The complete mitochondrial genome of the gall-forming fly, Fergusonina taylori(Nelson and Yeates) (Diptera: Fergusoninidae)

The monogeneric family Fergusoninidae consists of gall-forming flies that, together with Fergusobia (Tylenchida: Neotylenchidae) nematodes, form the only known mutualistic association between insects and nematodes. In this study, the entire 16,000 bp mitochondrial genome of Fergusonina taylori Nelson and Yeates was sequenced. The circular genome contains one encoding region including 27 genes and one non-coding A þT-rich region. The arrangement of the proteincoding, ribosomal RNA (rRNA) and transfer RNA (tRNA) genes was the same as that found in the ancestral insect. Nucleotide composition is highly A þ T biased. All of the protein initiation codons are ATN, except for nad1 which begins with TTT. All 22 tRNA anticodons of F. taylori match those observed in Drosophila yakuba, and all form the typical cloverleaf structure except for tRNA-Ser (AGN) which lacks a dihydrouridine (DHU) arm. Secondary structural features of the rRNA genes of Fergusonina are similar to those proposed for other insects, with minor modifications. The mitochondrial genome of Fergusonina presented here may prove valuable for resolving the sister group to the Fergusoninidae, and expands the available mtDNA data sources for acalyptrates overall.

Branch-length estimation bias misleads molecular dating for a vertebrate mitochondrial phylogeny

Despite recent methodological advances in inferring the time-scale of biological evolution from molecular data, the fundamental question of whether our substitution models are sufficiently well specified to accurately estimate branch-lengths has received little attention. I examine this implicit assumption of all molecular dating methods, on a vertebrate mitochondrial protein-coding dataset. Comparison with analyses in which the data are RY-coded (AG → R; CT → Y) suggests that even rates-across-sites maximum likelihood greatly under-compensates for multiple substitutions among the standard (ACGT) NT-coded data, which has been subject to greater phylogenetic signal erosion. Accordingly, the fossil record indicates that branch-lengths inferred from the NT-coded data translate into divergence time overestimates when calibrated from deeper in the tree. Intriguingly, RY-coding led to the opposite result. The underlying NT and RY substitution model misspecifications likely relate respectively to “hidden” rate heterogeneity and changes in substitution processes across the tree, for which I provide simulated examples. Given the magnitude of the inferred molecular dating errors, branch-length estimation biases may partly explain current conflicts with some palaeontological dating estimates.

Tinamous and moa flock together : mitochondrial genome sequence analysis reveals independent losses of flight among ratites

Ratites are large, flightless birds and include the ostrich, rheas, kiwi, emu, and cassowaries, along with extinct members, such as moa and elephant birds. Previous phylogenetic analyses of complete mitochondrial genome sequences have reinforced the traditional belief that ratites are monophyletic and tinamous are their sister group. However, in these studies ratite monophyly was enforced in the analyses that modeled rate heterogeneity among variable sites. Relaxing this topological constraint results in strong support for the tinamous (which fly) nesting within ratites. Furthermore, upon reducing base compositional bias and partitioning models of sequence evolution among protein codon positions and RNA structures, the tinamou–moa clade grouped with kiwi, emu, and cassowaries to the exclusion of the successively more divergent rheas and ostrich. These relationships are consistent with recent results from a large nuclear data set, whereas our strongly supported finding of a tinamou–moa grouping further resolves palaeognath phylogeny. We infer flight to have been lost among ratites multiple times in temporally close association with the Cretaceous–Tertiary extinction event. This circumvents requirements for transient microcontinents and island chains to explain discordance between ratite phylogeny and patterns of continental breakup. Ostriches may have dispersed to Africa from Eurasia, putting in question the status of ratites as an iconic Gondwanan relict taxon. [Base composition; flightless; Gondwana; mitochondrial genome; Palaeognathae; phylogeny; ratites.]

Isolation and characterisation of novel microsatellite and mitochondrial DNA markers for the Eastern Water Dragon (Physignathus lesueurii)

Habitat fragmentation as a result of urbanisation is a growing problem for native lizard species. The Eastern Water Dragon (Physignathus lesueurii) is a social arboreal agamid lizard, native to Australia. This species represents an ideal model species to investigate the effect of urbanisation because of their prominent abundance in the urban landscape. Here we describe the isolation and characterisation of a novel set of 74 di-, tri-, and tetramicrosatellites from which 18 were selected and optimised into two multiplexes. The 18 microsatellites generated a total 148 alleles across the two populations. The number of alleles per locus varied from 2 to 18 alleles and measures of Ho and He varied from 0.395 to 0.877 and from 0.441 to 0.880, respectively. We also present primers for four novel mitochondrial DNA (mtDNA) markers. The combined length of the four mtDNA marker pairs was 2,528 bp which included 15 nucleotides changes. In comparison to threatened species, which are generally characterised by small population sizes, the Eastern Water Dragon represents an ideal model species to investigate the effect of urbanisation on their behavioural ecology and connectivity patterns among populations.

Population structure of Bactrocera dorsalis s.s., B. papayae and B. philippinensis (Diptera: Tephritidae) in southeast Asia: evidence for a single species hypothesis using mitochondrial DNA and wingshape data

Background Bactrocera dorsalis s.s. is a pestiferous tephritid fruit fly distributed from Pakistan to the Pacific, with the Thai/Malay peninsula its southern limit. Sister pest taxa, B. papayae and B. philippinensis, occur in the southeast Asian archipelago and the Philippines, respectively. The relationship among these species is unclear due to their high molecular and morphological similarity. This study analysed population structure of these three species within a southeast Asian biogeographical context to assess potential dispersal patterns and the validity of their current taxonomic status. Results Geometric morphometric results generated from 15 landmarks for wings of 169 flies revealed significant differences in wing shape between almost all sites following canonical variate analysis. For the combined data set there was a greater isolation-by-distance (IBD) effect under a ‘non-Euclidean’ scenario which used geographical distances within a biogeographical ‘Sundaland context’ (r2 = 0.772, P < 0.0001) as compared to a ‘Euclidean’ scenario for which direct geographic distances between sample sites was used (r2 = 0.217, P < 0.01). COI sequence data were obtained for 156 individuals and yielded 83 unique haplotypes with no correlation to current taxonomic designations via a minimum spanning network. BEAST analysis provided a root age and location of 540kya in northern Thailand, with migration of B. dorsalis s.l. into Malaysia 470kya and Sumatra 270kya. Two migration events into the Philippines are inferred. Sequence data revealed a weak but significant IBD effect under the ‘non-Euclidean’ scenario (r2 = 0.110, P < 0.05), with no historical migration evident between Taiwan and the Philippines. Results are consistent with those expected at the intra-specific level. Conclusions Bactrocera dorsalis s.s., B. papayae and B. philippinensis likely represent one species structured around the South China Sea, having migrated from northern Thailand into the southeast Asian archipelago and across into the Philippines. No migration is apparent between the Philippines and Taiwan. This information has implications for quarantine, trade and pest management.

Australian lungfish (Neoceratodus forsteri: Dipnoi) have low genetic variation at allozyme and mitochondrial DNA loci : a conservation alert?

Genetic variation at allozyme and mitochondrial DNA loci was investigated in the Australian lungfish, Neoceratodus forsteri Krefft 1870. Tissue samples for genetic analysis were taken non-lethally from 278 individuals representing two spatially distinct endemic populations (Mary and Burnett rivers), as well as one population thought to be derived from an anthropogenic translocation in the 1890's (Brisbane river). Two of 24 allozyme loci resolved from muscle tissue were polymorphic. Mitochondrial DNA nucleotide sequence diversity estimated across 2,235 base pairs in each of 40 individuals ranged between 0.000423 and 0.001470 per river. Low genetic variation at allozyme and mitochondrial loci could be attributed to population bottlenecks, possibly induced by Pleistocene aridity. Limited genetic differentiation was detected among rivers using nuclear and mitochondrial markers suggesting that admixture may have occurred between the endemic Mary and Burnett populations during periods of low sea level when the drainages may have converged before reaching the ocean. Genetic data was consistent with the explanation that lungfish were introduced to the Brisbane river from the Mary river. Further research using more variable genetic loci is needed before the conservation status of populations can be determined, particularly as anthropogenic demands on lungfish habitat are increasing. In the interim we recommend a management strategy aimed at conserving existing genetic variation within and between rivers.

Antioxidant supplementation reduces skeletal muscle mitochondrial biogenesis

Purpose: Exercise increases the production of reactive oxygen species (ROS) in skeletal muscle, and athletes often consume antioxidant supplements in the belief they will attenuate ROS-related muscle damage and fatigue during exercise. However, exercise-induced ROS may regulate beneficial skeletal muscle adaptations, such as increased mitochondrial biogenesis. We therefore investigated the effects of long-term antioxidant supplementation with vitamin E and alpha-lipoic acid on changes in markers of mitochondrial biogenesis in the skeletal muscle of exercise-trained and sedentary rats. Methods: Male Wistar rats were divided into four groups: 1) sedentary control diet, 2) sedentary antioxidant diet, 3) exercise control diet, and 4) exercise antioxidant diet. Animals ran on a treadmill 4 d.wk(-1) at similar to 70% V (over dot)O(2max) for up to 90 min.d(-1) for 14 wk. Results: Consistent with the augmentation of skeletal muscle mitochondrial biogenesis and antioxidant defenses, after training there were significant increases in peroxisome proliferator-activated receptor F coactivator 1 alpha (PGC-1 alpha) messenger RNA (mRNA) and protein, cytochrome C oxidase subunit IV (COX IV) and cytochrome C protein abundance, citrate synthase activity, Nfe2l2, and SOD2 protein (P < 0.05). Antioxidant supplementation reduced PGC-1 alpha mRNA, PGC-1 alpha and COX IV protein, and citrate synthase enzyme activity (P < 0.05) in both sedentary and exercise-trained rats. Conclusions: Vitamin E and alpha-lipoic acid supplementation suppresses skeletal muscle mitochondrial biogenesis, regardless of training status.

A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue

A high-throughput method of isolating and cloning geminivirus genomes from dried plant material, by combining an Extract-n-Amp™-based DNA isolation technique with rolling circle amplification (RCA) of viral DNA, is presented. Using this method an attempt was made to isolate and clone full geminivirus genomes/genome components from 102 plant samples, including dried leaves stored at room temperature for between 6 months and 10 years, with an average hands-on-time to RCA-ready DNA of 15 min per 20 samples. While storage of dried leaves for up to 6 months did not appreciably decrease cloning success rates relative to those achieved with fresh samples, efficiency of the method decreased with increasing storage time. However, it was still possible to clone virus genomes from 47% of 10-year-old samples. To illustrate the utility of this simple method for high-throughput geminivirus diversity studies, six Maize streak virus genomes, an Abutilon mosaic virus DNA-B component and the DNA-A component of a previously unidentified New Word begomovirus species were fully sequenced. Genomic clones of the 69 other viruses were verified as such by end sequencing. This method should be extremely useful for the study of any circular DNA plant viruses with genome component lengths smaller than the maximum size amplifiable by RCA. © 2008 Elsevier B.V. All rights reserved.

Serine protease inhibition and mitochondrial dysfunction associated with cisplatin resistance in human tumor cell lines : targets for therapy

Indicators of mitochondrial function were studied in two different cell culture models of cis-diamminedichloroplatinum-II (CDDP) resistance: the intrinsically resistant human ovarian cancer cell line CI-80-13S, and resistant clones (HeLa-S1a and HeLa-S1b) generated by stable expression of the serine protease inhibitor—plasminogen activator inhibitor type-2 (PAI-2), in the human cervical cancer cell line HeLa. In both models, CDDP resistance was associated with sensitivity to killing by adriamycin, etoposide, auranofin, bis[1,2-bis(diphenylphosphino)ethane]gold(I) chloride {[Au(DPPE)2]Cl}, CdCl2 and the mitochondrial inhibitors rhodamine-123 (Rhl23), dequalinium chloride (DeCH), tetraphenylphosphonium (TPP), and ethidium bromide (EtBr) and with lower constitutive levels of ATP. Unlike the HeLa clones, CI-80-13S cells were additionally sensitive to chloramphenicol, 1-methyl-4-phenylpyridinium ion (MPP+), rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A, and showed poor reduction of 1-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), suggesting a deficiency in NADH dehydrogenase and/or succinate dehydrogenase activities. Total platinum uptake and DNA-bound platinum were slightly lower in CI-80-13S than in sensitive cells. The HeLa-S1a and HeLa-S1b clones, on the other hand, showed poor reduction of triphenyltetrazolium chloride (TTC), indicative of low cytochrome c oxidase activity. Total platinum uptake by HeLa-S1a was similar to HeLa, but DNA-bound platinum was much lower than for the parent cell line. The mitochondria of CI-80-13S and HeLa-S1a showed altered morphology and were fewer in number than those of JAM and HeLa. In both models, CDDP resistance was associated with less platinum accumulation and with mitochondrial and membrane defects, brought about one case with expression of a protease inhibitor which is implicated in tumor progression. Such markers may identify tumors suitable for treatment with gold phosphine complexes or other mitochondrial inhibitors.

The complete mitochondrial genome of the flesh fly, Sarcophaga impatiens Walker (Diptera: Sarcophagidae)

Approximately 2500 fly species comprise the Sarcophagidae family worldwide. The complete mitochondrial genome of the carrion-breeding, forensically important Sarcophaga impatiens Walker (Diptera: Sarcophagidae) from Australia was sequenced. The 15,169 bp circular genome contains the 37 genes found in a typical Metazoan genome: 13 protein-coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes. It also contains one non-coding A+T-rich region. The arrangement of the genes was the same as that found in the ancestral insect. All the protein initiation codons are ATN, except for cox1 that begins with TCG (encoding S). The 22 tRNA anticodons of S. impatiens are consistent with those observed in Drosophila yakuba, and all form the typical cloverleaf structure, except for tRNA-Ser(AGN) that lacks the DHU arm. The mitochondrial genome of Sarcophaga presented will be valuable for resolving phylogenetic relationships within the family Sarcophagidae and the order Diptera, and could be used to identify favourable genetic markers for species identifications for forensic purposes.

Serine protease inhibition and mitochondrial dysfunction associated with cisplatin resistance in human tumor cell lines: targets for therapy

Indicators of mitochondrial function were studied in two different cell culture models of cis-diamminedichloroplatinum-II (CDDP) resistance: the intrinsically resistant human ovarian cancer cell line CI-80-13S, and resistant clones (HeLa-S1a and HeLa-S1b) generated by stable expression of the serine protease inhibitor—plasminogen activator inhibitor type-2 (PAI-2), in the human cervical cancer cell line HeLa. In both models, CDDP resistance was associated with sensitivity to killing by adriamycin, etoposide, auranofin, bis[1,2-bis(diphenylphosphino)ethane]gold(I) chloride {[Au(DPPE)2]Cl}, CdCl2 and the mitochondrial inhibitors rhodamine-123 (Rhl23), dequalinium chloride (DeCH), tetraphenylphosphonium (TPP), and ethidium bromide (EtBr) and with lower constitutive levels of ATP. Unlike the HeLa clones, CI-80-13S cells were additionally sensitive to chloramphenicol, 1-methyl-4-phenylpyridinium ion (MPP+), rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A, and showed poor reduction of 1-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), suggesting a deficiency in NADH dehydrogenase and/or succinate dehydrogenase activities. Total platinum uptake and DNA-bound platinum were slightly lower in CI-80-13S than in sensitive cells. The HeLa-S1a and HeLa-S1b clones, on the other hand, showed poor reduction of triphenyltetrazolium chloride (TTC), indicative of low cytochrome c oxidase activity. Total platinum uptake by HeLa-S1a was similar to HeLa, but DNA-bound platinum was much lower than for the parent cell line. The mitochondria of CI-80-13S and HeLa-S1a showed altered morphology and were fewer in number than those of JAM and HeLa. In both models, CDDP resistance was associated with less platinum accumulation and with mitochondrial and membrane defects, brought about one case with expression of a protease inhibitor which is implicated in tumor progression. Such markers may identify tumors suitable for treatment with gold phosphine complexes or other mitochondrial inhibitors.

The evolution and biogeography of the austral horse fly tribe Scionini (Diptera: Tabanidae: Pangoniinae) inferred from multiple mitochondrial and nuclear genes

Phylogenetic relationships within the Tabanidae are largely unknown, despite their considerable medical and ecological importance. The first robust phylogenetic hypothesis for the horse fly tribe Scionini is provided, completing the systematic placement of all tribes in the subfamily Pangoniinae. The Scionini consists of seven mostly southern hemisphere genera distributed in Australia, New Guinea, New Zealand and South America. A 5757. bp alignment of 6 genes, including mitochondrial (COI and COII), ribosomal (28S) and nuclear (AATS and CAD regions 1, 3 and 4) genes, was analysed for 176 taxa using both Bayesian and maximum likelihood approaches. Results indicate the Scionini are strongly monophyletic, with the exclusion of the only northern hemisphere genus Goniops. The South American genera Fidena, Pityocera and Scione were strongly monophyletic, corresponding to current morphology-based classification schemes. The most widespread genus Scaptia was paraphyletic and formed nine strongly supported monophyletic clades, each corresponding to either the current subgenera or several previously synonymised genera that should be formally resurrected. Molecular results also reveal a newly recognised genus endemic to New Zealand, formerly placed within Scaptia. Divergence time estimation was employed to assess the global biogeographical patterns in the Pangoniinae. These analyses demonstrated that the Scionini are a typical Gondwanan group whose diversification was influenced by the fragmentation of that ancient land mass. Furthermore, results indicate that the Scionini most likely originated in Australia and subsequently radiated to New Zealand and South American by both long distance dispersal and vicariance. The phylogenetic framework of the Scionini provided herein will be valuable for taxonomic revisions of the Tabanidae.

A possible role for mitochondrial dysfunction in migraine

Migraine is a common neurological disorder characterised by debilitating head pain and an assortment of additional symptoms which can include nausea, emesis, photophobia, phonophobia and occasionally visual sensory disturbances. Migraine is a complex disease caused by an interplay between predisposing genetic variants and environmental factors. It affects approximately 12 % of studied Caucasian populations with affected individuals being predominantly female. Genes involved in neurological, vascular or hormonal pathways have all been implicated in predisposition towards developing migraine. All of these are nuclear encoded genes, but given the role of mitochondria in a number of neurological disorders and in energy production it is possible that mitochondrial variants may play a role in the pathogenesis of this disease. Mitochondrial DNA has been a useful tool for studying population genetics and human genetic diseases due to the clear inheritance shown through successive generations. Given the clear gender bias found in migraine patients it may be important to investigate X-linked inheritance and mitochondrial-related variants in this disorder. This paper explores the possibility that mitochondrial DNA changes may play a role in migraine. Few variants in the mitochondrial genome have so far been investigated in migraine and new studies should be aimed towards investigating the role of mitochondrial DNA in this common disorder.