Cryptic species diversity in a widespread bumble bee complex revealed using mitochondrial DNA RFLPs

Cryptic species diversity is thought to be common within the class Insecta, posing problems for basic ecological and population genetic studies and conservation management. Within the temperate bumble bee (Bombus spp.) fauna, members of the subgenus Bombus sensu stricto are amongst the most abundant and widespread. However, their species diversity is controversial due to the extreme difficulty or inability morphologically to identify the majority of individuals to species. Our character-based phylogenetic analyses of partial CO1 (700 bp) from 39 individuals spread across their sympatric European ranges provided unequivocal support for five taxa (3-22 diagnostic DNA base pair sites per species). Inclusion of 20 Irish specimens to the dataset revealed >= 2.3% sequence divergence between taxa and 200 m) whilst B. cryptarum was relatively more abundant at higher altitudes. Bombus magnus was rarely encountered at urban sites. Both B. lucorum and B. terrestris are nowadays reared commercially for pollination and transported globally. Our RFLP approach to identify native fauna can underpin ecological studies of these important cryptic species as well as the impact of commercial bumble bees on them.

Species boundaries and phylogenetic relationships within the green algal genus Codium (Bryopsidales) based on plastid DNA sequences

Despite the potential model role of the green algal genus Codium for studies of marine speciation and evolution, there have been difficulties with species delimitation and a molecular phylogenetic framework was lacking. In the present study, 74 evolutionarily significant units (ESUs) are delimited using 227 rbcL exon 1 sequences obtained from specimens collected throughout the genus' range. Several morpho-species were shown to be poorly defined, with some clearly in need of lumping and others containing pseudo-cryptic diversity. A phylogenetic hypothesis of 72 Codium ESUs is inferred from rbcL exon 1 and rps3-rp/16 sequence data using a conventional nucleotide substitution model (GTR + Gamma + I), a codon position model and a covariotide (covarion) model, and the fit of a multitude of substitution models and alignment partitioning strategies to the sequence data is reported. Molecular clock tree rooting was carried out because out-group rooting was probably affected by phylogenetic bias. Several aspects of the evolution of morphological features of Codium are discussed and the inferred phylogenetic hypothesis is used as a framework to study the biogeography of the genus, both at a global scale and within the Indian Ocean. (c) 2007 Elsevier Inc. All rights reserved.

Mitochondrial DNA haplotype analysis of liver fluke in bison from Bialowieza Primaeval Forest indicates domestic cattle as the likely source of infection

We have determined the mitochondrial genotype of liver fluke present in Bison (Bison bonasus) from the herd maintained in the Bialowieza National Park in order to determine the origin of the infection. Our results demonstrated that the infrapopulations present in the bison were genetically diverse and were likely to have been derived from the population present in local cattle. From a consideration of the genetic structure of the liver fluke infrapopulations we conclude that the provision of hay at feeding stations may be implicated in the transmission of this parasite to the bison. This information may be of relevance to the successful management of the herd. © 2012 Elsevier B.V.

REMA: A computer-based mapping tool for analysis of restriction sites in multiple DNA sequences

REMA is an interactive web-based program which predicts endonuclease cut sites in DNA sequences. It analyses Multiple sequences simultaneously and predicts the number and size of fragments as well as provides restriction maps. The users can select single or paired combinations of all commercially available enzymes. Additionally, REMA permits prediction of multiple sequence terminal fragment sizes and suggests suitable restriction enzymes for maximally discriminatory results. REMA is an easy to use, web based program which will have a wide application in molecular biology research. Availability: REMA is written in Perl and is freely available for non-commercial use. Detailed information on installation can be obtained from Jan Szubert (jan.szubert@gmail.com) and the web based application is accessible on the internet at the URL http://www.macaulay.ac.uk/rema. Contact: b.singh@macaulay.ac.uk. (C) 2007 Elsevier B.V. All rights reserved.

Mitochondrial DNA Damage Following Isolated Muscle Exercise: A Preliminary Investigation Into HO-1 Gene Expression

PURPOSE: This preliminary investigation was designed to test the hypothesis that high intensity single-leg exercise can cause extensive cell DNA damage, which subsequently may affect the expression of the HO-1 gene. METHODS: Six (n=6) apparently healthy male participants (age 27 + 7 yrs, stature 174 + 12 cm, body mass 79 + 4 kg and BMI 24 + 4 kg/m2) completed 100 isolated and continuous maximal concentric contractions (minimum force = 200 N, speed of contraction = 60°/sec) of the rectus femoris muscle. Using a spring-loaded and reusable Magnum biopsy gun with a 16-gauge core disposable biopsy needle, skeletal muscle micro biopsy tissue samples were extracted at rest and following exercise. mRNA gene expression was determined via two-step quantitative real-time PCR using GAPDH as a reference gene. RESULTS: The average muscle force production was 379 + 179 N. High intensity exercise increased mitochondrial 8-OHdG concentration (P < 0.05 vs. rest) with a concomitant decrease in total antioxidant capacity (P < 0.05 vs. rest). Exercise also increased protein oxidation as quantified by protein carbonyl concentration (P < 0.05 vs. rest). HO-1 expression increased (> 2-fold change vs. rest) following exercise, and it is postulated that this change was not significant due to low subject numbers (P > 0.05). CONCLUSION: These preliminary findings tentatively suggest that maximal concentric muscle contractions can cause intracellular DNA damage with no apparent disruption to the expression of the antioxidant stress protein HO-1. Moreover, it is likely that cell oxidant stress is required to activate the signal transduction cascade related to the expression of HO-1. A large-scale study incorporating a greater subject number is warranted to fully elucidate this relationship.

Global mitochondrial DNA phylogeography and population structure of the silky shark, Carcharhinus falciformis

Globally, sharks are under enormous pressure from fishing efforts. One such species is the silky shark, Carcharhinus falciformis, which occurs in all the Earth’s tropical oceans and is captured in large numbers in pelagic fisheries. Regionally, the silky shark is listed as Vulnerable to Near Threatened by the International Union for the Conservation of Nature due to high levels of direct and by catch exploitation. Despite major conservation concerns about this species, little is known about its genetic status and level of demographic or evolutionary connectivity among its regional distributions. We report a genetic assessment of silky sharks sampled across a major portion of the species’ global range. We sequenced the complete mitochondrial DNA control region from 276 individuals taken from the western Atlantic and Indo-Pacific Oceans and the Red Sea. Overall, haplotype and nucleotide diversities were relatively large (0.93 ± 0.01 and 0.61 ± 0.32 %, respectively). Nucleotide diversity in Indo-Pacific sharks, however, was significantly lower and about half that in Atlantic sharks. Strong phylogeographic partitioning occurred between ocean basins. Furthermore, shallow but significant pairwise statistical differentiation occurred among most regional samples within the Indo-Pacific, but not the western Atlantic. Overall, at least five mitochondrial DNA populations of silky sharks were identified globally. Despite historically large population sizes, silky sharks appear to be isolated on relatively small spatial scales, at least in the Indo-Pacific, indicating that conservation and management efforts will need to be exerted at relatively small scales in a pelagic and highly vagile species.

Unique mitochondrial DNA lineages in Irish stickleback populations: cryptic refugium or rapid recolonization?

Repeated recolonization of freshwater environments following Pleistocene glaciations has played a major role in the evolution and adaptation of anadromous taxa. Located at the western fringe of Europe, Ireland and Britain were likely recolonized rapidly by anadromous fishes from the North Atlantic following the last glacial maximum (LGM). While the presence of unique mitochondrial haplotypes in Ireland suggests that a cryptic northern refugium may have played a role in recolonization, no explicit test of this hypothesis has been conducted. The three-spined stickleback is native and ubiquitous to aquatic ecosystems throughout Ireland, making it an excellent model species with which to examine the biogeographical history of anadromous fishes in the region. We used mitochondrial and microsatellite markers to examine the presence of divergent evolutionary lineages and to assess broad-scale patterns of geographical clustering among postglacially isolated populations. Our results confirm that Ireland is a region of secondary contact for divergent mitochondrial lineages and that endemic haplotypes occur in populations in Central and Southern Ireland. To test whether a putative Irish lineage arose from a cryptic Irish refugium, we used approximate Bayesian computation (ABC). However, we found no support for this hypothesis. Instead, the Irish lineage likely diverged from the European lineage as a result of postglacial isolation of freshwater populations by rising sea levels. These findings emphasize the need to rigorously test biogeographical hypothesis and contribute further evidence that postglacial processes may have shaped genetic diversity in temperate fauna.

Results of a collaborative study of the EDNAP group regarding mitochondrial DNA heteroplasmy and segregation in hair shafts.

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the distribution of mitochondrial DNA (mtDNA) heteroplasmy amongst the hairs of an individual who displays point heteroplasmy in blood and buccal cells. A second aim of the exercise was to study reproducibility of mtDNA sequencing of hairs between laboratories using differing chemistries, further to the first mtDNA reproducibility study carried out by the EDNAP group. Laboratories were asked to type 2 sections from each of 10 hairs, such that each hair was typed by at least two laboratories. Ten laboratories participated in the study, and a total of 55 hairs were typed. The results showed that the C/T point heteroplasmy observed in blood and buccal cells at position 16234 segregated differentially between hairs, such that some hairs showed only C, others only T and the remainder, C/T heteroplasmy at varying ratios. Additionally, differential segregation of heteroplasmic variants was confirmed in independent extracts at positions 16093 and the poly(C) tract at 302-309, whilst a complete A-G transition was confirmed at position 16129 in one hair. Heteroplasmy was observed at position 16195 on both strands of a single extract from one hair segment, but was not observed in the extracts from any other segment of the same hair. Similarly, heteroplasmy at position 16304 was observed on both strands of a single extract from one hair. Additional variants at positions 73, 249 and the HVII poly(C) region were reported by one laboratory; as these were not confirmed in independent extracts, the possibility of contamination cannot be excluded. Additionally, the electrophoresis and detection equipment used by this laboratory was different to those of the other laboratories, and the discrepancies at position 249 and the HVII poly(C) region appear to be due to reading errors that may be associated with this technology. The results, and their implications for forensic mtDNA typing, are discussed in the light of the biology of hair formation.

Isolation of DNA sequences with homology to a degenerate FaRP oligonucleotide from the crayfish Procambarus clarkii

The neuropeptide Th1RFamide with the sequence Phe-Met-Arg-Phe-amide was originally isolated in the clam Macrocallista nimbosa (price and Greenberg, 1977). Since its discovery, a large family ofFl\1RFamide-related peptides termed FaRPs have been found to be present in all major animal phyla with functions ranging from modulation of neuronal activity to alteration of muscular contractions. However, little is known about the genetics encoding these peptides, especially in invertebrates. As FaRP-encoding genes have yet to be investigated in the invertebrate Malacostracean subphylum, the isolation and characterization ofFaRP-encoding DNA and mRNA was pursued in this project. The immediate aims of this thesis were: (1) to amplify mRNA sequences of Procambarus clarkii using a degenerate oligonucleotide primer deduced from the common amino acid sequence ofisolated Procambarus FaRPS, (2) to determine if these amplification products encode FaRP gene sequences, and (3) to create a selective cDNA library of sequences recognized by the degenerate oligonucleotide primer. The polymerase chain reaction - rapid amplification of cDNA ends (PCR-RACE) is a procedure in which a single gene-specific primer is used in conjunction with a generalized 3' or 5' primer to amplify copies ofthe region between a single point in the transcript and the 3' or 5' end of cDNA of interest (Frohman et aI., 1988). PCRRACE reactions were optimized with respect to primers used, buffer composition, cycle number, nature ofgenetic substrate to be amplified, annealing, extension and denaturation temperatures and times, and use of reamplification procedures. Amplification products were cloned into plasmid vectors and recombinant products were isolated, as were the recombinant plaques formed in the selective cDNA library. Labeled amplification products were hybridized to recombinant bacteriophage to determine ligated amplification product presence. When sequenced, the five isolated PCR-RACE amplification products were determined not to possess FaRP-encoding sequences. The 200bp, 450bp, and 1500bp sequences showed homology to the Caenorhabditis elegans cosmid K09A11, which encodes for cytochrome P450; transfer-RNA; transposase; and tRNA-Tyr, while the 500bp and 750bp sequences showed homology with the complete genome of the Vaccinia virus. Under the employed amplification conditions the degenerate oligonucleotide primer was observed to bind to and to amplify sequences with either 9 or 10bp of 17bp identity. The selective cDNA library was obselVed to be of extremely low titre. When library titre was increased, white. plaques were isolated. Amplification analysis of eight isolated Agt11 sequences from these plaques indicated an absence of an insertion sequence. The degenerate 17 base oligonucleotide primer synthesized from the common amino acid sequence ofisolated Procambarus FaRPs was thus determined to be non-specific in its binding under the conditions required for its use, and to be insufficient for the isolation and identification ofFaRP-encoding sequences. A more specific primer oflonger sequence, lower degeneracy, and higher melting temperature (TJ is recommended for further investigation into the FaRP-encoding genes of Procambarlls clarkii.

Isolation and characterization of genomic DNA sequences that enhance the stability of plasmid DNA in mammalian cells /

The ease of production and manipulation has made plasmid DNA a prime target for its use in gene transfer technologies such as gene therapy and DNA vaccines. The major drawback of plasmid however is its stability within mammalian cells. Plasmid DNA is usually lost by cellular mechanisms or as a result of mitosis by simple dilution. This study set out to search for mammalian genomic DNA sequences that would enhance the stability of plasmid DNA in mammalian cells.Creating a plasmid based genomic DNA library, we were able to screen the human genome by transfecting the library into Human Embryonic Kidney (HEK 293) Cells. Cells that contained plasmid DNA were selected, using G418 for 14 days. The resulting population was then screened for the presence of biologically active plasmid DNA using the process of transformation as a detector.A commercially available plasmid DNA isolation kit was modified to extract plasmid DNA from mammalian cells. The standardized protocol had a detection limit of -0.6 plasmids per cell in one million cells. This allowed for the detection of 45 plasmids that were maintained for 32 days in the HEK 293 cells. Sequencing of selected inserts revealed a significantly higher thymine content in comparison to the human genome. Sequences with high A/T content have been associated with Scaffold/Matrix Attachment Region (S/MAR) sequences in mammalian cells. Therefore, association with the nuclear matrix might be required for the stability of plasmids in mammalian cells.

Stocking may increase mitochondrial DNA diversity but fails to halt the decline of endangered Atlantic salmon populations

Over the last 50 years, Spanish Atlantic salmon (Salmo salar) populations have been in decline. In order to bolster these populations, rivers were stocked with fish of northern European origin during the period 1974-1996, probably also introducing the furunculosis-inducing pathogen, Aeromonas salmonicida. Here we assess the relative importance of processes influencing mitochondrial (mt)DNA variability in these populations from 1948 to 2002. Genetic material collected over this period from four rivers in northern Spain (Cantabria) was used to detect variability at the mtDNA ND1 gene. Before stocking, a single haplotype was found at high frequency (0.980). Following stocking, haplotype diversity (h) increased in all rivers (mean h before stocking was 0.041, and 0.245 afterwards). These increases were due principally to the dramatic increase in frequency of a previously very low frequency haplotype, reported at higher frequencies in northern European populations proximate to those used to stock Cantabrian rivers. Genetic structuring increased after stocking: among-river differentiation was low before stocking (1950s/1960s Phi(ST) = -0.00296-0.00284), increasing considerably at the height of stocking (1980s Phi(ST) = 0.18932) and decreasing post-stocking (1990s/2002 Phi(ST) = 0.04934-0.03852). Gene flow from stocked fish therefore seems to have had a substantial role in increasing mtDNA variability. Additionally, we found significant differentiation between individuals that had probably died from infectious disease and apparently healthy, angled fish, suggesting a possible role for pathogen-driven selection of mtDNA variation. Our results suggest that stocking with non-native fish may increase genetic diversity in the short term, but may not reverse population declines.

Rare and fleeting: an example of interspecific recombination in animal mitochondrial DNA

Recombination is thought to occur only rarely in animal mitochondrial DNA ( mtDNA). However, detection of mtDNA recombination requires that cells become heteroplasmic through mutation, intramolecular recombination or ' leakage' of paternal mtDNA. Interspecific hybridization increases the probability of detecting mtDNA recombinants due to higher levels of sequence divergence and potentially higher levels of paternal leakage. During a study of historical variation in Atlantic salmon ( Salmo salar) mtDNA, an individual with a recombinant haplotype containing sequence from both Atlantic salmon and brown trout ( Salmo trutta) was detected. The individual was not an F1 hybrid but it did have an unusual nuclear genotype which suggested that it was a later-generation backcross. No other similar recombinant haplotype was found from the same population or three neighbouring Atlantic salmon populations in 717 individuals collected during 1948 - 2002. Interspecific recombination may increase mtDNA variability within species and can have implications for phylogenetic studies.