Ana Domínguez

An evaluation of RAPD fragment reproducibility and nature

Random amplified polymorphic DNA (RAPD) fragment reproducibility was assayed in three animal species: red deer (Cervus elaphus), wild boar (Sus scrofa) and fruit fly (Drosophila melanogaster). Ten 10‐mer primers (Operon) were tested in two replicate reactions per individual under different stringency conditions (annealing temperatures of 35 °C or 45 °C). Two estimates were generated from the data: autosimilarity, which tests the reproducibility of overall banding patterns, and band repeatability, which tests the reproducibility of specific bands. Autosimilarity (the similarity of individuals with themselves) was lower than 1 for all three species ranging between values of 0.66 for Drosophila at 45 °C and 0.88 for wild boar at 35 °C. Band repeatability was estimated as the proportion of individuals showing homologous bands in both replicates. The fraction of repeatable bands was 23% for deer, 36% for boar and 26% for fruit fly, all at an annealing temperature of 35 °C. Raising the annealing temperature did not improve repeatability. Phage lambda DNA was subjected to amplification and the pattern of bands compared with theoretical expectations based on nucleotide sequence. Observed fragments could not be related to expected ones, even if a 2bp mismatch is allowed. Therefore, the nature of genetic variation uncovered by the RAPD method is unclear. These data demonstrate that prudence should guide inferences about population structure and nucleotide divergence based on RAPD markers.

Phylogeography of chamois (Rupicapra spp.) inferred from microsatellites.

Evolutionary relationships among populations of chamois (Rupicapra spp.) across their current range from the Caucasus to the Cantabrian Mountains were investigated. The allelic variation in 23 microsatellite loci was assessed in eight geographical populations, recognised as subspecies of the two closely related species R. pyrenaica and R. rupicapra. Analysis of variance in allele frequencies (Fst, statistics) and in repeat numbers (Rst, statistics) showed these data to be highly structured. Two genetic distances between pairs of populations, Ds and (deltamu)(2), were computed and phylogenetic trees were constructed. Similar patterns were produced by the different statistics. All trees indicate a deep divergence between the two recognised species, which is compatible with archaeological data that place their split in the Riss-Würm interglacial period. Genetic distances between pairs of populations are highly correlated with geographical distance. This suggests that the history of the genus during Pleistocene glacial-interglacial periods was dominated by expansions and contractions within limited geographic regions, leading to alternate contact and isolation of contiguous populations. In addition, the alpine barrier has played a substantial role in West-East differentiation.

Estimation of microsatellite mutation rates in Drosophila melanogaster

Microsatellite mutations were studied in a set of 175 mutation accumulation lines, all of them independently derived from a completely homozygous population of Drosophila melanogaster and maintained under strong inbreeding during 80 generations. We assayed 28 microsatellites and detected two mutations. One mutation consisted of a single addition of a dinucleotide repeat and the other was a deletion of five trinucleotide repeats. The average mutation rate was 5.1 x 10(-6), in full agreement with previous estimates from two different sets of mutation accumulation lines.

Integrating phylogeographic patterns of microsatellite and mtDNA divergence to infer the evolutionary history of chamois (genus Rupicapra).

BackgroundThe chamois, distributed over most of the medium to high altitude mountain ranges of southern Eurasia, provides an excellent model for exploring the effects of historical and evolutionary events on diversification. Populations have been grouped into two species, Rupicapra pyrenaica from southwestern Europe and R. rupicapra from eastern Europe. However, a previous study of cytochrome b revealed that the two proposed species were non-monophyletic. The reconstruction of phylogenetic relationships between animal species often depends on the markers studied. To further elucidate the evolutionary history of chamois, we extended earlier studies by analysing DNA sequences of four mitochondrial regions (ND1, 12S, tRNApro and Control Region) and microsatellites (20 loci) to include all subspecies and cover its entire distribution range.ResultsWe found discordant microsatellite (μsat) and mitochondrial (mt) DNA phylogenies. Mitochondrial phylogenies form three clades, West, Central and East (mtW, mtC and mtE), at variance with taxonomic classification. Our divergence age estimates indicate an initial separation into branches mtW-mtC and mtE 1.7 million years ago (mya), in the late Pliocene-early Pleistocene, quickly followed by the split of clades mtW and mtC. Clade mtW contains haplotypes from the Iberian peninsula and the western Alps, Clade mtC includes haplotypes from the Apennines and the Massif of Chartreuse and Clade mtE comprises populations to the east of the Alps. Divergence among populations within these three major clades is recent (< 0.5 mya). New microsatellite multilocus genotypes added to previously published data revealed differences between every pair of subspecies, forming three well defined groups (μsatW, μsatC and μsatE) also with a strong geographic signature. Grouping does not correspond with the mitochondrial lineages but is closer to morphology and taxonomic classification. Recent drastic reductions in population size can be noted for the subspecies ornata as an extremely low diversity.ConclusionsThe phylogeographic patterns for mtDNA and microsatellites suggest an evolutionary history with limited range contractions and expansions during the Quaternary period and reflect a major effect of the Alpine barrier on west-east differentiation. The contrasting phylogenies for mtDNA and microsatellites indicate events of hybridization among highly divergent lineages in the central area of distribution. Our study points to the importance of reticulate evolution, with periods of isolation and reduction of population size followed by expansions and hybridizations, in the diversification at the level of close species or subspecies.

Practical Application of DNA Fingerprinting To Trace Beef

DNA fingerprinting allows the verification of conventional methods used to implement beef traceability. At any point along the supply chain, the identity of an animal or piece of meat can be checked by comparison of its DNA profile with an initial sample. Practical application of DNA fingerprinting to trace beef requires a choice of DNA markers as well as the optimization of sampling methods. This has been achieved as the result of collaboration between meat technicians and geneticists over a period of 4 years. The discrimination power of nine highly polymorphic microsatellite markers was evaluated. We propose that three markers (with a 0.001 probability that two individual profiles match by chance) are adequate for routine tests. Two key points along the production-commercialization chain where sampling must be systematic were defined: (i) the tagging of the calf (identity control) and (ii) after slaughter (slaughter control), before the animal loses its external appearance. The identity control was blood collected on a filter paper adapted to the ear tag; the slaughter control was the tagged ear itself. These constituted the control samples, which were archived with a code matching the individual tag number. Test samples were obtained on a random basis from live animals, carcasses, and pieces of meat at cutting halls and at the retail outlet and in cases when the verification of identity was needed. The DNA profiles of the test samples and the controls were then obtained and compared, to verify either an individual identity or the origin of a piece of meat from the stated animal.

RATES OF MOVEMENT OF TRANSPOSABLE ELEMENTS IN DROSOPHILA MELANOGASTER

Mobilization rates of nine families of transposable elements (P, hobo, FB, gypsy, 412, copia, blood, 297, andjockey) were estimated by using 182 lines. Lines were started from a completely isogenic population ofDrosophila melanogaster, carrying the markersepia as an indicator of possible contamination, and have been accumulating spontaneous mutations independently for 80 generations of brother-sister (or two double-first-cousin) matings. Transposable element movements have been analyzed in complete genomes by the Southern technique. Mobilization was a rare event, with an average rate of 10−5 per site per generation. The most active element wasFB. In contrast, the retroelementsgypsy andblood did not move at all. Most changes in restriction patterns were consistent with rearrangements rather than with true transposition. The euchromatic or heterochromatic location of elements was tested by comparing insertion patterns from adults and salivary glands. Certain putative rearrangements involved heterochromatic copies of the retroelements412, copia or297. Clustering of movement across families was observed, suggesting that movement of different families may be non-independent. An association between modified insertion patterns and mutant effects on quantitative traits shows that spontaneous transposition events cause continuous variation.

A comparative framework to infer landscape effects on population genetic structure: Are habitat suitability models effective in explaining gene flow?

ContextMost current methods to assess connectivity begin with landscape resistance maps. The prevailing resistance models are commonly based on expert opinion and, more recently, on a direct transformation of habitat suitability. However, habitat associations are not necessarily accurate indicators of dispersal, and thus may fail as a surrogate of resistance to movement. Genetic data can provide valuable insights in this respect.ObjectivesWe aim at directly comparing the utility of habitat suitability models for estimating landscape resistance versus other approaches based on actual connectivity data.MethodsWe develop a framework to compare landscape resistance models based on (1) a genetic-based multi model optimization and (2) a direct conversion of habitat suitability into landscape resistance. We applied this framework to the endangered brown bear in the Cantabrian Range (NW Spain).ResultsWe found that the genetic-based optimization produced a resistance model that was more related to species movement than were models produced by direct conversion of habitat suitability. Certain land cover types and transport infrastructures were restrictive factors for species occurrence, but did not appear to impede the brown bear movements that determined observed genetic structure.ConclusionsIn this study case, habitat suitability is not synonymous with permeability for dispersal, and does not seem to provide the best way to estimate actual landscape resistance. We highlight the general utility of this comparative approach to provide a comprehensive and practical assessment of factors involved in species movements, with the final aim of improving the initiatives to enhance landscape connectivity in conservation planning.

Cytochrome b Phylogeography of Chamois (Rupicapra spp.). Population Contractions, Expansions and Hybridizations Governed the Diversification of the Genus

The chamois provides an excellent model for exploring the effect of historical and evolutionary events on diversification. We investigate cytochrome b (cytb) sequences in the 10 recognized subspecies of Rupicapra classified within 2 species: Rupicapra pyrenaica, with the subspecies parva, pyrenaica, and ornata, and Rupicapra rupicapra, with cartusiana, rupicapra, tatrica, carpatica, balcanica, asiatica, and caucasica. A fragment of 349 bp of the cytb was sequenced in 189 individuals. We identified 3 cytb lineages: Clade West in Iberia and Western Alps; Clade Central in the Apennines and the Massif of Chartreuse; and Clade East present in populations to the east of the Alps. The 2 proposed species were polyphyletic; the clades West and Central are represented in both, whereas the Clade East is restricted to R. rupicapra. In contrast to the current systematic, cytb phylogenies suggest the classification of the 10 subspecies of chamois into a single species, R. rupicapra. Phylogeny and geographical distribution of the 3 lineages show the effects of limited latitudinal range expansions, contractions, and hybridizations among highly divergent lineages, along with a major role of the glacial ice sheets of the Alps and the Pyrenees as barriers to gene flow, on the diversification of extant taxa.

Evidence for improved connectivity between Cantabrian brown bear subpopulations

Abstract The brown bear (Ursus arctos) population in the Cantabrian Mountains of northwest Spain is among the most endangered bear populations worldwide. It is divided into 2 isolated and genetically differentiated subpopulations. We present evidence of recent male migration between the subpopulations based on genetic identification of hair and scats samples gathered between 2004 and 2007. Of 76 identified individuals, our analysis assigned 3 males sampled in the eastern subpopulation to the western subpopulation. As well, 1 male genetically belonging to the eastern subpopulation was repeatedly sampled along his way to the western subpopulation during April to November 2006 (a linear distance of 144 km). This bears path may help identify natural corridors, which could be improved through restoration management. In addition, we identified 2 genetically admixed individuals during 2008 in the Western limit of the eastern subpopulation range. Connectivity between subpopulations and gene flow appears to be improving after a long isolation.

Y-chromosome phylogeny in the evolutionary net of chamois (genus Rupicapra )

BackgroundThe chamois, distributed over most of the medium to high altitude mountain ranges of southern Eurasia, provides an excellent model for exploring the effects of historical and evolutionary events on diversification. Populations have been grouped into two species, Rupicapra pyrenaica from southwestern Europe and R. rupicapra from eastern Europe. The study of matrilineal mitochondrial DNA (mtDNA) and biparentally inherited microsatellites showed that the two species are paraphyletic and indicated alternate events of population contraction and dispersal-hybridization in the diversification of chamois. Here we investigate the pattern of variation of the Y-chromosome to obtain information on the patrilineal phylogenetic position of the genus Rupicapra and on the male-specific dispersal of chamois across Europe.ResultsWe analyzed the Y-chromosome of 87 males covering the distribution range of the Rupicapra genus. We sequenced a fragment of the SRY gene promoter and characterized the male specific microsatellites UMN2303 and SRYM18. The SRY promoter sequences of two samples of Barbary sheep (Ammotragus lervia) were also determined and compared with the sequences of Bovidae available in the GenBank. Phylogenetic analysis of the alignment showed the clustering of Rupicapra with Capra and the Ammotragus sequence obtained in this study, different from the previously reported sequence of Ammotragus which groups with Ovis. Within Rupicapra, the combined data define 10 Y-chromosome haplotypes forming two haplogroups, which concur with taxonomic classification, instead of the three clades formed for mtDNA and nuclear microsatellites. The variation shows a west-to-east geographical cline of ancestral to derived alleles.ConclusionsThe phylogeny of the SRY-promoter shows an association between Rupicapra and Capra. The position of Ammotragus needs a reinvestigation. The study of ancestral and derived characters in the Y-chromosome suggests that, contrary to the presumed Asian origin, the paternal lineage of chamois originated in the Mediterranean, most probably in the Iberian Peninsula, and dispersed eastwards through serial funding events during the glacial-interglacial cycles of the Quaternary. The diversity of Y-chromosomes in chamois is very low. The differences in patterns of variation among Y-chromosome, mtDNA and biparental microsatellites reflect the evolutionary characteristics of the different markers as well as the effects of sex-biased dispersal and species phylogeography.