bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment of the stocked or wild origin of anadromous brown trout (Salmo trutta L.) in a Danish river system, using mitochondrial DNA RFLP analysis
Authors: HANSEN M. M. , HYNES R. A. , LOESCHCKE V. , RASMUSSEN G.
Published: April 2008

Declines in the number of anadromous brown trout in the Karup River in Denmark, due to environmental degradation, led to the stocking of large numbers of hatchery trout during the 1980s. This practice was gradually replaced by stocking with the offspring of electrofished local trout The genetic contribution of the hatchery fish to the current population of anadromous trout in the river was estimated by restriction fragment length polymorphism analysis of mitochondrial DNA, using seven restriction endonucleases. Fish from the hatchery strain as well as from five locations in the river system, and from a further unstocked river were screened. Eight haplotypes were observed. The distribution and frequencies of the observed haplotypes revealed little genetic differentiation among stocked populations. The hatchery strain differed significantly from the stocked populations. One haplotype which was found at a high frequency in the hatchery strain was almost absent from the stocked populations. This suggests that the genetic contribution of the hatchery trout to the current population is much less than would be expected from the number of stocked fish. The possible reasons for the failure of the hatchery trout to contribute to the gene pool, and also the implications for conservation biology, are discussed.
Molecular diversity and derivations of populations of Silene acaulis and Saxifraga oppositifolia from the high Arctic and more southerly latitudes
Authors: ABBOTT R. J. , CHAPMAN H. M. , CRAWFORD R. M. M. , FORBES D. G.
Published: April 2008

A survey of allozyme diversity within and between populations of Silene acaulis from Spitsbergen, Norway, Iceland and Scotland, showed that populations from the high Arctic (Spitsbergen, > 76°N) contained high levels of diversity and were genetically similar to populations from more southern locations. Indirect measures of gene flow (Nm), calculated from Wrigh's F indicated that there had been extensive gene flow between Spitsbergen and some Norwegian populations. A restriction site analysis of chloroplast DNA (cpDNA) in S. acaulis revealed that all populations contained a single identical cpDNA haplotype, except one population from Norway which also contained a second haplotype. In contrast, five different cpDNA haplotypes were distinguished in a more limited survey of cpDNA variation in Saxifraga oppositifolia, with all five haplotypes present in one of two Spitsbergen populations surveyed. The contrasting cpDNA results for the two species suggest that whereas high‐Arctic populations of Silene acaulis have most likely been derived from immigrants which arrived from the south after the last glacial period, high‐Arctic populations of Saxifraga oppositifolia may be derived, in part, from ancient northern stocks which survived the last glaciation in high‐Arctic refugia.
Diversity and genetic structure of a natural population of Rhizobium leguminosarum bv. trifolii isolated from Trifolium subterraneum L.
Authors: DEMEZAS D. H. , REARDON T. B. , STRAIN S. R. , WATSON J. M. , GIBSON A. H.
Published: April 2008

A collection of 121 isolates of Rhizobium leguminosarum biovar (bv.) trifolii was obtained from root nodules of Trifolium subterraneum L. (subclover) plants growing in an established pasture. The collection consisted of a single isolate from each of 18 plants sampled from seven microplots. The following year, a further 28 and 27 isolates were collected from the first and seventh sampling points, respectively. Analysis of restriction fragment length polymorphisms (RFLPs) of both chromosomal and Sym (symbiotic) plasmid DNA and multilocus enzyme electrophoresis (MLEE) were used to assess the diversity, genetic relationships and structure of this population. Symbiotic effectiveness tests were used to examine the symbiotic phenotype of each isolate collected in the first year. Analysis of RFLPs of the first year isolates revealed 13 chromosomal types and 25 Sym plasmid types. Similar Sym plasmid types were grouped into 14 families containing 1–6 members. No new chromosomal types and six new Sym plasmid types were detected in the second year. The symbiotic effectiveness of the first year isolates of the same Sym plasmid type was similar. Significant differences in symbiotic effectiveness were detected between different Sym plasmid types in the same plasmid family. Representative isolates of each chromosomal type Sym plasmid type identified in the first year were analysed using multilocus enzyme electrophoresis. Mean genetic diversity per locus was high (0.559). Enzyme electrophoresis revealed 17 electrophoretic types (ETs). Ouster analysis of the enzyme data revealed large genetic diversity amongst the ETs. Strong linkage disequilibrium was observed for the population as a whole, i.e. clonal population structure, but significantly less disequilibrium was observed among a cluster of ETs suggesting that recombination occurred between ETs within the cluster. Our results revealed that a population of naturally occurring isolates of Rhizobium leguminosarum bv. trifolii can be genetically diverse and support the possibility that recombination plays a role in generating new genotypes.
Glucose utilization by 6pgd genotypes in Lolium perenne
Authors: RAINEY‐FOREMAN D. , MITTON J. B.
Published: April 2008

To gain a deeper understanding of the mechanisms underlying associations between allozyme genotypes and rates of respiration in Lolium perenne, Vmax Km and rates of glucose flux through glycolysis and the pentose phosphate pathway were estimated for the three genotypes of the 6pgd locus. Km Vmax and Vmax/Km differed significantly among genotypes. Values of Km for the 11, 12, and 22 genotypes were 0.29, 0.25, 0.13, while the values of Vmax/Km for die 11, 12, and 22 genotypes were 3.79, 3.85, 6.70. Flux through the pentose shunt did not differ among genotypes at 20 °C, but at 35 °C the rates of flux for the 11, 12, and 22 genotypes were 0.15, 0.25 and 0.42, respectively. Thus, the 6PGD allozyme genotypes differ markedly in both enzyme kinetic characteristics and in flux through a metabolic pathway. These associations reveal potentially causal relationships between allozyme genotypes and rates of respiration.
Natal dispersal and genetic structure in a population of the European wild rabbit (Oryctolagus cuniculus)
Authors: WEBB N. J. , IBRAHIM K. M. , BELL D. J. , HEWITT G. M.
Published: April 2008

A combination of behavioural observation, DNA fingerprinting, and allozyme analysis were used to examine natal dispersal in a wild rabbit population. Rabbits lived in territorial, warren based social groups. Over a 6‐year period, significantly more male than female rabbits moved to a new social group before the start of their first breeding season. This pattern of female philopatry and male dispersal was reflected in the genetic structure of the population. DNA fingerprint band‐sharing coefficients were significantly higher for females within the same group than for females between groups, while this was not the case for males. Wrighfs inbreeding coefficients were calculated from fingerprint band‐sharing values and compared to those obtained from allozyme data. There was little correlation between the relative magnitudes of the F‐statistics calculated using the two techniques for comparisons between different social groups. In contrast, two alternative methods for calculating FST from DNA fingerprints gave reasonably concordant values although those based on band‐sharing were consistently lower than those calculated by an ‘allele’ frequency approach. A negative FIS value was obtained from allozyme data. Such excess heterozygosity within social groups is expected even under random mating given the social structure and sex‐biased dispersal but it is argued that the possibility of behavioural avoidance of inbreeding should not be discounted in this species. Estimates of genetic differentiation obtained from allozyme and DNA fingerprint data agreed closely with reported estimates for the yellow‐bellied marmot, a species with a very similar social structure to the European rabbit.
Nonamplifying alleles at microsatellite loci: a caution for parentage and population studies
Authors: PEMBERTON J. M. , SLATE J. , BANCROFT D. R. , BARRETT J. A.
Published: April 2008

While genotyping wild red deer (Cervus elaphus) at microsatellite loci for paternity assignment, we found three loci (MAP65, BOVIRBP and CelJP23) with segregating nonamplifying alleles. Nonamplifying alleles were detected through mismatches between known mother‐offspring pairs and by significant deviations from Hardy‐Weinberg equilibria. In a wide range of molecular ecology applications, and especially in parentage assignment, the possible existence of undetectable alleles must be taken into account; this may be particularly important for microsatellite data.
Differentiation of the endemic New Zealand greenheaded and brownheaded leafroller moths by restriction fragment length variation in the ribosomal gene complex
Authors: SIN F. Y. T. , SUCKLING D. M. , MARSHALL J. W.
Published: April 2008

This study identified markers in the ribosomal RNA gene complex (rDNA) and evaluated their utility in the differentiation of four endemic New Zealand species of leafroller moth, Planotortrix excessana (Walker), P. octo (Dugdale), Ctenopseustis herana (Felder and Rogenhofer) and C. obliquana (Walker). Pooled genomic DNA from laboratory‐reared specimens of C. herana and C. obliquana could be distinguished with a single restriction enzyme (e.g. ClaI), while P. excessana and P. octo could be satisfactorily distinguished using double digestions with either ClaI/PvuII or ClaI/BglII. It is concluded mat these markers may be used to identify moths from wild populations.
Transposon signatures: species‐specific molecular markers that utilize a class of multiple‐copy nuclear DNA
Authors: PURUGGANAN M. D. , WESSLER S. R.
Published: April 2008

Transposable elements are mobile sequences found in nuclear genomes and can potentially serve as molecular markers in various phylogenetic and population genetic investigations. A PCR‐based method that utilizes restriction site variation of element copies within a genome is developed. These patterns of site variation, referred to as transposon signatures, are useful in differentiating between closely related groups. Signature data using the magellan retrotransposon, for example, is useful in examining relationships within the genus Zea and Tripsacum. This method allows transposable elements, or even other multiple‐copy nuclear DNA sequences, to be generally utilized as molecular markers in discriminating between other closely related species and subspecies.
Colonization history of north European field voles (Microtus agrestis) revealed by mitochondrial DNA
Authors: JAAROLA M. , TEGELSTRÖM H.
Published: April 2008

The genetic structure of field vole (Microtus agrestis) populations from northern Europe was examined by restriction fragment length polymorphisms of mitochondrial DNA (mtDNA) in 150 individuals from 67 localities. A total of 83 haplotypes was observed, most of which were rare and highly localized geographically. Overall nucleotide diversity was high (134%), but showed a tendency to decrease with higher latitude. Two major mtDNA lineages differing by 2% in nucleotide sequence were identified. A southern mtDNA lineage was observed in field voles from Britain, Denmark and southern and central Sweden, whereas voles from Finland and northern Sweden belonged to a northern lineage. The strict phylogeographic pattern suggests that the present population generic structure in field voles reflects glacial history: the two groups are derived from different glacial refugia, and recolonized Fennoscandia from two directions. A 150–200‐km‐wide secondary contact zone between the two mtDNA groups was found in northern Sweden. Distinct phylogeographic substructuring was observed within both major mtDNA groups.
Use of RAPD analysis in devising conservation strategies for the rare and endangered Grevillea scapigera (Proteaceae)
Authors: ROSSETTO M. , WEAVER P. K. , DIXON K. W.
Published: April 2008

Extensive human impact in south western Australia has resulted in a high incidence of rarity throughout the highly endemic flora of the region. Grevillea scapigera (Proteaceae) is a typical example, with 27 plants (represented by four extant populations) remaining in the wild. In order to devise an appropriate strategy for the conservation of this species, its population genetics were studied using RAPD analysis, which enabled the discrimination of individual plants and the detection of a relatively high amount of variability (V = 0.32) within G. scapigera. This variability was found to be evenly distributed within the plants analysed despite the clear distinction between most populations (87% of the variability being attributable to single plant difference and 13% to population difference). Finally, RAPD analysis was used to select a small group of plants that captured maximum genetic variability to be used in the recovery program of the species. Because of the low genetic difference between populations, the mixing of these selected plants during the recovery process should not create genetic imbalances. The methods used in this study provide a useful model for future projects involving the recovery of rare flora.
Phylogeographic structure in mitochondrial DNA of a South‐east Asian freshwater fish, Hemibagrus nemurus (Siluroidei; Bagridae) and Pleistocene sea‐level changes on the Sunda shelf
Authors: DODSON J. J. , COLOMBANI F. , NG P. K. L.
Published: April 2008

We postulated that the biogeographical history of South‐east Asia contributed to extensive admixture during Pleistocene low sea levels of genetic groups of an obligate freshwater fish (the river catfish, Hemibagrus nemurus) isolated during periods of high sea levels. During Pleistocene glacial maxima, the sea level was lower than at present and the islands of the Sunda shelf (Sumatra, Borneo and Java) and the Asian mainland were connected by lowlands traversed by rivers. Restriction fragment length polymorphisms in mitochondrial DNA were documented for 140 putative H. nemurus analysed from 13 sampling sites resulting in the definition of 35 haplotypes. The high level of haplotype differentiation (mean P × 100 = 2.22, SD = 1.33) indicates that the subdivision of the ancestral H. netnurus group was extensive and probably occurred early in the Pleistocene. The occurrence of some genetically divergent groups of the H. netnurus complex occurring in sympatry in widely separated locations supports the proposition that low sea levels aided the dispersion and mingling of genetic groups. Based on both genetic and morphological evidence, the main H. nemurus line gave rise to three regional groups: (1) a morphologically distinct ‘Indochinese’ group composed of two mtDNA clades overlapping in east peninsular Malaysia; (2) a ‘Sundaic’ group composed of various lineages of differing morphology and genetic identity; (3) a genetically distinct ‘Sarawak’ group in west Borneo, similar in morphology to the ‘Sundaic’ and ‘Indochinese’ groups, but including a small, golden colour morph as a distinct dade. The morphologically similar Sundaic forms from west Java, Sumatra and west Borneo show some degree of genetic divergence, but their phylogenetic relationships are poorly resolved. The most genetically and morphologically distinct Sundaic dade, assigned to H. hoevenii, colonized the Kapuas river (west Borneo), east Sumatra and south peninsular Malaysia. Contrary to our original hypothesis and present biogeographical theory, little exchange of genetic groups has apparently occurred between the mainland and the Sunda Islands during recent glaciations.
A comparison of mtDNA restriction sites vs. control region sequences in phylogeographic assessment of the musk turtle (Sternotherus minor)
Authors: WALKER D. , BURKE V. J. , BARÁK I. , AVISE J. C.
Published: April 2008

A total of nearly 800 base pairs of mitochondrial DNA sequence was assayed in each of 52 musk turtles (Sternotherus minor) collected across the species' range in the south‐eastern USA. About one‐half of the sequence information in effect was accessed by conventional recognition‐site assays of the entire mtDNA molecule; the remainder came from direct sequence assays of a normally hypervariable 5′ section of the noncoding control region. The two assay methods produced essentially nonoverlapping sets of variable character states that were compared with respect to magnitudes and phylogeographic patterns of mtDNA variation. The two assay procedures yielded nearly identical outcomes with regard to: (a) total levels of species‐wide mtDNA genetic variation; (b) mean levels of within‐locale variation; (c) extremely high population genetic structure; (d) a phylogenetcally significant separation of samples from the north‐western half of the species' range vs. those in the south‐eastern segment; and (e) considerably lower genetic variability within the north‐western clade. The micro‐ and macro‐phylogeographic mtDNA patterns in the musk turtle are consistent with a low‐dispersal natural history, and with a suspected longer‐term biogeographic history of the species, respectively.
Mitochondrial DNA variation among populations of Oedura reticulata (Gekkonidae) in remnant vegetation: implications for metapopulation structure and population decline
Authors: SARRE S.
Published: April 2008

Restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA was used to examine the regional population structure of a species of gecko (Oedura reticulata) in vegetation remnants within the Western Australian wheatbelt. The species exhibited considerable polymorphism within and between populations with 22 haplotypes recognized among 12 populations. Phylogenetic analysis of haplotypes and clustering of nucleotide divergence among populations demonstrated little regional structure within the species with several haplotypes present in all three regions surveyed. This contrasted markedly with variation in haplotype frequency among populations which showed a high degree of independence between populations indicating that current levels of maternal gene flow are low and that the populations are too small to prevent genetic drift. This conclusion is supported by generally lower numbers of haplotypes in remnant populations than in nearby nature reserves. These findings, combined with demographic data from a previous study, suggest that post‐fragmentation populations of O. reticulata are unable to form a metapopulation structure in the habitat that remains and that stochastic extinction forces alone will be sufficient to severely reduce the regional distribution of this species. This study demonstrates that mtDNA is a useful tool for detecting contemporary population phenomena and can provide qualitative information of practical importance to wildlife managers.
Outgroup heteroduplex analysis using temperature gradient gel electrophoresis: high resolution, large scale, screening of DNA variation in the mitochondrial control region
Authors: CAMPBELL N. J. H. , HARRISS F. C. , ELPHINSTONE M. S. , BAVERSTOCK P. R.
Published: April 2008

The ability of DNA screening techniques such as Temperature Gradient Gel Electrophoresis (TGGE) and Heteroduplex Analysis to provide resolution approaching that provided by DNA sequencing for a fraction of the time, effort and expense point to them as the logical successor to allozyme electrophoresis for population genetics. Here we present a novel alternative to the standard TGGE/Heteroduplex Analysis protocol ‐ Outgroup Heteroduplex Analysis (OHA). We assess this technique's sensitivity in comparison to previous screening approaches using a known hierarchy of sequence differences. Our data show that Outgroup Heteroduplex Analysis has greatly increased sensitivity for screening DNA variants from that of TGGE used alone and is easily applicable to large numbers of samples. Using this technique we can consistently detect differences of as small as one base change in a 433‐base‐pair fragment of Control Region mitochondrial DNA from Melomys cerbinipes (an Australian rodent). The approach should easily be extendable to nuclear loci and is not necessarily dependent on the use of a denaturing gradient When combined with a targeted sequencing effort, OHA provides a sensitive and simple means of obtaining allele/haplotype frequencies and their phylogenies for population and phylogeographic studies in molecular ecology.
Mitochondrial DNA variation, population structure, and evolution of mountain sheep in the south‐western United States and Mexico
Authors: II R. R. RAMEY
Published: April 2008

In the south‐western United States and Mexico, mountain sheep were found to have low levels of mtDNA sequence divergence among haplotypes and low overall nucleotide diversity. Potential causes for this low diversity are discussed. Significant differences in mtDNA haplotype distributions over short distances and high values of NST on a local scale appear to be a result of the tendency of female mountain sheep to disperse less frequently and over shorter distances than males. A lack of concordance between mtDNA haplotype distributions and assumed subspecies boundaries suggest that some taxonomic labels need to be revised.
Molecular structure of the Frankia spp. nifD—K intergenic spacer and design of Frankia genus compatible primer
Authors: NALIN R. , DOMENACH A. M. , NORMAND P.
Published: April 2008

The nifD—K intergenic spacer (IGS) of ArI3 and ACoN24d were found to have a length 265 and 199 nucleotides, respectively. They are markedly less conserved than the two neighbouring genes and have, in some instances, a repeated structure reminiscent of an insertion event The repeated sequence and the IGSs have no detectable homology with sequences in DNA databanks. The IGS has a stem‐loop structure with a low folding energy, lower than that between nifH and nifD. No convincing alignment of IGS sequences could be obtained among Frankia strains. Only between ACoN24d and ArI3, which belong to the same genomic species, was the alignment good enough to permit detection of a doubly repeated structure. No promoter could be detected in the IGSs. The putative nifK open reading frame (ORF) in Frankia strain ArI3 has a length of 1587 nucleotides, starting with a GTG codon, preceded by a ribosome binding site of a structure similar to that of nifH (GGAGGN7). The codon usage was similar to that of previously sequenced Frankia genes with a strong bias toward G‐ and C‐ending codons except in the case of glycine where GGT is frequent. Alignment of the three Frankia nifK sequences (EUN1f; ArI3 and ACoN24d) with those of other nitrogen‐fixing bacteria permitted detection of a sequence conserved among the three Frankia strains but absent in the other sequences. A primer targeted to that region in combination with FGPD807‐85 amplified the nifD—K IGS sequences of all Frankia strains (except the non‐nitrogen‐fixing Frankia strains CN3 and AgB1‐9) and yet failed to amplify DNA of all other nitrogen–fixing bacteria. Conversely, the failure of primer FGPK700′‐92 to amplify Alnus‐infective strains could be explained by point mutations in the 3′ part of the primer.
Use of the PCR and fluorescent probes to recover SSU rRNA gene sequences from single cells of the ciliate protozoon Spathidiutn
Authors: DYAL P. L. , HOPE S. , ROBERTS D. M. , EMBLEY T. M.
Published: April 2008

A two‐stage heminested PCR approach was developed to amplify small subunit (SSU) rDNA sequences, via two overlapping fragments, from single cells of microbial eucary‐otes. The method was evaluated using the ciliate protozoon Spathidiutn when PCR products were obtained from nine of 10 cells tested. Southern blotting demonstrated that all fragments contained the same sequence in a region of SSU rDNA which is normally highly variable between species. A fluorescent oligonucleotide probe was used to demonstrate that this sequence also occurred in fixed cells of Spathidiutn. Fixatives containing mercuric salts preserved cell shape and allowed probe binding with little background auto‐fluorescence. The Spathidiutn sequence is closely related to that from the haptorid Homalozoon vermiculare.
Nucleic‐acid‐based methods for monitoring the performance of in situ bioremediation
Authors: BROCKMAN F. J.
Published: April 2008

Over the last decade, major advancements have occurred in the application of nucleic‐acid‐based methods to detect and determine the levels of catabolic genes in environmental samples. Studies have focused on validating methods in microcosms, studying changes in the structure and expression of microbial communities in response to contaminants, and improving the sensitivity of the methods. Only in the last few years have these methods transitioned from development and validation to efforts to apply these methods for monitoring in situ bioremediation. Methods that analyse nucleic acids extracted from environmental samples are of value to bioremediation because they allow analysis independent of the artefacts that can arise from laboratory biodegradative potential assays and laboratory culture‐based enumerations and from the inability to culture a large proportion of the micro‐organisms in the environment In theory, these methods enable a more comprehensive perspective, and a more defensible interpretation, of the microbial community response to intrinsic and engineered bioremediation processes. Results from the first studies applying nucleic‐acid‐based methods to intrinsic or engineered bioremediation indicate that these methods have both potential and limitations. The rapidly increasing number of cloned and sequenced catabolic genes, methodological advancements such as the ability to track specific micro‐organisms without prior sequence data, and the potential use of bioaugmentation in the field suggest that the utility of these methods for in situ bioremediation will increase in the coming years.
Extraction and purification of microbial DNA from petroleum‐contaminated soils and detection of low numbers of toluene, octane and pesticide degraders by multiplex polymerase chain reaction and Southern analysis
Authors: KNAEBEL D. B. , CRAWFORD R. L.
Published: April 2008

We investigated the use of multiplex polymerase chain reaction (FCR) techniques coupled with Southern analysis to detect xenobiotic‐degrading organisms that had been added to three soils. Two soils highly contaminated with petroleum hydrocarbons and a less contaminated control soil were amended with tenfold dilutions of Pseudomonas putida mt‐2 (pWWO), P. oleovorans (OCT), and Alcaligenes eutrophus JMP134 (pJP4), or, for controls, phosphate buffer alone. Total DNA was then isolated from the soils and purified using a sequential precipitation and dissolution purification procedure. This DNA was subjected to multiplex polymerase chain reaction (PCR) using primers that amplify regions of xylM (PCR product = 631 bp), alkB (546 bp) and tfdA (710 bp), which are found on pWWO, OCT and pJP4, respectively. The sizes of the amplified DNA fragments were designed to permit simultaneous amplification and detection of the target genes. Ethidium bromide‐stained gels of the initial PCR reaction indicated detectable amplification of between 10* to 10* cells per gram soil, depending on the soil and the target gene. Southern analysis of the PCR amplified DNA improved detection limits to between 1 and 10 cells of each target species per gram of soil, and confirmed the identity of the PCR products. For some samples that were initially resistant to PCR, dilution of the environmental DNA resulted in positive PCR results. This treatment presumably overcame the inhibition of the PCR by diluting coextracted contaminants in the environmental DNA. A second PCR on an aliquot (1 μL) of the first reaction increased the ethidium bromide‐based detection limits for one of the soils to six cells per gram of soil; it did not increase the detection limits for the other soils. Therefore, the DNA extraction procedure and multiplex PCR permitted the simultaneous detection of three types of biodegradarJve cells, at a lower detection limit of = > 10 cells per gram of highly contaminated, organic soil. However, due to kinetic limitations of multiplex PCR, the amplified signals did not follow a close dose response to the numbers of added target cells.
Genetic diversity within mer genes directly amplified from communities of noncultivated soil and sediment bacteria
Authors: BRUCE K. D. , OSBORN A. M. , PEARSON A. J. , STRIKE P. , RITCHIE D. A.
Published: April 2008

Individual merRTΔP regions were amplified from DNA directly isolated from soil and sediment samples using consensus primers derived from the conserved mer sequences of Tn501, Tn21 and pMER419. Soil and sediment samples were taken from four sites in the British Isles; one ‘pristine’ (SB) and three polluted (SO, SE, T2) with respect to mercury. The sizes of the PCR products amplified (= 1 kb) were consistent with their generation from mer determinants related to the archetypal elements found in Gram negative bacteria. Forty‐five individual clones of sequences obtained from these four sites were isolated which hybridized (> 70% homology) to a merRTΔP probe from Tn501. The diversity of these amplified mer genes was analysed using Restriction Fragment Length Polymorphism (RFLP) profiling. Fourteen RFLP classes were distinguished, 12 of which proved to be novel and only two of which had been identified in an earlier study of 40 Gram negative mercury resistant bacteria cultured from the same four sites. UPGMA analysis was used to examine the relationships between the 22 classes of determinant identified. The T2 site, which has the longest history of mercury exposure, was found to have the greatest level of diversity in terms of numbers of classes of determinant, while the SO site, which had the highest mercury levels showed relatively low variation. Variation of mer genes within and between the sequences from cultivated bacteria and from total bacterial DNA shows clearly that analysing only sequences from cultivated organisms results in a gross underestimation of genetic variation.
Gene transfer from a bacterium injected into an aquifer to an indigenous bacterium
Authors: ZHOU J. Z. , TIEDJE J. M.
Published: April 2008

Two novel 3‐chlorobenzoate‐degrading bacteria were previously isolated from an aquifer in which no such bacteria could be enriched prior to the introduction of the 3‐chlorobenzoate‐degrading strain, Pseudomonas sp. B13. To understand the origin of 3‐chlorobenzoate‐degrading genes in the two novel isolates, the 16S ribosomal RNA, clcD (dienelactone hydrolase) and clcA (chlorocatechol oxygenase) genes from these bacteria were amplified and sequenced. The partial 16S rRNA gene sequences and REP‐PCR patterns showed that these two novel isolates were identical but differed from strain B13. Phylogenetic analyses revealed that the novel isolates were closely related to Alcaligenes eutrophus in the beta subclass of the Proteobacteria, whereas strain B13 was related to Pseudomonas aeruginosa and P. mendocina in the gamma subclass of the Proteobacteria. In contrast, the clcD and clcA gene sequences were identical on strain B13 and these two isolates, indicating that the 3‐chlorobenzoate‐degrading genes were transferred from strain B13 to these isolates. What cannot be established is when this transfer occurred.
Microsatellite variation in grey seals (Halichoerus grypus) shows evidence of genetic differentiation between two British breeding colonies
Authors: ALLEN P. J. , AMOS W. , POMEROY P. P. , TWISS S. D.
Published: April 2008

Eight highly variable microsatellite loci were used to examine the genetic variability and differentiation of grey seals (Halichoerus grypus) at two widely spaced British breeding colonies. Samples were collected from adults and pups on the island of North Rona, off the north‐west coast of Scotland, and on the Isle of May, situated at the mouth of the Firth of Forth on the east coast Highly significant differences in allele frequencies between these two sites were found for all eight loci, indicating considerable genetic differentiation. Thus, although grey seals are known to range over very large areas outside the breeding season, site fidelity of adults and philopatry of pups for these breeding colonies must be sufficiently common to have effects, through genetic drift, at the sub‐population level. Migration rate was estimated using Wrighf's fixation index (FST), Slatkin's private alleles model and the new statistic, RST, which is analogous to (FST) but which takes into account the process of microsatellite mutation. An almost 8‐fold discrepancy between the values we obtained provides cautionary evidence that microsatellite loci may contravene one or more of the assumptions on which these methods are based.
A PCR test for avian malaria in Hawaiian birds
Authors: FELDMAN R. A. , FREED L. A. , CANN R. L.
Published: April 2008

The decline of native Hawaiian forest birds since European contact is attributed to factors ranging from habitat destruction to interactions with introduced species. Remaining populations of Hawaiian honeycreepers (Fringillidae: Drepanidinae) are most abundant and diverse in high elevation refuges above the normal range of disease‐carrying mosquitoes. Challenge experiments suggest that honeycreepers are highly susceptible to avian malaria (Plasmodium sp.) but resistance exists in some species. In order to detect low levels of malarial infection and quantify prevalence of Plasmodium in high elevation natural populations of Hawaiian birds, a polymerase chain reaction (PCR) based diagnostic test was developed that identifies rRNA genes of Plasmodium in avian blood samples. Quantitative competitive PCR (QC‐PCR) experiments indicate that the detection limit of our test is an order of magnitude greater than that reported for human malaria DNA blot tests. Compared with standard histological methods, the PCR test detected a higher prevalence of diseased birds at mid‐elevations. Malaria was detected in three species of native birds living in a high elevation wildlife refuge on the island of Hawaii and in four species from Maui. Our results show that avian malaria is more widespread in Hawaiian forests than previously thought, a finding that has important conservation implications for these threatened species.
Genetic differentiation in Fomitopsis pinicola (Schwarts: Fr.) Karst studied by means of arbitrary primed‐PCR
Authors: HÖGBERG N. , STENLID J. , KARLSSON J. O.
Published: April 2008

Generic variation within and among one Finnish and three Swedish populations of Fomitopsis pinicola (Schwarts: Fr.) Karst. were studied by amplifying DNA from hap‐loid isolates originating from single spore cultures using two arbitrary primers. Analysis offspring from single fruit bodies revealed only three pairs of codominant alleles among 42 variable genetic markers, the remaining 38 segregated independently. Genetic similarity was measured in terms of Euclidean distance. Individuals in the Finnish population tended to form a distinct cluster in the principal component analysis. Variation within and among populations/regions was partitioned by Analysis of Molecular Variance — AMOVA. Within population variation accounted for 91.6% of the total genetic variation. The remaining 7.68% was accounted for by variation between the Finnish population and each of the three Swedish ones. Variation among the Swedish populations accounted for only 0.72% of the total variation. Wright's Fst was 0.17 for all four populations and 0.13 for the three Swedish populations. These relatively low values indicate that there is gene flow among all populations or that they are derived from a common ancestral population. The observed pattern of genetic variation is probably the result of effective spore dispersal and the continuous distribution of this common early successional species.
The effects on genetic variability following a recent colonization event: the Australian sheep blowfly, Lucilia cuprina arrives in New Zealand
Authors: GLEESON D. M.
Published: April 2008

The Australian sheep blowfly, Lucilia cuprina, was first identified in New Zealand in 1988 and is now found to have spread throughout many sheep‐farming regions. L. cuprina is estimated to have been present in New Zealand < 20 years, while in Australia L. cuprina has been estimated present > 100 years. The aim of this study was to determine the genetic effects of colonization of L. cuprina and to compare populations of L. cuprina from these two countries in terms of genetic variability and differentiation. Allozyme electrophoresis was used which revealed variability at six loci. 1680 blowflies were examined from 56 sites throughout L. cuprina's range in both countries. Genetic variability at each locus in terms of allele composition was found to be high and genetic differentiation varied considerably in New Zealand in comparison to Australia. Temporal sampling in New Zealand suggests seasonal fluctuations of population size in the recently colonized region of the South Island.
Intraspecific phylogeography of the gopher tortoise, Gopherus polyphemus: RFLP analysis of amplified mtDNA segments
Authors: OSENTOSKI M. F. , LAMB T.
Published: April 2008

The slow rate of mtDNA evolution in turtles poses a limitation on the levels of intraspecific variation detectable by conventional restriction fragment surveys. We examined mtDNA variation in the gopher tortoise (Gopherus polyphemus) using an alternative restriction assay, one in which PCR‐amplified segments of the mitochondrial genome were digested with tetranucleotide‐site endonucleases. Restriction fragment polymorphisms representing four amplified regions were analysed to evaluate population genetic structure among 112 tortoises throughout the species' range. Thirty‐six haplotypes were identified, and three major geographical assemblages (Eastern, Western, and Mid‐Florida) were resolved by UPGMA and parsimony analyses. Eastern and Western assemblages abut near the Apalachicola drainage, whereas the Mid‐Florida assemblage appears restricted to the Brooksville Ridge. The Eastern/Western assemblage boundary is remarkably congruent with phylogeographic profiles for eight additional species from the southeastern U.S., representing both freshwater and terrestrial realms.
Using chloroplast DNA to trace postglacial migration routes of oaks into Britain
Authors: FERRIS C. , OLIVER R. P. , DAVY A. J. , HEWITT G. M.
Published: April 2008

Postglacial migration is a major factor responsible for the patterns of genetic variation we see in natural populations. Fossil pollen data indicate that early postglacial colonists such as oak, were able to take both western and eastern migration routes into Britain. Analysis at a finer level is now permitted by the use of modern molecular techniques. A 13‐bp duplication in the chloroplast tRNALeul intron occurs in natural populations of East Anglian oaks, but is not found in other parts of Britain or from mainland Europe. The distribution of this marker suggests that the mutation occurred either in southern England, or during migration from the mainland, and became fixed in a source population from which East Anglia was colonized. Planting of non‐native trees for roadside boundaries and in the grounds of old houses and estates, explains the absence of the marker from some East Anglian oaks.
Handicapped males and extrapair paternity in pied flycatchers: a study using microsatellite markers
Authors: ELLEGREN H. , LIFJELD J. T. , SLAGSVOLD T. , PRIMMER C. R.
Published: April 2008

We report an attempt to induce extrapair copulations and fertilizations in a species with a low intensity of sperm competition, the pied flycatcher Ficedula hypoleuca. Shortly after pair formation males were made less attractive to females by removing certain wing and tail feathers. Earlier research has shown that this manipulation reduces a male's pairing success. The idea was to test whether females mated to such males (N= 9) were more likely to obtain extrapair fertilizations than females mated to unmanipulated controls (N= 9). Paternity testing was carried out on all 98 young in the 18 broods, using a set of six microsatellite markers isolated from the species. Extrapair fertilizations were revealed in only three (17%) broods; two broods of handicapped males and one of a control male. A total of seven (7%) offspring were not genetically related to their putative father, a level which agrees well with results of other studies of this and other populations. We conclude that there was no evidence to suggest that the fertilization pattern was altered by the experimental manipulation. One reason for the lack of response could be that female mate choice in this species is based on male phenotypic, and not genotypic, quality.
Mitochondrial DNA variation and population genetic structure of the northern redbelly dace (Phoxinus eos)
Authors: TOLINE C. A. , BAKER A. J.
Published: April 2008

Two sections of the control region and the genes coding for NADH dehydrogenase sub‐units 5 and 6 (ND‐5/6) of mitochondrial DNA (mtDNA) were amplified from Phoxinus eos with the polymerase chain reaction. Both sections of the control region were sequenced directly while the ND‐5/6 fragment was sequenced in from each end only. Additionally, the entire ND‐5/6 fragment was examined for sequence variation using RFLP analysis. No sequence variation was detected in the control region among 70 individuals sampled from 18 populations across three Ontario regions (Spanish River, Madawaska R. and Cataraqui R.). To examine ND‐5/6 variation, a total of 75 individuals were sampled from five populations representing two of the three regions (Madawaska River and Cataraqui R.). Six haplotypes were detected by direct sequencing and four by RFLP analysis. Estimates of population subdivision from RFLP data, sequence analysis, and the two data sets combined for the ND‐5/6 fragment, suggest that gene flow is restricted within and between regions. However, estimates of sequence divergence for both sequence and RFLP analysis of this fragment suggested that populations were either founded by already differentiated populations or that populations were founded by a single stock and divergence between regions occurred prior to isolation of populations within regions. These estimates of population structure are much greater than those obtained from allozyme analysis. Additionally, high levels of heterozygosity in nuclear DNA, but low mtDNA diversity suggests that populations have experienced reductions in population size sufficient to reduce only mtDNA variation. Random lineage extinction and limited time for the accumulation of new mutations are likely responsible for low levels of mtDNA variation in ND‐5/6 and the control region, while functional constraints may limit variation more than expected in the control region in dace and other fishes.
Site directed chromosomal marking of a fluorescent pseudomonad isolated from the phytosphere of sugar beet; stability and potential for marker gene transfer.
Authors: BAILEY M. J. , LILLEY A. K. , THOMPSON I. P. , RAINEY P. B. , ELLIS R. J.
Published: April 2008

A plasmid‐free, non‐pathogenic, ribosomal RNA group 1 fluorescent pseudomonad, Pseudomonas fluorescens SBW25, was selected from the microflora of sugar beet (Beta vulgaris) and modified to contain constitutively expressed marker genes. By site directed homologous recombination a KX cassette [kanamycin resistance (kanτ) and catechol 2,3 dioxygenase (xylE)] and a ZY cassette [lactose utilization (lacZY, β‐galactosidase, lactose permease)] were introduced at least 1 Mbp apart on the 6.6 Mbp bacterial chromosome. Separate sites were selected to provide sensitive detection methods and allow assessments of marker gene stability of the genetically modified micro‐organism (GMM), SBW25EeZY6KX, when it colonized the leaves and roots of sugar beet plants following seed inoculation.
Genetic monogamy in single‐male groups of acorn woodpeckers, Melanerpes formicivorus
Authors: DICKINSON J. , HAYDOCK J. , KOENIG W. , STANBACK M. , PITELKA F.
Published: April 2008

Acorn woodpeckers have one of the most complex social systems of any bird species. Breeding units range in size from monogamous pairs to groups of 15 birds that include multiple breeding males and females as well as nonreproductive helpers‐at‐the‐nest. Groups form when young remain at their natal nest to help their parents breed or when single‐sex coalitions of siblings disperse to fill a reproductive vacancy on another territory. Plural breeding and helping behaviour are thought to be favoured through indirect fitness benefits for individuals that would otherwise be unable to breed due to a shortage of reproductive vacancies on territories with acorn stores. We report the results of multi‐locus DNA fingerprinting of 51 offspring from 18 nests of 16 socially monogamous pairs of acorn woodpeckers. If socially monogamous females mate outside the pair‐bond, indirect fitness benefits for cobreeders and helpers will be significantly reduced. Monogamous pairs accounted for all but one of the 51 offspring we tested; the single exception was apparently sired by the putative father, but the putative mother was excluded from maternity. Our results indicate that individuals remaining on their natal territories as helpers are generally the genetic offspring of the pair they help. They also suggest that single‐sex coalitions offspring dispersing together from nests of socially monogamous pairs will be full‐siblings.
Molecular identification of six species of scale insects (Quadraspidiotus sp.) by RAPD‐PCR: assessing the field‐specificity of pheromone traps
Authors: FREY J. E. , FREY B.
Published: April 2008

The San‐José Scale Quadraspidiotus perniciosus (SJS) is a quarantine pest in Switzerland. Its occurrence is monitored by trapping males on glue traps treated with artificial female sex pheromone that is supposed to be species‐specific. However, large numbers of males were caught in locations where, for ecological reasons, this species was not expected to occur, suggesting that the pheromone is attractive for one or several otrter scale species. Because no morphological species determination key is described for males of this genus, it was not possible to test this hypothesis until now. We used randomly amplified polymorphic DNA (RAPD‐PCR) to establish a molecular identification key for six European Quadraspidiotus species. Because the glue used on the traps proved to be an excellent preservative, this key enabled us to identify males caught on pheromone traps in the field and to assess the species‐specificity of the SJS‐pheromone.
Field application of the polymerase chain reaction (PCR) to the detection and characterization of trypanosomes in Glossina longipalpis (Diptera: Glossinidae) in Côte d'Ivoire
Authors: SOLANO P. , ARGIRO L. , REIFENBERG J. M. , YAO YAO , DUVALLET G.
Published: April 2008

The Polymerase Chain Reaction (PCR) technique was used for the identification of natural trypanosome infections in Glossina longipalpis (Diptera: Glossinidae) in Côte d'Ivoire. A total number of 139 flies were examined microscopically for the presence of trypanosomes. Out of them 50 were detected positive and were subsequently prepared for the PCR using primers specific for Trypanosoma (Nannomonas) congolense of Savannah, Riverine‐Forest, Kilifi, and Tsavo types, T. (N.) simiae, T. (Duttonella) vivax and Trypanozoon. Almost 90% of the infections detected by the PCR were attributed to Nannomonas, especially T. congolense Savannah and Riverine‐Forest types, with many infections in which both of these two types were present T. simiae and T. vivax were also detected in some flies. The sequence specificity of the PCR products was confirmed by hybridization with parasite‐type specific DNA probes. Differences between parasitological and PCR results are discussed.