Mitochondrial DNA differentiation between two forms of trout Salmo letnica, endemic to the Balkan Lake Ohrid, reflects their reproductive isolation

Mitochondrial haplotype diversity in sympatric populations of Ohrid trout, Salmo letnica was investigated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mtDNA control region and ND1, ND3/4, ND5/6 segments. A 310 bp fragment at the 5' end, and a 340-572 bp fragment at the 3' end of the control region were sequenced from representatives of the populations studied. Based on pairwise comparison of the sequences, five new haplotypes were identified plus one identical with the brown trout Andalusian haplotype from the southern Iberian Peninsula. The combination of both RFLP and sequence data sets yielded a total of 10 composite haplotypes. A high degree of genetic subdivision between S. letnica typicus and S. letnica aestivalis populations was observed. The notion of a sympatric origin for the two morphs is discussed. Length variation of the mtDNA control region due to the presence of an 82 bp unit, tandemly repeated one to four times, in the region between the conserved sequence block-3 (CSB-3) and the gene for phenylalanine tRNA is reported. Further, we demonstrate that a single duplication of the approximately 82 bp repeat unit is a common element of the salmonid mitochondrial control region. The unique genetic structure of Ohrid trout represents a highly valuable genetic resource that deserves appropriate management and conservation.     

A regional approach to plant DNA barcoding provides high species resolution of sedges (Carex and Kobresia, Cyperaceae) in the Canadian Arctic Archipelago

Previous research on barcoding sedges (Carex) suggested that basic searches within a global barcoding database would probably not resolve more than 60% of the world’s some 2000 species. In this study, we take an alternative approach and explore the performance of plant DNA barcoding in the Carex lineage from an explicitly regional perspective. We characterize the utility of a subset of the proposed protein-coding and noncoding plastid barcoding regions (matK, rpoB, rpoC1, rbcL, atpF-atpH, psbK-psbI) for distinguishing species of Carex and Kobresia in the Canadian Arctic Archipelago, a clearly defined eco-geographical region representing 1% of the Earth’s landmass. Our results show that matK resolves the greatest number of species of any single-locus (95%), and when combined in a two-locus barcode, it provides 100% species resolution in all but one combination (matK + atpFH) during unweighted pair-group method with arithmetic mean averages (UPGMA) analyses. Noncoding regions were equally or more variable than matK, but as single markers they resolve substantially fewer taxa than matK alone. When difficulties with sequencing and alignment due to microstructural variation in noncoding regions are also considered, our results support other studies in suggesting that protein-coding regions are more practical as barcoding markers. Plastid DNA barcodes are an effective identification tool for species of Carex and Kobresia in the Canadian Arctic Archipelago, a region where the number of co-existing closely related species is limited. We suggest that if a regional approach to plant DNA barcoding was applied on a global scale, it could provide a solution to the generally poor species resolution seen in previous barcoding studies.     

Differential patterns of mitochondrial,chloroplastic and nuclear DNA synthesis in the synchronous cell cycle of Chlamydomonas reinhardtii

Nuclear DNA (ncDNA) synthesis in Chlamydomonas reinhardtii was measured by both ³²P[or-thophosphoric acid] (³²P) and [¹⁴C]adenine incorporation and found to be highly synchronous. Ca. 85% of incorporation was confined to the first 6 h of the dark period of a synchronized regime consisting of an alternating light-dark period of 12 h each. In contrast, no such synchronous incorporation pattern was found for chloroplast (cp) and mitochondrial (mt) DNAs in the same cell population. These two organellar DNAs also exhibited different ³²P-incorporation patterns in the cell cycle. Considerable amounts of ³²P were incorporated into cpDNA throughout the light-dark synchronous cycle under both mixo- and phototrophic growth conditions, although the second 6-h light period under phototrophy showed an increase not apparent under mixotrophy. This change in growth conditions did not affect ³²P incorporation into mtDNA, which was found throughout the cell cycle, with a modest peak in the first 6-h of the dark period. The pattern of [³H]thymidine incorporation into cpDNA was also determined. Under synchronous phototrophic conditions, this pattern was quite different from that obtained with ³²P. Most [³H]thymidine incorporation occurred during the light period of the synchronous cycle; this period had been shown previously by density transfer experiments to be the time of cpDNA duplication. Such preferential [³H]thymidine incorporation into cpDNA in the light period was not observed under mixotrophic synchronous growth conditions; in these, [³H]thymidine incorporation was detected throughout the cell cycle. This lack of coincidence between the patterns of ³²P- and of [³H]thymidine incorporation into cpDNA during the synchronous cell cycle indicates that in addition to replication, the considerably reiterated organelle-DNA molecules may also regularly undergo an extensive repair process during each cell cycle.     

Dps proteins, an efficient detoxification and DNA protection machinery in the bacterial response to oxidative stress

The proteins belonging to the Dps (DNA-binding proteins from starved cells) family play an important role within the bacterial defence system against oxidative stress. They act on Fe(II) and hydrogen peroxide that are potentially toxic in the presence of air. Fe(II) forms spontaneously insoluble Fe(III) and reacts with molecular oxygen or its reduced forms to yield the highly damaging hydroxyl radicals. All Dps proteins have the distinctive capacity to annul the toxic combination of iron and hydrogen peroxide as they use the latter compound to oxidise Fe(II). In addition to this intrinsic DNA protection capacity, several members of the family, including the archetypical Escherichia coli Dps, protect DNA physically by shielding it in large Dps-DNA complexes. The structural and functional characteristics that endow Dps proteins with the chemical and physical protection mechanism are presented and discussed also in the framework of the varied situations that may be encountered in different bacterial species.      

Between an ocean and a high place: coastal drainage isolation generates endemic cryptic species in the Cape kurper Sandelia capensis (Anabantiformes: Anabantidae), Cape Region,South Africa

This study investigated the range-wide phylogenetics and biogeography of the Cape kurper Sandelia capensis, a primary freshwater fish endemic to and widespread within the Cape Floristic Region (CFR) of South Africa. Maximum likelihood, Bayesian phylogenetic and haplotype network analyses, based on two mitochondrial and two nuclear genes, revealed the existence of three reciprocally monophyletic, deeply divergent and allopatric clades that probably represent cryptic species. The West Coast Clade is largely confined to the Langvlei, Verlorenvlei, Berg and Diep Rivers, the Klein River Clade is endemic to the Klein River and the South Coast Clade is found everywhere else in the range of S. capensis sensu lato. It was hypothesised that divergences within S. capensis sensu lato probably occurred because of isolation of coastal drainages by persistent drainage divides or vicariance of current tributaries by the drowning of their confluences by high sea levels. The current distribution of lineages could be due to historical range expansion and gene flow via river capture or some other mode of transdivide dispersal or dispersal during periods of low sea level via palaeoriver confluences of currently isolated coastal rivers. Comparison of BEAST2 estimated divergence times with the timing of climatic, geological and geomorphological events supported long-term coastal drainage isolation, punctuated by rare transdivide dispersal events and limited palaeoriver dispersal, as the best explanation of current phylogeographic and divergence patterns in S. capensis. Hydrological barriers that block upstream passage in palaeotributaries could hypothetically explain why S. capensis failed to disperse through certain palaeoriver confluences. There were several sites where biogeographic patterns have likely been confounded by human translocation of S. capensis. Alien fish predators and water extraction may threaten the three cryptic species more severely than previously realised, due to their smaller population sizes and inhabitation of only a portion of the range previously ascribed to S. capensis sensu lato. The preponderance of cryptic diversity and endemism in the CFR suggests that additional undescribed cryptic species of obligate freshwater fishes may be found in short coastal river systems around the world, especially in regions with a history of geological stability and a narrow continental shelf.     

Phylogeography, population structure and dispersal patterns of the beluga whale Delphinapterus leucas in the western Nearctic revealed by mitochondrial DNA

The recent evolutionary history, population structure and movement patterns of beluga whales in the western Nearctic were inferred from an analysis of mitochondrial DNA control region sequence variation of 324 whales from 32 locations representing five summer concentration areas in Alaska and north-west Canada. Phylogenetic relationships among haplotypes were inferred from parsimonious networks, and genetic subdivision was examined using haplotypic frequency-based indices and an analysis of variance method modified for use with interhaplotypic distance data. MtDNA relationships were characterized by a series of star-like phylogenies which, when viewed in conjunction with information on haplotype frequency and distribution, suggested a rapid radiation of beluga whales into the western Nearctic following the Pleistocene, and an early divergence of the Beaufort Sea from the Chukchi and Bering Seas subpopulations. Overall nucleotide diversity was low (0.51%) yet all major summering concentrations were significantly differentiated (Φ_ST= 0.33) from one another. Stratification of samples by gender and age from the three northernmost subpopulations suggested that female cohorts from neighbouring subpopulations were more differentiated than males. Further stratification of adult animals by age revealed that older adults were substantially less subdivided among locations than younger adults, particularly for males, suggesting that dispersal, although limited, is biased toward older adult males. Overall, the patterns of mtDNA variation in beluga whales indicated that the summering concentrations are demographically, if not phyletically distinct. Population structure appears to be maintained primarily by natal homing behaviour, while asymmetries in dispersal may be associated with the type of mating system.     

Influence of DNA isolation from historical otoliths on nuclear-mitochondrial marker amplification and age determination in an overexploited fish, the common sole (Solea solea L.)

Historical otolith collections are crucial in assessing the evolutionary consequences of natural and anthropogenic changes on the demography and connectivity of commercially important fish species. Hence, it is important to define optimal protocols for purifying DNA from such valuable information sources while avoiding any damage to the physical structure of the otolith. Before being able to conclude on the harmlessness of a method, it is important to validate protocols on different kinds of otoliths by testing purification methodologies under standardized conditions. Here we compare the effect of two DNA extraction methods on the success in identifying the age in an overexploited marine fish, the common sole (Solea solea L.). To ensure optimal future population genetic and demographic analyses, we assessed DNA quantity and tested the DNA quality by investigating the amplification success of a mitochondrial and nuclear marker. Our results show that the choice of the DNA extraction method had a significant effect on the success of using these otoliths in age and growth analyses. Standard commercial and published protocols resulted in a severe damaging of the otolith structure, hampering accurate preparation and analyses of the morphological structures of the otoliths. Shortening the lysis time and lowering the EDTA (ethylene diamine tetraacetic acid) and SDS (sodium dodecylsulphate) concentration turned out to be beneficial for the stability of otolith structure, while maintaining an overall high DNA quality measured through polymerase chain reaction amplification success. We therefore recommend that care should be taken when choosing the extraction method for a molecular study on archived samples, in order to enable the maximal use of information embedded in historical material.     

Ancestry to an endemic radiation in Lake Tanganyika? Evolution of the viviparous gastropod Potadomoides Leloup, 1953 in the Congo River system (Caenogastropoda,Cerithioidea, Paludomidae)

Providing another spectacular model for understanding speciation and radiation, the origin of the gastropod species flock in Lake Tanganyika (with an estimated age of approximately 12 Myr) remained enigmatic to date. Although, for a long time, an in situ radiation was assumed, Lake Tanganyika could have functioned as a reservoir for ancient African lineages, implying that the now lacustrine taxa originiated elsewhere. However, the fluviatile gastropod fauna of adjacent river systems in Central and East Africa is only poorly known. Here, we provide conchological, anatomical, phylogenetical, and biogeographical data on the fluviatile genus Potadomoides Leloup, 1953, which was hitherto regarded as ancestral to the entire Tanganyika gastropod radiation. The type species Potadomoides pelseneeri is restricted to the delta region of the Malagarasi River east of Lake Tanganyika, whereas three congeneric species (Potadomoides bequaerti, Potadomoides hirta, and Potadomoides schoutedeni) inhabit the Congo River with its tributaries Lualaba and Luvua, west of the Tanganyikan Rift. We describe and document, with scanning electron microscopy, the ontogenetic development of embryos of this uterine brooder as well as the detailed reproductive anatomy. Phylogenetic analysis of 44 morphological characters (including adult and embryonic shell, operculum, radula, reproductive tract) for 15 paludomid taxa could not support monophyly of the Tanganyika species flock. Instead, we found two major lineages that colonized Lake Tanganyika independently, one comprising the Nassopsinae Kesteven, 1903 (= Lavigeriinae Thiele, 1925) with the riverine Potadomoides plus the lacustrine Lavigeria and Vinundu, the second comprising the riverine Cleopatra together with the rest of the lacustrine species (except for Tiphobia horei). The analysis identifies Potadomoides as paraphyletic, with the uterine brooder P. pelseneeri being the sister taxon to the uterine brooder Lavigeria plus the oviparous Vinundu, but not to the entire Tanganyika species flock. We reconstruct the independent evolution of an fluviolacustrine taxon Nassopsinae for which we evaluate the synapomorphic characters, in particular those of reproductive biology, and discuss systematic and evolutionary implications of repeated origin of (ovo‐)viviparity in these limnic Cerithioidea. Finally, we outline a hypothesis on the evolutionary history of Potadomoides in the context of the gastropod radiation in Lake Tanganyika. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 92 , 367–401.     

Haldane's rule in an avian system: using cline theory and divergence population genetics to test for differential introgression of mitochondrial, autosomal, and sex-linked loci across the Passerina bunting hybrid zone

Using cline fitting and divergence population genetics, we tested a prediction of Haldane's rule: autosomal alleles should introgress more than z-linked alleles or mitochondrial haplotypes across the Passerina amoena/Passerina cyanea (Aves: Cardinalidae) hybrid zone. We screened 222 individuals collected along a transect in the Great Plains of North America that spans the contact zone for mitochondrial (two genes), autosomal (four loci) and z-linked (two loci) markers. Maximum-likelihood cline widths estimated from the mitochondrial (223 km) and z-linked (309 km) datasets were significantly narrower on average than the autosomal cline widths (466 km). We also found that mean coalescent-based estimates of introgression were larger for the autosomal loci (0.63 genes/generation, scaled to the mutation rate mu) than for both the mitochondrial (0.27) and z-linked loci (0.59). These patterns are consistent with Haldane's rule, but the among-locus variation also suggests many independently segregating loci are required to investigate introgression patterns across the genome. These results provide the first comprehensive comparison of mitochondrial, sex-linked, and autosomal loci across an avian hybrid zone and add to the body of evidence suggesting that sex chromosomes play an important role in the formation and maintenance of reproductive isolation between closely related species.     

Restriction fragment length polymorphisms in mitochondrial DNA and ribosomal RNA gene complexes as an aid to the characterization of species and sub-species populations in the genus Verticillium

Abstract Genomic DNA was extracted from seven species of Verticillium and digested with the restriction endonucleases Eco RI or Hae III. Hybridization with an homologous V. albo-atrum ribosomal RNA gene probe revealed restriction fragment length polymorphisms (RFLPs) which could differentiate V. lateritium, V. lecanii, V. nigrescens, V. nubilum and V. tricorpus . Digestion with Eco RI did not provide RFLPs which could distinguish between V. albo-atrum and V. dahliae . Digestion of genomic and mitochondrial DNA with Hae III showed distinctive patterns on ethidium bromide gels which allowed each species to be distinguished. Some intra-species variation in patterns occurred and a combination of mitochondrial and ribosomal RNA gene complex RFLPs has potential as an aid for the characterization of species and sub-species populations in the genes Verticillium .     

Analysis of mitochondrial DNA in Bolivian llama,alpaca and vicuna populations: a contribution to the phylogeny of the South American camelids

The objectives of this work were to assess the mtDNA diversity of Bolivian South American camelid (SAC) populations and to shed light on the evolutionary relationships between the Bolivian camelids and other populations of SACs. We have analysed two different mtDNA regions: the complete coding region of the MT‐CYB gene and 513 bp of the D‐loop region. The populations sampled included Bolivian llamas, alpacas and vicunas, and Chilean guanacos. High levels of genetic diversity were observed in the studied populations. In general, MT‐CYB was more variable than D‐loop. On a species level, the vicunas showed the lowest genetic variability, followed by the guanacos, alpacas and llamas. Phylogenetic analyses performed by including additional available mtDNA sequences from the studied species confirmed the existence of the two monophyletic clades previously described by other authors for guanacos (G) and vicunas (V). Significant levels of mtDNA hybridization were found in the domestic species. Our sequence analyses revealed significant sequence divergence within clade G, and some of the Bolivian llamas grouped with the majority of the southern guanacos. This finding supports the existence of more than the one llama domestication centre in South America previously suggested on the basis of archaeozoological evidence. Additionally, analysis of D‐loop sequences revealed two new matrilineal lineages that are distinct from the previously reported G and V clades. The results presented here represent the first report on the population structure and genetic variability of Bolivian camelids and may help to elucidate the complex and dynamic domestication process of SAC populations.     

Integrating DNA data and traditional taxonomy to streamline biodiversity assessment: an example from edaphic beetles in the Klamath ecoregion, California, USA

Conservation and land management decisions may be misguided by inaccurate or misinterpreted knowledge of biodiversity. Non‐systematists often lack taxonomic expertise necessary for an accurate assessment of biodiversity. Additionally, there are far too few taxonomists to contribute significantly to the task of identifying species for specimens collected in biodiversity studies. While species level identification is desirable for making informed management decisions concerning biodiversity, little progress has been made to reduce this taxonomic deficiency. Involvement of non‐systematists in the identification process could hasten species identification. Incorporation of DNA sequence data has been recognized as one way to enhance biodiversity assessment and species identification. DNA data are now technologically and economically feasible for most scientists to apply in biodiversity studies. However, its use is not widespread and means of its application has not been extensively addressed. This paper illustrates how such data can be used to hasten biodiversity assessment of species using a little‐known group of edaphic beetles. Partial mitochondrial cytochrome oxidase I was sequenced for 171 individuals of feather‐wing beetles (Coleoptera: Ptiliidae) from the Klamath ecoregion, which is part of a biodiversity hotspot, the California Floristic Province. A phylogram of these data was reconstructed via parsimony and the strict consensus of 28,000 equally parsimonious trees was well resolved except for peripheral nodes. Forty‐two voucher specimens were selected for further identification from clades that were associated with many synonymous and non‐synonymous nucleotide changes. A ptiliid taxonomic expert identified nine species that corresponded to monophyletic groups. These results allowed for a more accurate assessment of ptiliid species diversity in the Klamath ecoregion. In addition, we found that the number of amino acid changes or percentage nucleotide difference did not associate with species limits. This study demonstrates that the complementary use of taxonomic expertise and molecular data can improve both the speed and the accuracy of species‐level biodiversity assessment. We believe this represents a means for non‐systematists to collaborate directly with taxonomists in species identification and represents an improvement over methods that rely solely on parataxonomy or sequence data.     

Combining mitochondrial DNA sequences and morphological data to infer species boundaries: phylogeography of lanceheaded pitvipers in the Brazilian Atlantic forest,and the status of Bothrops pradoi (Squamata: Serpentes: Viperidae)

Phylogeographic studies using mitochondrial DNA sequence information are frequently used as the principal source of evidence to infer species boundaries. However, a critical analysis of further evidence is essential to test whether different haplotype clades identify different species. We demonstrate a hypothesis‐testing approach, using a combination of phylogeographic methods, multivariate morphometrics and matrix association tests, to investigate species boundaries in eastern Brazilian pitvipers conventionally assigned to the species Bothrops leucurus and B. pradoi. Two basal haplotype clades with partly overlapping geographical distributions are identified, which could either represent two partly sympatric species, or multiple haplotypes within one organismal lineage. We use partial Mantel matrix association tests to verify whether generalized morphology, or any of four supposedly diagnostic characters for the two species, show any association with mtDNA variation. Negative results lead to the conclusion that the haplotype clades do not denote independently evolving organismal lineages, and do not constitute separate species under any criterion.     

Temperate phages TP901-1 and phiLC3, belonging to the P335 species, apparently use different pathways for DNA injection in Lactococcus lactis subsp. cremoris 3107

Five mutants of Lactococcus lactis subsp. cremoris 3107 resistant to phage TP901-1 were obtained after treatment with ethyl methanesulfonate. Two of the mutants were also resistant to phage phiLC3. The remaining three mutants were as sensitive as 3107. Mutants E46 and E100 did not adsorb the two phages. Mutants E119, E121 and E126 adsorbed phage phiLC3 as well as 3107 but phage TP901-1 with significantly reduced efficiency. All, except E46, could be lysogenized with phage TP901-BC1034, a derivative of TP901-1 harboring an erythromycin-resistance marker. However, the lysogenization frequency was 10(3)-10(4) fold higher for 3107 than for the mutants. Mitomycin C induction of lysogenized mutants 3107 indicated that phage propagation was not affected in these four mutants. Electron microscopy and analysis of total DNA of infected cells showed that DNA was liberated from the phage particle during infection of strain 3107 with TP901-1 and that intracellular phage DNA replication occurred. This was not the case for mutants E121 and E126. This strongly suggests that some step starting with triggering DNA release and ending with DNA injection is impaired during infection with TP901-1. As such impairment was not seen when infecting E119, E121 and E126 with phiLC3, we conclude that TP901-1 and phiLC3 either are differently triggered by their receptor or utilize different pathways of injection.     

Correlation of the cytotoxic activity of four different alkaloids, from Chelidonium majus (greater celandine), with their DNA intercalating properties and ability to induce breaks in the DNA of NK/Ly murine lymphoma cells

The purpose of this study was to examine the relationship between the DNA intercalating characteristics and the DNA damaging capacity of four alkaloids extracted from Chelidonium majus L, as well as their toxicity towards murine NK/Ly lymphoma cells. Chelerythrine, sanguinarine and coptisine were found to be intercalated into the DNA isolated from NK/Ly cells, meanwhile, chelidonine exhibited no affinity to DNA. Sanguinarine exhibited the greatest toxicity toward NK/Ly cells, and the toxicity of the other three decreased in descending order: chelerythrine, coptisine and chelidonine. Chelerythrine and sanguinarine caused DNA damage, illustrated by the formation of comets of the third class. Coptisine was less toxic than chelerythrine and sanguinarine, and affected the formation the same class of comets in higher concentration. The quantity of comets induced by chelidonine were negligible, a finding consistent with its inability to intercalate into DNA structure. The ability of four main alkaloids of Chelidonium majus L., to intercalate into DNA isolated from murine NK/Ly lymphoma cells, correlated with their ability to induce breaks in cellular DNA and with their toxic effect towards those cells.     

Immunization with a poly (lactide co-glycolide) encapsulated plasmid DNA expressing antigenic regions of HPV 16 and 18 results in an increase in the precursor frequency of T cells that respond to epitopes from HPV 16, 18, 6 and 11

A phase II trial was conducted in subjects with human papillomavirus (HPV) associated high-grade cervical dysplasia testing the safety and efficacy of a microparticle encapsulated pDNA vaccine. Amolimogene expresses T cell epitopes from E6 and E7 proteins of HPV types 16 and 18. An analysis was performed on a subset of HLA-A2+ subjects to test whether CD8+ T cells specific to HPV 16, 18, 6 and 11 were increased in response to amolimogene immunization. Of the 21 subjects receiving amolimogene, 11 had elevated CD8+ T cell responses to HPV 16 and/or 18 peptides and seven of these also had increases to corresponding HPV 6 and/or 11 peptides. In addition, T cells primed and expanded in vitro with an HPV 18 peptide demonstrated cross-reactivity to the corresponding HPV 11 peptide. These data demonstrate that treatment with amolimogene elicits T cell responses to HPV 16, 18, 6 and 11.