG from a C:8-oxoG pair. We find here that about half of human mtDNA molecules are cleaved by another 8-oxoG-recognizing enzyme, an adenine DNA glycosylase or MutY, which excises adenine from an A:8-oxoG pair. The cleavage sites are mapped to adenines. The calculated number of MutY-sensitive sites in mtDNA is approximately 1.4/10(4) G. This value roughly corresponds with the electrochemically measured amount of 8-oxoG in mtDNA (2.2/10(4) G), raising the possibility that 8-oxoG mainly accumulates as an A:8-oxoG pair.     

Role of mtDNA haplogroups in COPD susceptibility in a southwestern Han Chinese population

The interplay of a complex genetic basis with the environmental factors of chronic obstructive pulmonary disease (COPD) may account for the differences in individual susceptibility to COPD. Mitochondrial DNA (mtDNA) contributes to an individual's ability to resist oxidation, an important determinant that affects COPD susceptibility. To investigate whether mtDNA haplogroups play important roles in COPD susceptibility, the frequencies of mtDNA haplogroups and an 822-bp mtDNA deletion in 671 COPD patients and 724 control individuals from southwestern China were compared. Multivariate logistic regression analysis revealed that, whereas mtDNA haplogroups A and M7 might be associated with an increased risk for COPD (OR=1.996, 95% CI=1.149-2.831, p=0.006, and OR=1.754, 95% CI=1.931-2.552, p=0.021, respectively), haplogroups F, D, and M9 might be associated with a decreased risk for COPD in this population (OR=0.554, 95% CI=0.390-0.787, p=0.001; OR=0.758, 95% CI=0.407-0.965, p=0.002; and OR=0.186, 95% CI=0.039-0.881, p=0.034, respectively). Additionally, the increased frequency of the 822-bp mtDNA deletion in male cigarette-smoking subjects among COPD patients and controls of haplogroup D indicated that haplogroup D might increase an individual's susceptibility to DNA damage from external reactive oxygen species derived from heavy cigarette smoking. We conclude that haplogroups A and M7 might be risk factors for COPD, whereas haplogroups D, F, and M9 might decrease the COPD risk in this Han Chinese population.     

Variation of the mitochondrial genome in the evolution of Drosophila

The evidence on mitochondrial genome variation and its role in evolution of the genus Drosophila are reviewed. The mitochondrial genome is represented by a circular double-stranded DNA molecule 16 to 19 kb in length. The genome contains no introns involved in recombination. The entire mitochondrial genome can be arbitrarily divided into three parts: (1) protein-coding genes; (2) genes encoding rRNA and tRNA; and (3) the noncoding regulatory region (A + T region). The selective importance of mutations within different mtDNA regions is therefore unequal. In Drosophila, the content of the A + T pairs in mtDNA is extremely low and a pattern of nucleotide substitution is characterized by a low transition/transversion ratio (and a low threshold of mutation saturation). The deletions and duplications are of common occurrence in the mitochondrial genome. However, this genome lacks such characteristic for the nuclear genome aberrations as the inversions and transpositions. The phenomena of introgression and heteroplasmy provide an opportunity to study the adaptive role of the mitochondrial genome and its role in speciation. Analysis of evidence concerning mtDNA variation in different species of the genus Drosophila made it possible to ascertain data on phylogenetic relationships among species obtained by studying nuclear genome variation. In some species, mtDNA variation may serve as a reliable marker for population differentiation within a species, although evidence on the population dynamics of the mtDNA variation is very scarce.