Mitochondrial Sequence Variation Research Articles

Mitochondrial DNA-control region sequence variation in the NE Portuguese Jewish community

The cultural phenomenon of Crypto-Judaism, defined as the secret adherence to Judaism while publicly professing another faith, arose in Portugal in the beginning of the 16th century after the Decree of Expulsion and the establishment of the Inquisition. Surprisingly, the scientific community acknowledged the persistence of Crypto-Judaic communities at the beginning of the 20th century in central and north-eastern regions of the country (e.g. Bragança). In the present work, we have sampled 56 unrelated individuals from the Bragança Jewish community aiming to characterize their maternal lineage. A 3348 bp mtDNA fragment was amplified and sequenced using mitochondrial-specific primers in order to obtain the entire control region. Haplogroup classification was performed according to current nomenclature. High frequencies were found for haplogroups H, HV0, T2, U2 and N1, indicating some degree of European admixture along with a remarkable signature of a Near East ancestry. These data confirm that the Crypto-Jews from Bragança were able to maintain not only their cultural identity but also some ancestral genetic identity, showing a significant population substructure within Portugal, with forensic relevance.

Occasional males in parthenogenetic populations of Asobara japonica (Hymenoptera: Braconidae): low Wolbachia titer or incomplete coadaptation?

Wolbachia are endosymbiotic bacteria known to manipulate the reproduction of their hosts. Some populations of the parasitoid wasp Asobara japonica are infected with Wolbachia and reproduce parthenogenetically, while other populations are not infected and reproduce sexually. Wolbachia-infected A. japonica females regularly produce small numbers of male offspring. Because all females in the field are infected and infected females are not capable of sexual reproduction, male production seems to be maladaptive. We investigated why these females nevertheless produce males. We tested three hypotheses: high rearing temperatures could result in higher offspring sex ratios (more males), low Wolbachia titer of the mother could lead to higher offspring sex ratios and/or the Wolbachia infection is of relatively recent origin and not enough time has passed to allow complete coadaptation between Wolbachia and host. In all, 33% of the Wolbachia-infected females produced males and 56% of these males were also infected with Wolbachia. Neither offspring sex ratio nor male infection frequency was significantly affected by rearing temperature or Wolbachia concentration of the mother. The mitochondrial DNA sequence of one of the uninfected populations was identical to that of two of the infected populations. Therefore, the initial Wolbachia infection of A. japonica must have occurred recently. Mitochondrial sequence variation among the infected populations suggests that the spread of Wolbachia through the host populations involved horizontal transmission. We conclude that the occasional male production by Wolbachia-infected females is most likely a maladaptive side effect of incomplete coevolution between symbiont and host in this relatively young infection.

Discovery of Pseudocalanus moultoni (Frost, 1989) in Northeast Atlantic waters based on mitochondrial COI sequence variation

The genus Pseudocalanus (Copepoda, Calanoida) consists of seven species, all of which are known to co-occur with two or more sibling species in some areas of their geographic ranges. Despite the ecological importance of this abundant genus, there is no available method that can reliably and accurately identify Pseudocalanus species without knowledge of origin. We present evidence of several observations of Pseudocalanus moultoni [Frost (1989) Can. J. Zool., 67, 525–551] in fjords of Svalbard and northern Norway; this species has previously been known to occur only on the east and west coasts of North America. Patterns of DNA sequence variation of the mitochondrial cytochrome oxidase subunit I (COI) gene allow us to confidently identify the species, discriminate it from co-occurring sibling species and infer relationships among the newly discovered and previously sampled P. moultoni populations. Our observations suggest that NE Atlantic populations of P. moultoni are self-sustaining and we discuss potential source populations and pathways of transport. In light of recent reports of climate-driven shifts in distributional ranges of marine zooplankton, accurate species identification is essential for monitoring and understanding marine ecosystems.

Application of the precautionary principle to taxa of uncertain status: the case of the Bellinger River turtle

Legislative and regulatory frameworks for conserving biodiversity often focus on the species as a fundamental unit for protection. In cases where the taxonomic or conservation status of a species is uncertain, the precautionary principle may be invoked in listing suspected but as yet undescribed taxa as vulnerable or endangered. In this paper, we present an evaluation of the taxo- nomic status of what has been regarded as a distinctive but as yet undescribed species of freshwater turtle, declared endangered in the Action Plan for Australian Reptiles and vulnerable in the sched- ules of state and federal conservation acts. Using mitochondrial sequence variation, we show that the Bellinger River turtle is an unremarkable population of a common and widespread species, Emydura macquarii. In addition, we present evidence suggesting that it may have been recently introduced to, or may be a recent invader of, the Bellinger River (New South Wales, Australia) where it may come to compete with Myuchelys georgesi, an endemic found only in the Bellinger River. Our study illus- trates the need to couple fundamental research with on-ground action early in an adaptive manage- ment context, particularly where taxonomic status of the target species is uncertain. Short-term cost savings of failing to do so may come to be greatly exceeded by longer-term opportunity loss where conservation dollars are limited.

Molecular Evidence for Ancient Asexuality in Timema Stick Insects

Asexuality is rare in animals in spite of its apparent advantage relative to sexual reproduction, indicating that it must be associated with profound costs [1-9]. One expectation is that reproductive advantages gained by new asexual lineages will be quickly eroded over time [3, 5-7]. Ancient asexual taxa that have evolved and adapted without sex would be "scandalous" exceptions to this rule, but it is often difficult to exclude the possibility that putative asexuals deploy some form of "cryptic" sex, or have abandoned sex more recently than estimated from divergence times to sexual relatives [10]. Here we provide evidence, from high intraspecific divergence of mitochondrial sequence and nuclear allele divergence patterns, that several independently derived Timema stick-insect lineages have persisted without recombination for more than a million generations. Nuclear alleles in the asexual lineages displayed significantly higher intraindividual divergences than in related sexual species. In addition, within two asexuals, nuclear allele phylogenies suggested the presence of two clades, with sequences from the same individual appearing in both clades. These data strongly support ancient asexuality in Timema and validate the genus as an exceptional opportunity to attack the question of how asexual reproduction can be maintained over long periods of evolutionary time.

Flyway evolution is too fast to be explained by the modern synthesis: proposals for an ‘extended’ evolutionary research agenda

In this paper, I argue that to fully grasp the generation and maintenance of variation in the migratory phenotypes of (shore-)birds we need to expand our scientific search image and include developmental processes and non-genetic pathways of inheritance in the explanatory frameworks. Traditionally, studies of micro-evolution of migratory phenotypes were restricted to comparative studies on migratory versus non-migratory taxa, and artificial selection and heritability experiments on quantitative behavioural traits related to migration. Such studies had a focus on the genetic axis of inheritance and were restricted to songbirds. In avian groups such as the shorebird families Scolopacidae and Charadriidae, all but a few island species are migrants, which precludes comparative studies at the species level. Like other taxa, shorebirds have geographically separate breeding populations (either or not recognized as subspecies on the basis of morphological differences) which differentiate with respect to the length, general direction and timing of migration, including the use of fuelling at staging sites and the timing of moult. However, their breeding systems preclude artificial selection and heritability experiments on quantitative traits. This would seem to limit the prospects of evolutionary analysis until one realizes that the speed of evolutionary innovation in shorebird migratory life-histories may be so fast as to necessitate other avenues of explanation and investigation. According to our best current estimates based on mitochondrial gene sequence variation, in Red Knots Calidris canutus considerable phenotypic variation has evolved since the Last Glacial Maximum ca. 20,000 years ago, to the extent that six subspecies are currently recognized. This would be too short a time for the origin of the qualitatively and quantitatively distinct and non-overlapping traits to be explained by random point mutations followed by natural selection, although we cannot dismiss the possibility of previously unexpressed (standing) genetic variation followed by selection. I argue that, to understand the flyway evolution of such shorebirds in the ‘extended’ evolutionary framework, we need to give due attention to developmental versatility and broad-sense epigenetic evolutionary mechanisms. This means that experimental studies at the phenotypic level are now necessary. This could involve a combination of observational studies in our rapidly changing world, common garden experiments, and even experiments involving global-scale displacements of particular migratory phenotypes at different phases of development. I provide suggestions on how such experiments could be carried out.

Putative origin of clonal lineages of Amylostereum areolatum, the fungal symbiont associated with Sirex noctilio, retrieved from Pinus sylvestris, in eastern Canada

The Eurasian Sirex noctilio– Amylostereum areolatum complex was discovered and has become established close to the North American Great Lakes in the 2000s. This invasive forest insect pest represents a very high risk to native and exotic pines in North America. We investigated the geographical origin of clonal lineages of the fungal symbiont A. areolatum in the recently pest-colonized eastern Canadian region by analyzing mitochondrial and nuclear sequence variations and comparing the genetic diversity of a worldwide collection of fungal symbionts among six countries where the Sirex complex is native and four countries from which the insect–fungal complex has been introduced. In total, 102 isolates were analyzed. While 12 multilocus genotypes (MLGs) are observed in the areas where S. noctilio is native, only two MLGs are retrieved from areas where S. noctilio is not native, indicating the wide spread of clonal lineages in the introduced fungal symbiont of S. noctilio. MLG2 comprises 26 % of the Canadian isolates and is also observed in Chile and South Africa, where the insect–fungal complex has also been introduced. MLG3 comprises 74 % of the Canadian isolates and is also observed in the USA, but nowhere else in our worldwide collection. Thus, at least one of the Canadian clonal lineages shares a common origin with A. areolatum isolates from the Southern Hemisphere. The source of the second clonal lineage is still unknown, but phylogenetic analyses show that MLG3 is isolated. More extended sampling is necessary to determine the origin of this fungal clonal lineage and investigate its probable symbiotic association with native North American Sirex.

The molecular systematics of the Carasobarbus species from Iran and adjacent areas, with comments on Carasobarbus albus (Heckel, 1843)

The diversity of cytochrome b sequences of Carasobarbus species from Iran and adjacent areas was investigated. Two divergent haplotype groups were found for C. luteus. The first one is widespread and congruent with the biogeographical hypothesis of a recent isolation among the various populations as a consequence of rising sea levels following the last Pleistocene glaciation. The second one is restricted to the Khabur River in Syria. The possibility that one of these groups corresponds to C. albus is discussed, but we conclude that it is more likely that C. luteus is a single species that retained two divergent mitochondrial lineages. The mitochondrial sequence diversity found for C. kosswigi and C. sublimus is high, possibly due to small population size and consequent genetic drift. Carasobarbus kosswigi is paraphyletic with respect to C. sublimus, indicating a recent speciation event of these taxa.

Detection of Somatic Mutations and Germline Polymorphisms in Mitochondrial DNA of Uterine Fibroids Patients

To identify the role of mitochondrial DNA (mtDNA) mutations in uterine fibroids patients, genomic DNA isolated from paired myometrium and fibroid tissues was screened for mutations. The present study represents the first investigation to report that 10.4% of uterine fibroids cases had either mtDNA mutations or polymorphisms or both. Among the 14 mitochondrial sequence variants identified, seven are somatic mutations (A3327C, G3352A, G3376A, G3380A, G3421A, T15312G, and C15493G) and the remaining (G3316A, C3342A, C3442T, T10205A, A10188G, A10229C, and A10301T) are gene polymorphisms. Somatic mutations were both homo- and heteroplasmic in nature. Of the seven somatic mutations located in the MTND1 and MTCYB genes, five (71.42%) are nonsynonymous in nature, whereas four (57.14%) of the polymorphisms located in MTND1 and MTND3 genes are found to be nonsynonymous. Sequence variants such as G3380A, G3421A, T15312G, G3376A, and G3316A have been earlier described in different human pathologies, but the remaining are novel ones. Mitochondrial somatic mutations and polymorphisms may predispose women to an earlier onset of degenerative cellular processes, which impair oxidative phosphorylation capacity and thereby promote tumorigenesis in uterine smooth muscle cells. Detection of mtDNA sequence variations in fibroid patients raises the need for larger case-control studies to screen the whole mitochondrial genome and evaluate as a future diagnostic biomarker in fibroid patients.

Neutral mitochondrial heteroplasmy and the influence of aging

The development and maintenance of mitochondrial heteroplasmy has important consequences for both health and heredity. Previous studies using pathogenic mutations have shown considerable variability between maternally related individuals and studies of several D-loop polymorphisms have suggested a relationship between heteroplasmy and somatic aging. To broadly explore the variation of human heteroplasmy and to clarify the dynamics of somatic heteroplasmy over the course of lifespan, we analyzed mitochondrial sequence variation across a range of ages. We utilized array-generated single-nucleotide polymorphism data that were well correlated with independent measures of heteroplasmy. Significant levels of heteroplasmy were identified at 0.24% of sites evaluated. By examining mother-child pairs, we found that heteroplasmy was inherited (30%) but could occur de novo in offspring or, conversely, be present in mothers but eliminated in their children (70%). Cumulatively, mitochondrial heteroplasmy across the genome increased significantly with advanced age (r = 0.224, P =8 × 10(-30)). Surprisingly, changes in heteroplasmy were not uniform with some sites demonstrating a loss of variation (increased homoplasmy) with aging. These data suggest that both mutation and selective pressure affect blood mitochondrial DNA sequence over the course of the human lifespan and reveal the unexpectedly dynamic nature of human heteroplasmy.

Radiation of European Eubosmina (Cladocera) from Bosmina(E.) longispina—concordance of multipopulation molecular data with paleolimnology

We investigated the evolutionary radiation of a freshwater zooplankter that possesses a mixed breeding system and a detailed, dated, subfossil record. We estimated the phylogenetic relationships among the proposed species with complete nicotinamide adenine dinucleotide dehydrogenase subunit 2 mitochondrial gene sequence variation. We sequenced 374 Eubosmina specimens representing 10 out of 11 distinct morphospecies from 86 water bodies in three separate Holarctic regions. As expected for a Holocene radiation, there was a lack of monophyly for the proposed species and rare sharing of derived haplotypes among some species. Nevertheless, the proposed species exhibited little or no sharing of mitochondrial deoxyribonucleic acid haplotypes. Moreover, the phylogenetic and haplotype network results revealed a radiation pattern that is concordant with the subfossil record—with an older Bosmina (E.) longispina radiating into several new forms. Our results bolster the subfossil, morphometric, experimental, and within‐population genetic evidence that B. (E.) longispina has radiated into several incipient species during the Holocene.

Evolution of reproductive mode variation and host associations in a sexual-asexual complex of aphid parasitoids

BackgroundThe Lysiphlebus fabarum group is a taxonomically poorly resolved complex of aphid parasitoids, presently split into three described species that comprise sexual (arrhenotokous) and asexual (thelytokous) lineages of unknown relationship. Specifically, it is unclear how asexuals evolved from sexuals in this system, to what extent reproductive modes are still connected by genetic exchange, how much the complex is structured by geography or by host-associated differentiation, and whether species designations are valid. Using a combination of population genetic and phylogenetic approaches, we addressed these issues in a comprehensive sample of parasitoid wasps from across Europe.ResultsAsexual reproduction predominated in parasitoids of the L. fabarum group, with asexual populations exhibiting high genotypic diversity. Sexual populations were only common in southern France; elsewhere sexual reproduction was restricted to specific aphid hosts. Although reproductive modes were aggregated on the mitochondrial genealogy and significantly differentiated at nuclear microsatellite loci, there was clear evidence for genetic exchange, especially on hosts attacked by sexual and asexual parasitoids. The microsatellite data further revealed that parasitoids collected from certain host aphids were significantly differentiated, yet the mitochondrial sequence variation across the entire L. fabarum group did not exceed 1.32% and exhibited a very shallow topology. Morphological characters used for delineation of described species were found to be phylogenetically non-conservative.ConclusionsOur results suggest that the sexual-asexual L. fabarum group represents a young complex of lineages with incomplete isolation between reproductive modes. We propose three mechanisms of genetic exchange that may jointly explain the high genotypic diversity observed in asexual parasitoids: (i) the formation of new asexual lineages via 'contagious parthenogenesis', (ii) introgression from sexual lineages through matings between sexual males and thelytokous females, and (iii) 'cryptic sex' within asexuals, mediated by rare males that thelytokous lines are known to produce spontaneously. The partially strong differentiation among wasps collected from different aphids suggests that host specialization can evolve readily in these parasitoids. Finally, we conclude that in the light of our data, the current taxonomic division of the L. fabarum group into three species cannot be upheld.

Introgression of mitochondrial DNA among Myodes voles: consequences for energetics?

BackgroundIntrogression of mitochondrial DNA (mtDNA) is among the most frequently described cases of reticulate evolution. The tendency of mtDNA to cross interspecific barriers is somewhat counter-intuitive considering the key function of enzymes that it encodes in the oxidative-phosphorylation process, which could give rise to hybrid dysfunction. How mtDNA reticulation affects the evolution of metabolic functions is, however, uncertain. Here we investigated how morpho-physiological traits vary in natural populations of a common rodent (the bank vole, Myodes glareolus) and whether this variation could be associated with mtDNA introgression. First, we confirmed that M. glareolus harbour mtDNA introgressed from M. rutilus by analyzing mtDNA (cytochrome b, 954 bp) and nuclear DNA (four markers; 2333 bp in total) sequence variation and reconstructing loci phylogenies among six natural populations in Finland. We then studied geographic variation in body size and basal metabolic rate (BMR) among the populations of M. glareolus and tested its relationship with mtDNA type.ResultsMyodes glareolus and its arctic neighbour, M. rutilus, are reciprocally monophyletic at the analyzed nuclear DNA loci. In contrast, the two northernmost populations of M. glareolus have a fixed mitotype that is shared with M. rutilus, likely due to introgressive hybridization. The analyses of phenotypic traits revealed that the body mass and whole-body, but not mass corrected, BMR are significantly reduced in M. glareolus females from northern Finland that also have the introgressed mitotype. Restricting the analysis to the single population where the mitotypes coexist, the association of mtDNA type with whole-body BMR remained but those with mass corrected BMR and body mass did not. Mitochondrial sequence variation in the introgressed haplotypes is compatible with demographic growth of the populations, but may also be a result of positive selection.ConclusionOur results show that the phenotypic traits vary markedly along the north-south axis of populations of M. glareolus. This variation may be related to adaptation to local environments and coincides with the gradient of genome reticulation between M. glareolus and M. rutilus, which was assessed by mtDNA introgression. Introgression of mtDNA may have affected morpho-physiological traits but do not show strong effects on either body mass or basal metabolic rate alone. We discuss the causes and biological meaning of our results and the means to clarify these questions in future research.

Insular arctic ground squirrels (Spermophilus parryii) of the North Pacific: indigenous or exotic?

We lack critical information for oceanic archipelagos worldwide related to the origin and status of insular faunas. In southwestern Alaska, in particular, a need exists to tease apart whether specific insular populations are naturally occurring or are the result of exotic introductions by humans. We analyzed variation in mitochondrial sequences of the cytochrome-b gene and 8 nuclear microsatellite loci across 215 individuals representing 17 populations (12 insular) to refine our understanding of the history of the previously identified Southwest clade of arctic ground squirrels (Spermophilus parryii). We found significant geographic structure that suggests long-term isolation and diversification (Ushugat Island and Cold Bay), but we also documented closely related populations that are likely the result of human-mediated introductions. The latter instances (Kavalga and Unalaska islands) corroborate reports from early Alaska explorers. DOI: 10.1644/09-MAMM-A386.1.

Species authentication of commercial beef jerky based on PCR-RFLP analysis of the mitochondrial 12S rRNA gene

In this study, we determined species-specific variations by analyzing the mitochondrial 12S rRNA gene sequence variation (∼440 bp) in 17 newly obtained sequences and 90 published cattle, yak, buffalo, goat, and pig sequences, which represent 62 breeds and 17 geographic regions. Based on the defined species-specific variations, two endonucleases, Alu I and Bfa I, were selected for species authentication using raw meat/tissue samples and the PCR-RFLP method. Goat and pig were identified using the Alu I enzyme, while cattle, yak, and buffalo were identified by digestion with Bfa I. Our approach had relatively high detection sensitivity of cattle DNA in mixed cattle and yak products, with the lowest detectable threshold equaling 20% of cattle DNA in a mixed cattle/yak sample. This method was successfully used to type commercial beef jerky products, which were produced by different companies utilizing various processing technologies. Our results show that several yak jerky products might be implicated in commercial fraud by using cattle meat instead of yak meat.

Potential pitfalls in MitoChip detected tumor-specific somatic mutations: a call for caution when interpreting patient data

BackgroundSeveral investigators have employed high throughput mitochondrial sequencing array (MitoChip) in clinical studies to search mtDNA for markers linked to cancers. In consequence, a host of somatic mtDNA mutations have been identified as linked to different types of cancers. However, closer examination of these data show that there are a number of potential pitfalls in the detection tumor-specific somatic mutations in clinical case studies, thus urging caution in the interpretation of mtDNA data to the patients. This study examined mitochondrial sequence variants demonstrated in cancer patients, and assessed the reliability of using detected patterns of polymorphisms in the early diagnosis of cancer.MethodsPublished entire mitochondrial genomes from head and neck, adenoid cystic carcinoma, sessile serrated adenoma, and lung primary tumor from clinical patients were examined in a phylogenetic context and compared with known, naturally occurring mutations which characterize different populations.ResultsThe phylogenetic linkage analysis of whole arrays of mtDNA mutations from patient cancerous and non-cancerous tissue confirmed that artificial recombination events occurred in studies of head and neck, adenoid cystic carcinoma, sessile serrated adenoma, and lung primary tumor. Our phylogenetic analysis of these tumor and control leukocyte mtDNA haplotype sequences shows clear cut evidence of mixed ancestries found in single individuals.ConclusionsOur study makes two prescriptions: both in the clinical situation and in research 1. more care should be taken in maintaining sample identity and 2. analysis should always be undertaken with respect to all the data available and within an evolutionary framework to eliminate artifacts and mix-ups.

Haplogroup relationships between domestic and wild sheep resolved using a mitogenome panel

Five haplogroups have been identified in domestic sheep through global surveys of mitochondrial (mt) sequence variation, however these group classifications are often based on small fragments of the complete mtDNA sequence; partial control region or the cytochrome B gene. This study presents the complete mitogenome from representatives of each haplogroup identified in domestic sheep, plus a sample of their wild relatives. Comparison of the sequence successfully resolved the relationships between each haplogroup and provided insight into the relationship with wild sheep. The five haplogroups were characterised as branching independently, a radiation that shared a common ancestor 920,000 ± 190,000 years ago based on protein coding sequence. The utility of various mtDNA components to inform the true relationship between sheep was also examined with Bayesian, maximum likelihood and partitioned Bremmer support analyses. The control region was found to be the mtDNA component, which contributed the highest amount of support to the tree generated using the complete data set. This study provides the nucleus of a mtDNA mitogenome panel, which can be used to assess additional mitogenomes and serve as a reference set to evaluate small fragments of the mtDNA.

Population Structure and Comparative Phylogeography of Jack Species (Caranx ignobilis and C. melampygus) in the High Hawaiian Islands

Members of the family Carangidae are top-level predators and highly prized food and sport fishes. Although ecologically and economically important, little is known about the biology of numerous species in the family. This is particularly true of the jacks Caranx ignobilis and C. melampygus, which have experienced recent population reductions around the high Hawaiian Islands due to overfishing. Previous studies have documented territorial tendencies as well as cases of long-distance excursions in both species, suggesting populations may exhibit a range of structure at the genetic level. To explore this possibility, mitochondrial DNA ATPase6 and ATPase8 gene sequence variation was assessed from 91 individuals (33 C. ignobilis and 58 C. melampygus) spanning the islands of Kaua'i, O'ahu, Moloka'i, Maui, and Hawai'i. Although a total of 20 distinct haplotypes (8 for C. ignobilis; 12 for C. melampygus) were recovered, no evidence of population structure was found for either species across the examined geographic range. However, distinct demographic patterns were identified, implying differing evolutionary histories and/or population dynamics. Additionally, ∼ 6% of the examined C. ignobilis were C. ignobilis × C. melampygus hybrids because they harbored mitochondrial haplotypes typical of C. melampygus. These hybrids contribute to measurable gene flow between the species and may play a significant role in the evolution of the genus.

Gene and species trees of a Neotropical group of treefrogs: Genetic diversification in the Brazilian Atlantic Forest and the origin of a polyploid species

The Neotropical Phyllomedusa burmeisteri treefrog group includes four diploid ( P. bahiana, P. burmeisteri, P. distincta and P. iheringii) and one tetraploid ( P. tetraploidea) forms. Here we use mitochondrial and nuclear sequence variation from across its range to verify if recognized morphospecies correspond to phylogenetic clades, examine the origin of the polyploid P. tetraploidea, and compare range wide patterns of diversification to those of other BAF organisms. We compared single gene trees with one Bayesian multi-gene tree, and one Bayesian species tree inferred under a coalescent framework. Our mtDNA phylogenetic analyses showed that P. bahiana, P. burmeisteri and P. iheringii correspond to monophyletic clades, while P. distincta and P. tetraploidea were paraphyletic. The nuclear gene trees were concordant in revealing two moderately supported groups including (i) P. bahiana and P. burmeisteri (northern species) and (ii) P. distincta, P. tetraploidea and P. iheringii (southern species). The multi-gene tree and the species tree retrieved similar topologies, giving high support to the northern and southern clades, and to the sister-taxa relationship between P. tetraploidea and P. distincta. Estimates of t MRCA suggest a major split within the P. burmeisteri group at ≈5 Myr (between northern and southern groups), while the main clades were originated between ≈0.4 and 2.5 Myr, spanning the late Pliocene and Pleistocene. Patterns of geographic and temporal diversification within the group were congruent with those uncovered for other co-distributed organisms. Independent paleoecological and geological data suggest that vicariance associated with climatic oscillations and neotectonic activity may have driven lineage divergence within the P. burmeisteri group. P. tetraploidea probably originated from polyploidization of P. distincta or from a common ancestor.

NRTIs’ effect on the sequence of mitochondrial DNA HV 2 in HIV infected patients

Potent combination antiretroviral therapy (cART) has significantly improved the life expectancy of people living with human immunodeficiency virus (HIV), but it has many side effects such as lipodystrophy (LD), hepatic steatosis, and lactic acidosis. Nucleoside reverse transcriptase inhibitors (NRTIs) could damage the mitochondria by inhibiting the human DNA polymerase gamma, leading to mtDNA deletion. However, it remains uncertain whether NRTIs could induce the hypervariable region (HV) mutations of the D loop of mitochondria in Chinese HIV/AIDS patients, and whether that effect is different between individuals with and without LD. Hereby, 30 Chinese AIDS patients who were receiving antiretroviral drugs were recruited, among which 16 had symptomatic LD and 14 did not. Blood samples were collected prior to and after 96 weeks of treatment. Total DNA was extracted from peripheral blood mononuclear cells (PBMCs). Fragments of 728 bp in length containing HV2 were amplified by standard polymerase chain reaction (PCR). Direct DNA-sequencing analysis techniques were used to detect mitochondrial sequence variants between paired longitudinal samples. Alterations were compared with the revised Cambridge Reference Sequence (rCRS) to determine mutation or polymorphism. Results showed that two years after ART, totally seven cases exhibited sequence variations, ve individuals showed 73 → G revised variation (two with and three without LD), while two cases of LD were found to have other nucleotide alterations. There was no new alteration in individuals without LD. In conclusion, NRTIs could induce mutation of mtDNA HV2, which might contribute to the development of LD.