Mitochondrial Sequence Variation Research Articles

Identification of a novel member in the family Albulidae (bonefishes)

A novel Caribbean species Albula sp. cf. vulpes in the family Albulidae (bonefishes) was diagnosed through genetic and morphometric study. Phylogenies derived from 16S rRNA sequences revealed deeply separated lineages among Caribbean bonefishes. Mitochondrial DNA sequence divergences indicated a separation between 3.0 and 5.2 million years before present (b.p.). Cytochrome b phylogenies further supported the classification of A. sp. cf. vulpes as a novel albulid. Morphological variability revealed several differences between A. sp. cf. vulpes and other Caribbean species. A microsatellite library was developed to discern hybridization rates among the species. Microsatellite analyses revealed low levels of hybridization between some members in the complex. One instance of backcrossing was found between A. vulpesxA. sp. B and a pure A. sp. B, indicating that hybrids may have reduced fitness or may be reproductively isolated due to temporal-spatial spawning habitat differences.

A distinctive new species of moss salamander (Caudata: Plethodontidae: Nototriton) from an imperiled Honduran endemism hotspot

A new species of Nototriton is described from Refugio de Vida Silvestre Texiguat in northern Honduras, and is presented in a new phylogenetic hypothesis for the genus Nototriton. This species differs from all other congeners by a number of morphological characteristics, including but not limited to large nares, syndactylous hands and feet with pointed toe tips, and a pale ventral surface with light mottling. Mitochondrial sequence divergence distinguishes this species from all other Nototriton (3.6–6.0% at 16S and 10.2–15.1% at cytochrome b). Refugio de Vida Silvestre Texiguat harbors a remarkable herpetofauna, including 20% of all endemic amphibian and reptile species in Honduras, among which are five species (one anuran, one lizard, and three snakes) known only from the immediate vicinity of the type locality of the new species. Despite the evolutionary and conservation importance of this reserve, it remains one of the most imperiled cloud forests in Honduras due to persistent illegal logging and forest clearing.

Investigation of mitochondrial sequence variants associated with aminoglycoside-induced ototoxicity in South African TB patients on aminoglycosides

A known side effect of aminoglycoside antibiotics is the development of permanent hearing loss. As South Africa is currently facing a tuberculosis (TB) epidemic, with an increasing number of multi-drug resistant tuberculosis (MDR-TB) infections, the use of aminoglycosides is on the increase. It is therefore important to determine whether the mitochondrial mutations associated with aminoglycoside-induced hearing loss occur at high frequencies in particular ethnic groups in our population. A total of 115 mainly MDR-TB patients all on aminoglycosides and 439 controls representative of the main ethnic groups in South Africa were screened for six mutations using the SNaPshot technique. Furthermore, the mitochondrial genomes of eight patients with ototoxicity were sequenced. Homoplasmic mutations were found in controls (A1555G in 0.9% of Black controls and A827G in 1.1% of Afrikaner controls) which reveal that a significant proportion of the South African population is genetically predisposed to developing aminoglycoside-induced hearing loss. The 961delT+insC (n) and T961G variants were found at frequencies of >1% indicating that both are probably non-pathogenic polymorphisms. Sequencing of the entire mitochondrial genome in eight patients did not reveal any mutations in the MT-RNR1 gene. However, two potentially pathogenic variants, T10114C (I19T in MT-ND3) and T15312C (I189T in MT-CYB) were found that may impact on the oxidative phosphorylation capacity and warrant further investigation for their possible role in this disorder. It is imperative that the genetic basis of this potentially preventable condition be investigated, particularly in countries where aminoglycosides are still commonly used, in order to identify individuals and/or ethnic groups who are at risk for this type of hearing loss.

Phylogeography of Nasonia vitripennis (Hymenoptera) indicates a mitochondrial–Wolbachia sweep in North America

Here we report evidence of a mitochondrial-Wolbachia sweep in North American populations of the parasitoid wasp Nasonia vitripennis, a cosmopolitan species and emerging model organism for evolutionary and genetic studies. Analysis of the genetic variation of 89 N. vitripennis specimens from Europe and North America was performed using four types of genetic markers: a portion of the mitochondrial cytochrome oxidase I gene, nine polymorphic nuclear microsatellites, sequences from 11 single-copy nuclear markers and six Wolbachia genes. The results show that the European populations have a sevenfold higher mitochondrial sequence variation than North American populations, but similar levels of microsatellite and nuclear gene sequence variation. Variation in the North American mitochondria is extremely low (pi=0.31%), despite a highly elevated mutation rate (approximately 35-40 times higher than the nuclear genes) in the mitochondria of Nasonia. The data are indicative of a mitochondrial sweep in the North American population, possibly due to Wolbachia infections that are maternally co-inherited with the mitochondria. Owing to similar levels of nuclear variation, the data could not resolve whether N. vitripennis originated in the New or the Old World.

Germline HVR-II mitochondrial polymorphisms associated with breast cancer in Tunisian women

A high incidence of somatic mtDNA polymorphisms has been reported in a wide variety of human cancers; some of them have been proposed as markers for the early detection of breast cancer. However, little attention has been paid to the potential of germline mitochondrial sequence variations as genetic risk factors for cancer. We performed a case-control study of 70 unrelated Tunisian women with breast cancer and 80 healthy age- and gender-matched blood donors, taking into account clinicopathological data, to evaluate germline polymorphism of mitochondrial HVR-II region as a genetic risk factor for breast cancer. Through direct sequencing, we detected 351 polymorphisms in controls and 248 variants in patients, with 47 and 39 segregating sites, respectively. In both groups, more than 50% of the polymorphisms were due to four variants: 315 ins C, 309 ins C, 263 A>G, and 73 A>G. The HVR-II sequences were also classified into haplotypes on the basis of the polymorphisms. Fifty-nine different haplotypes were found, 20 of them shared between patients and controls. Both groups had specific haplotypes, 18 in breast cancer patients and 21 in controls. Statistical analysis revealed a weak protective effect against breast cancer risk for two mitochondrial polymorphisms - 152 T>C (odds ratio (OR) = 0.33, 95% confidence interval (CI) = 0.12-0.91) and 263 A>G (OR = 0.17, 95%CI = 0.06-0.47). In contrast, an increased risk of breast cancer was detected for the 315+C haplotype (OR = 11.66, 95%CI = 1.44-252.23). We conclude that mitochondrial variants can affect breast cancer risk. More extensive studies, involving different types of cancer and patients with different genetic makeup, will be required to improve our understanding of the effects of germline mtDNA polymorphisms on carcinogenesis.

MitoVariome: a variome database of human mitochondrial DNA

BackgroundMitochondrial sequence variation provides critical information for studying human evolution and variation. Mitochondrial DNA provides information on the origin of humans, and plays a substantial role in forensics, degenerative diseases, cancers, and aging process. Typically, human mitochondrial DNA has various features such as HVSI, HVSII, single-nucleotide polymorphism (SNP), restriction enzyme sites, and short tandem repeat (STR).ResultsWe present a variome database (MitoVariome) of human mitochondrial DNA sequences. Queries against MitoVariome can be made using accession numbers or haplogroup/continent. Query results are presented not only in text but also in HTML tables to report extensive mitochondrial sequence variation information. The variation information includes repeat pattern, restriction enzyme site polymorphism, short tandem repeat, disease information as well as single nucleotide polymorphism. It also provides a graphical interface as Gbrowse displaying all variations at a glance. The web interface also provides the tool for assigning haplogroup based on the haplogroup-diagnostic system with complete human mitochondrial SNP position list and for retrieving sequences that users query against by using accession numbers.ConclusionMitoVariome is a freely accessible web application and database that enables human mitochondrial genome researchers to study genetic variation in mitochondrial genome with textual and graphical views accompanied by assignment function of haplogrouping if users submit their own data. Hence, the MitoVariome containing many kinds of variation features in the human mitochondrial genome will be useful for understanding mitochondrial variations of each individual, haplogroup, or geographical location to elucidate the history of human evolution.

Mitochondrial sequence variation in ancient horses from the Carpathian Basin and possible modern relatives

Movements of human populations leave their traces in the genetic makeup of the areas affected; the same applies to the horses that move with their owners This study is concerned with the mitochondrial control region genotypes of 31 archaeological horse remains, excavated from pre-conquest Avar and post-conquest Hungarian burial sites in the Carpathian Basin dating from the sixth to the tenth century. To investigate relationships to other ancient and recent breeds, modern Hucul and Akhal Teke samples were also collected, and mtDNA control region (CR) sequences from 76 breeds representing 921 individual specimens were combined with our sequence data. Phylogenetic relationships among horse mtDNA CR haplotypes were estimated using both genetic distance and the non-dichotomous network method. Both methods indicated a separation between horses of the Avars and the Hungarians. Our results show that the ethnic changes induced by the Hungarian Conquest were accompanied by a corresponding change in the stables of the Carpathian Basin.

Origin and genetic diversity of mosquitofish (Gambusia holbrooki) introduced to Europe

We provide mitochondrial sequence variation of the invasive fish Gambusiaholbrooki from 24 European populations, from Portugal to Greece. Phylogeographic structure in Europe was compared with genetic data from native samples (USA) and historical records were reviewed to identify introduction routes. Overall, data agree with records of historical introductions and translocations, and indicate that the most abundant haplotype throughout Europe originated from North Carolina and corresponded to the first introduction in 1921 to Spain, being transferred to Italy in 1922 and to many countries afterwards. Our results also show that at least another independent introduction occurred first in France and subsequently from France to Greece. Haplotypes of G. affinis were not detected in our European sampling effort but historical records and other data suggest that this species was introduced to Italy in 1927 and it might be present. At the continental scale, there is less diversity in Europe than in North America, in agreement with the low number of introduced fish. At the local scale, some European populations gained diversity from multiple introductions and from “de novo” mutations.

Taxonomic status and evolutionary history of the Saxicola torquata complex

We explored variation in mitochondrial ND2 gene sequences from 171 stonechats ( Saxicola torquata complex) collected from 27 Eurasian and 3 African localities. We found that two widespread neighboring Eurasian subspecies of Siberian stonechat, Saxicola maura maura and S. m. stejnegeri, although very similar in appearance, are not each other’s closest relatives. Eastern Asian S. m. stejnegeri appears to have split from other Palearctic, African, and island stonechats well before differentiation occurred among the latter taxa. Our data indicate that European ( S. t. rubicola), central Palearctic ( S. m. maura) and eastern Palearctic ( S. m. stejnegeri) are independently evolving, and could each warrant separate species status, as assumed earlier for S. rubicola, S. maura and S. torquata. However, we found three localities at which individuals from different major groups occurred. Thus, although these are likely phylogenetic species it is unclear whether they are biological species. There was little phylogeographic structure within the three major Palearctic clades, although samples from Spain might be showing incipient divergence. We maintain that the topology of a mtDNA gene tree is a valid means to discern taxonomic limits.

Utilization of a new host in the screaming cowbird Molothrus rufoaxillaris, a host specialist brood parasite: host switch or host acquisition?

The screaming cowbird Molothrus rufoaxillaris has been long known as a host specialist brood parasite. However, in the past years, the utilization of two new hosts has been documented. We examined the variation in mitochondrial control region sequences from screaming cowbird chicks found in the nests of two hosts, the bay-winged cowbird (Agelaioides badius), which is its regular host, and the chopi blackbird (Gnorimopsar chopi), which is a new host, in Formosa Province, Argentina. If a group of females switched to this new host, we expected to find an association between host use and haplotype frequency distribution, indicating the presence of host-specific female lineages, whereas we expected no such association if the cowbird population incorporated this new host and females use both hosts simultaneously. Haplotype frequency distributions differed between cowbird chicks from the nests of both hosts. This indicates that nest choice by females of this brood parasite is not random and that they preferentially parasitize the nests of the same host species.

Mitochondrial D-loop sequence variation among autochthonous horse breeds in Croatia

Genetic variation in three Croatian coldblood horse populations was analysed using a sequence analysis of the proximal part (nt 15 498–15 821) of the D-loop region of mtDNA. Twenty unrelated horses were chosen from the Posavina horse and the Croatian Coldblood breeds and fifteen horses from the Murinsulaner horse population. Sequencing of the proximal part of the mtDNA D-loop region revealed 26 polymorphic sites representing thirty haplotypes which were clustered into eight haplogroups. A wide variety of mitochondrial haplotypes of the analysed horse breeds were clustered into eight different haplogroups. Two haplogroups (D and F) were specific to Posavina horse, five haplogroups were shared among the Croatian Coldblood and Murinsulaner horse, the fact that can be explained by selection strategy at the beginning of the 20<sup>th</sup> century and possible gene flow between the two populations. These results indicate the presence of many ancient maternal lineages with high diversity in mtDNA. The genetic information based on mtDNA typing has a great importance for the future breed conservation strategy, especially for the critically endangered breed such as Murinsulaner horse.

Deep genetic divergences among morphologically similar and parapatric Skistodiaptomus (Copepoda: Calanoida: Diaptomidae) challenge the hypothesis of Pleistocene speciation

We used mitochondrial [cytochrome c oxidase subunit I (CO I), cytochrome b, and 16S] and nuclear [internal transcribed spacer (ITS) phylogenies of Skistodiaptomus copepods to test hypotheses of Pleistocene divergence and speciation within the genus. Mitochondrial (mt)DNA sequence divergences do not support hypotheses for Pleistocene speciation and instead suggest much more ancient speciation events in the genus. Skistodiaptomus oregonensis and Skistodiaptomus pygmaeus (i.e. two morphologically similar and parapatric species) exhibited uncorrected mtDNA sequence divergences exceeding 20%. Similarly, we identified three divergent clades of Skistodiaptomus pallidus that exhibited mtDNA sequence divergences exceeding 15%, suggesting that even intraspecific divergence within this morphospecies predates the Pleistocene. We found clear evidence of CO I pseudogenes in S. pygmaeus, but their presence did not lead to significant overestimates of sequence divergences for this gene. Substitution saturation and strong purifying selection have most likely led to underestimates of sequence divergences and divergence times among Skistodiaptomus. The widespread phenomenon of morphological stasis among genetically divergent copepod groups indicates that speciation often occurs with little or no morphological change. Instead, morphological evolution may occur idiosyncratically after speciation and create discordant patterns of morphological similarity, shared ancestry and divergence time. Cryptic species complexes are therefore common in copepods, and morphological species concepts underestimate their true species diversity. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96, 150‐165. ADDITIONAL KEYWORDS: cryptic species ‐ genitalia ‐ intrinsic incompatibility ‐ molecular clocks ‐ molecular phylogeny ‐ morphological stasis ‐ Pleistoscenario ‐ pseudogenes ‐ reproductive isolation ‐ sexual selection.

Bone‐eating Osedax females and their ‘harems’ of dwarf males are recruited from a common larval pool

Extreme male dwarfism occurs in Osedax (Annelida: Siboglinidae), marine worms with sessile females that bore into submerged bones. Osedax are hypothesized to use environmental sex determination, in which undifferentiated larvae that settle on bones develop as females, and subsequent larvae that settle on females transform into dwarf males. This study addresses several hypotheses regarding possible recruitment sources for the males: (i) common larval pool--males and females are sampled from a common pool of larvae; (ii) neighbourhood--males are supplied by a limited number of neighbouring females; and (iii) arrhenotoky--males are primarily the sons of host females. Osedax rubiplumus were sampled from submerged whalebones located at 1820-m and 2893-m depths in Monterey Bay, California. Immature females typically did not host males, but mature females maintained male 'harems' that grew exponentially in the number of males as female size increased. Allozyme analysis of the females revealed binomial proportions of nuclear genotypes, an indication of random sexual mating. Analysis of mitochondrial DNA sequences from the male harems and their host females allowed us to reject the arrhenotoky and neighbourhood hypotheses for male recruitment. No significant partitioning of mitochondrial diversity existed between the male and female sexes, or between subsamples of worms collected at different depths or during different years (2002-2007). Mitochondrial sequence diversity was very high in these worms, suggesting that as many as 10(6) females contributed to a common larval pool from which the two sexes were randomly drawn.

Contrasting patterns of natural variation in global Drosophila melanogaster populations

Despite the popularity of Drosophila melanogaster in functional and evolutionary genetics, the global pattern of natural variation has not yet been comprehensively described in this species. For the first time, we report a combined survey using neutral microsatellites and mitochondrial sequence variation jointly. Thirty-five populations originating from five continents were compared. In agreement with previous microsatellite studies, sub-Saharan African populations were the most variable ones. Consistent with previous reports of a single 'out of Africa' habitat expansion, we found that non-African populations contained a subset of the African alleles. The pattern of variation detected for the mitochondrial sequences differed substantially. The most divergent haplotypes were detected in the Mediterranean region while Africa harboured most haplotypes, which were all closely related. In the light of the well-established African origin of D. melanogaster, our results cast severe doubts about the suitability of mtDNA for biogeographic inference in this model organism.

Mitochondrial cytochromebgene sequence diversity among Steller’s sea lion rookeries in the Kuril Islands and the Sea of Okhotsk

1 National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan 2 Primate Research Institute, Kyoto University, Inuyama 484-8506, Japan 3 National Marine Mammal Laboratory, AFSC, NMFS, NOAA 7600 Sand Point Way, NE, Building 4, Seattle, WA 98115, USA 4 Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai, Fuchu, Tokyo 183-8509, Japan 5 Graduate school of Fisheries Sciences, Division of Marine Environment and resources, Hokkaido University, Hakodate 041-8611, Japan 6 Laboratory of Wildlife Ecology, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan

Purifying selection of mtDNA and its implications for understanding evolution and mitochondrial disease

Mutations of mitochondrial DNA (mtDNA) are frequent in humans and are implicated in many different types of pathology. The high substitution rate and the maternal, asexual mode of transmission of mtDNA make it more likely to accumulate deleterious mutations. Here, we discuss recent evidence that mtDNA transmission is subject to strong purifying selection in the mammalian female germ line, limiting the accumulation of such mutations. This process shapes mitochondrial sequence diversity and is therefore probably of fundamental importance for animal evolution and in human mitochondrial disease.

A new species of African Forest Robin from Gabon (Passeriformes: Muscicapidae: Stiphrornis)

We describe a new species of forest robin from the Gamba Complex in southwest Gabon. This common bird, Stiphrornis pyrrholaemus sp. nov., inhabits primary lowland forest and forages on or near the ground like the other members of the genus Stiphrornis of central and western Africa. Unique phenotypic features of the new species include the male’s bright orange chin, throat, and breast, creamy yellow belly, olive green back and rump, and gray flanks. Mitochondrial sequence divergence corroborates our assessment based on its distinct physical characteristics that this is a new species, and suggest that Stiphrornis erythrothorax is likely the most closely related congener.

A taxonomic reappraisal of the European Daphnia longispina complex (Crustacea, Cladocera, Anomopoda)

The Daphnia longispina complex contains some of the most common water flea species in the northern hemisphere, and has been a model organism for many ecological and evolutionary studies. Nevertheless, the systematics and nomenclature of this group, in particular its Palaearctic members, have been in flux for the past 150 years; this hinders the correct interpretation of scientific results and promotes the erroneous use of species names. We revise the systematics of this species complex based on mitochondrial sequence variation (12S rDNA and COI) of representative populations across Europe, with a special focus on samples from type localities of the respective taxa. Combining genetic evidence and morphological assignments of analysed individuals, we propose a comprehensive revision of the European members of the D. longispina complex. We show that D. hyalina and D. rosea morphotypes have evolved several times independently, and we find no evidence to maintain these morphotypes as distinct biological species. Alpine individuals described as D. zschokkei are conspecific with the above‐mentioned lineage. We suggest that this morphologically and ecologically plastic but genetically uniform hyalina–rosea–zschokkei clade should be identified as D. longispina (O. F. Müller, 1776). The valid name of Fennoscandian individuals labelled D. longispina sensu stricto in the recent literature is D. lacustris G. O. Sars, 1862. Additionally, we discovered another divergent lineage of this group, likely an undescribed species, in southern Norway. Our results present a solution for several prevailing taxonomic problems in the genus Daphnia, and have broad implications for interpretation of biogeographical patterns, and ecological and evolutionary studies.

The Spectrum of Mitochondrial Mutation Differs across Species

Mutations are ubiquitous, and many arise during the very process of replicating and transmitting genomes. This process is the source of the genetic variation that provides the raw material for both evolutionary novelty and human disease. Mutation rates are known to vary among nucleotides, across genomic regions, and between taxa. It is conventional wisdom that animal mitochondrial DNA (mtDNA) is one genomic region that has a particularly high mutation rate. Until recently, this high rate of mutation has been predominantly inferred from high levels of mitochondrial sequence divergence between species. However, the apparently simple process of mutation and sequence divergence is intriguingly complex in mitochondria, due to the unique biology of these extrachromosomal genomes. Unlike nuclear DNA, where a new mutation arises on one of four possible DNA strands that can be passed to a diploid offspring, a new mtDNA mutation exists on one of many thousands of mtDNA strands that might (or might not) get incorporated into an egg. With a complex cellular pedigree of mtDNA molecules per mitochondrion, mitochondria per egg cell, egg cells per female, and an even more complex pedigree of females per population, it is a complicated path from mtDNA mutation to fixed mtDNA difference between species [1]. The basic biology of this problem was sketched out more than 30 years ago in a pioneering study of mtDNA sequence variation in sheep and goats by Upholt and Dawid [2]. They recognized the clonal nature of mtDNA inheritance, the random drift process acting on mutations within cytoplasms, and the likelihood that mutations may contribute to variation within species but not become fixed substitutions between species. In short order, mtDNA became a powerful tool of population and evolutionary biologists when it was realized that the rapid rate of mitochondrial mutation and evolution was useful for evolutionary inference [3,4]. In the mid-1980s, mtDNA mutations became candidates for human disease as several papers attributed a variety of disorders to specific point mutations and deletions in the mitochondrial genome [5–7]. In the ensuing years, mutation in the mitochondrial genome has been studied intensively by two different camps: evolutionary biologists, who assumed that mtDNA mutations had no significant functional effects and would serve as reliable neutral markers, and molecular and cell biologists, who saw mtDNA mutations as an underappreciated source of human pathologies. However, it is becoming increasingly popular to apply evolutionary models to problems in mitochondrial disease [8,9] and to examine molecular mechanisms of mutation among strains of model organisms that have been allowed to mutate and evolve in the lab. What we are learning after three decades of extensive study is that the spectrum of mitochondrial mutations varies widely across taxa, with important consequences for the mutation-selection balance maintaining nucleotide composition. However, a new flurry of papers quantifying mitochondrial mutation rates in mutation accumulation studies across model organisms is showing us just how much we still have to learn about mtDNA mutation, variation, and evolution.

Taxonomic implications for diaptomid copepods based on contrasting patterns of mitochondrial DNA sequence divergences in four morphospecies

Morphological identification methods do not provide reliable and meaningful species identi- fications for taxa where morphological differences among distinct species are either absent or over- looked (i.e., cryptic species). For example, due to the minute nature of the morphological characters used to delineate diaptomid copepod species and the apparent potential for copepod speciation to occur with little or no morphological change (i.e., morphological stasis), morphological identifications of diaptomid species may not adequately capture their true species diversity. Here, we present results from a geographic survey of mtDNA sequences from populations across the geographic ranges of four North American diaptomid species— Leptodiaptomus minutus, Skistodiaptomus pallidus, Skistodiaptomus reighardi, and Onychodiaptomus sanguineus. Shallow mitochondrial DNA sequence divergences (maximum of 1.1%) among haplotypes of L. minutus from across its geographic range suggest that current morphological identification techniques reliably identify this species. In contrast, we found large mitochondrial DNA sequence divergences (14-22%) among populations within the currently recognized morphospecies of S. palli- dus, S. reighardi, and O. sanguineus. However, pairwise sequence divergences within four distinct S. pallidus clades and within populations of S. reighardi and O. sanguineus were similarly low (maximum of 1.5%) as found within L. minutus as a whole. Thus, the S. pallidus, S. reighardi, and O. sanguineus morphospecies may be considered best as cryptic species complexes. Our study therefore indicates that morphological identifica- tions, while sufficient for some species, likely underestimate the true species diversity of diapto- mid copepods. As such, we stress the need for extensive taxonomic revision that integrates genetic, morphological, reproductive, and ecological analy- ses of this diverse and important group of freshwater zooplankton. Furthermore, we believe an extensive taxonomic revision will shed important insight into major questions regarding the roles of geography, phylogeny, and habitat on the frequency of cryptic species on earth.