Microsatellites can be misleading: an empirical and simulation study.

It has been long recognized that highly polymorphic genetic markers can lead to underestimation of divergence between populations when migration is low. Microsatellite loci, which are characterized by extremely high mutation rates, are particularly likely to be affected. Here, we report genetic differentiation estimates in a contact zone between two chromosome races of the common shrew (Sorex araneus), based on 10 autosomal microsatellites, a newly developed Y-chromosome microsatellite, and mitochondrial DNA. These results are compared to previous data on proteins and karyotypes. Estimates of genetic differentiation based on F- and R-statistics are much lower for autosomal microsatellites than for all other genetic markers. We show by simulations that this discrepancy stems mainly from the high mutation rate of microsatellite markers for F-statistics and from deviations from a single-step mutation model for R-statistics. The sex-linked genetic markers show that all gene exchange between races is mediated by females. The absence of male-mediated gene flow most likely results from male hybrid sterility.

Methylation profile of TP53 regulatory pathway and mtDNA alterations in breast cancer patients lacking TP53 mutations.

The present study investigated promoter hypermethylation of TP53 regulatory pathways providing a potential link between epigenetic changes and mitochondrial DNA (mtDNA) alterations in breast cancer patients lacking a TP53 mutation. The possibility of using the cancer-specific alterations in serum samples as a blood-based test was also explored. Triple-matched samples (cancerous tissues, matched adjacent normal tissues and serum samples) from breast cancer patients were screened for TP53 mutations, and the promoter methylation profile of P14(ARF), MDM2, TP53 and PTEN genes was analyzed as well as mtDNA alterations, including D-loop mutations and mtDNA content. In the studied cohort, no mutation was found in TP53 (DNA-binding domain). Comparison of P14(ARF) and PTEN methylation patterns showed significant hypermethylation levels in tumor tissues (P < 0.05 and <0.01, respectively) whereas the TP53 tumor suppressor gene was not hypermethylated (P < 0.511). The proportion of PTEN methylation was significantly higher in serum than in the normal tissues and it has a significant correlation to tumor tissues (P < 0.05). mtDNA analysis revealed 36.36% somatic and 90.91% germline mutations in the D-loop region and also significant mtDNA depletion in tumor tissues (P < 0.01). In addition, the mtDNA content in matched serum was significantly lower than in the normal tissues (P < 0.05). These data can provide an insight into the management of a therapeutic approach based on the reversal of epigenetic silencing of the crucial genes involved in regulatory pathways of the tumor suppressor TP53. Additionally, release of significant aberrant methylated PTEN in matched serum samples might represent a promising biomarker for breast cancer.

Phylogeographical footprints of the Strait of Gibraltar and Quaternary climatic fluctuations in the western Mediterranean: a case study with the greater white-toothed shrew, Crocidura russula (Mammalia: Soricidae).

We used mitochondrial cyt b sequences to investigate the phylogenetic relationships of Crocidura russula (sensu lato) populations across the Strait of Gibraltar, western Europe, Maghreb, and the Mediterranean and Atlantic islands. This revealed very low genetic divergence between European and Moroccan populations. The application of a molecular clock previously calibrated for shrews suggested that the separation of European from Moroccan lineages occurred less than 60 000 bp, which is at least 5 million years (Myr) after the reopening of the Strait of Gibraltar. This means that an overwater dispersal event was responsible for the observed phylogeographical structure. In contrast, genetic analyses revealed that Moroccan populations were highly distinct from Tunisian ones. According to the molecular clock, these populations separated about 2.2 million years ago (Ma), a time marked by sharp alternations of dry and humid climates in the Maghreb. The populations of the Mediterranean islands Ibiza, Pantelleria, and Sardinia were founded from Tunisian populations by overwater dispersal. In conclusion, overwater dispersal across the Strait of Gibraltar, probably assisted by humans, is possible for small terrestrial vertebrates. Moreover, as in Europe, Quaternary climatic fluctuations had a major effect on the phylogeographical structure of the Maghreb biota.

Translocation of the yeast dolichol-phosphate-mannose synthase into microsomal membranes.

Dolichol-phosphate-mannose synthase catalyzes the formation of Dolichol-phosphate-mannose from Dolichol-phosphate and GDP-mannose. Analysis of the primary amino acid sequence of the yeast enzyme predicts a luminal orientation of the enzyme in the endoplasmic reticulum. We analysed the translocation of the Dolichol-phosphate-mannose synthase into dog pancreatic microsomal membranes: resistance to proteolytic attack provides evidence of its luminal orientation and asks for a reevaluation of the topology of the reaction.

Origine des populations de rainette verte (Hyla spp.) de l'ouest de la Suisse

DNA-based techniques are important tools for species assignment, in particular when identification with morphological criteria is difficult. The aim of this study was to genetically determine the species identity of tree frogs (Hyla spp.) populations from western and northern Switzerland (Swiss Plateau), this area being frequently subjected to introductions of species or sub-species from south of the Alps. We sequenced 261 base pairs of the mitochondrial DNA cytochrome b gene from 24 samples of tree frogs from the Swiss Plateau, Ticino (southern Switzerland) and the Dombes region (Ain, France), and compared them with homologous sequences retrieved from DNA databases. The phylogenetic analyses revealed two distinct clades. The first one is represented by samples of Green tree frog (Hyla arborea) from the Swiss Plateau, France, Germany and Greece, confirming the current knowledge about the species' distribution. The second clade includes samples belonging to the Italian tree frog (Hyla intermedia) from south of the Alps (Ticino and Italy), and unexpectedly from the Grangettes site in western Switzerland. These results suggest the introduction of the Italian tree frog H. intermedia north of the Alps, and raise questions about the management of the Grangettes protected area.

Low gene copy number shows that arbuscular mycorrhizal fungi inherit genetically different nuclei.

Arbuscular mycorrhizal fungi (AMF) are ancient asexually reproducing organisms that form symbioses with the majority of plant species, improving plant nutrition and promoting plant diversity. Little is known about the evolution or organization of the genomes of any eukaryotic symbiont or ancient asexual organism. Direct evidence shows that one AMF species is heterokaryotic; that is, containing populations of genetically different nuclei. It has been suggested, however, that the genetic variation passed from generation to generation in AMF is simply due to multiple chromosome sets (that is, high ploidy). Here we show that previously documented genetic variation in Pol-like sequences, which are passed from generation to generation, cannot be due to either high ploidy or repeated gene duplications. Our results provide the clearest evidence so far for substantial genetic differences among nuclei in AMF. We also show that even AMF with a very large nuclear DNA content are haploid. An underlying principle of evolutionary theory is that an individual passes on one or half of its genome to each of its progeny. The coexistence of a population of many genomes in AMF and their transfer to subsequent generations, therefore, has far-reaching consequences for understanding genome evolution.

A vertebrate reproductive system involving three ploidy levels : hybrid origin of triploids in a contact zone of diploid and tetraploid green toads (Bufo viridis subgroup)

The rise and consequences of polyploidy in vertebrates, whose origin was associated with genome duplications, may be best studied in natural diploid and polyploid populations. In a diploid/tetraploid (2n/4n) geographic contact zone of Palearctic green toads in northern Kyrgyzstan, we examine 4ns and triploids (3n) of unknown genetic composition and origins. Using mitochondrial and nuclear sequence, and nuclear microsatellite markers in 84 individuals, we show that 4n (Bufo pewzowi) are allopolyploids, with a geographically proximate 2n species (B. turanensis) being their maternal ancestor and their paternal ancestor as yet unidentified. Local 3n forms arise through hybridization. Adult 3n mature males (B. turanensis mtDNA) have 2n mothers and 4n fathers, but seem distinguishable by nuclear profiles from partly aneuploid 3n tadpoles (with B. pewzowi mtDNA). These observations suggest multiple pathways to the formation of triploids in the contact zone, involving both reciprocal origins. To explain the phenomena in the system, we favor a hypothesis where 3n males (with B. turanensis mtDNA) backcross with 4n and 2n females. Together with previous studies of a separately evolved, sexually reproducing 3n lineage, these observations reveal complex reproductive interactions among toads of different ploidy levels and multiple pathways to the evolution of polyploid lineages.

Genes associated with retinitis pigmentosa and allied diseases are frequently mutated in the general population.

BACKGROUND: Retinitis pigmentosa and other hereditary retinal degenerations (HRD) are rare genetic diseases leading to progressive blindness. Recessive HRD are caused by mutations in more than 100 different genes. Laws of population genetics predict that, on a purely theoretical ground, such a high number of genes should translate into an extremely elevated frequency of unaffected carriers of mutations. In this study we estimate the proportion of these individuals within the general population, via the analyses of data from whole-genome sequencing. METHODOLOGY/PRINCIPAL FINDINGS: We screened complete and high-quality genome sequences from 46 control individuals from various world populations for HRD mutations, using bioinformatic tools developed in-house. All mutations detected in silico were validated by Sanger sequencing. We identified clear-cut, null recessive HRD mutations in 10 out of the 46 unaffected individuals analyzed (∼22%). CONCLUSIONS/SIGNIFICANCE: Based on our data, approximately one in 4-5 individuals from the general population may be a carrier of null mutations that are responsible for HRD. This would be the highest mutation carrier frequency so far measured for a class of Mendelian disorders, especially considering that missenses and other forms of pathogenic changes were not included in our assessment. Among other things, our results indicate that the risk for a consanguineous couple of generating a child with a blinding disease is particularly high, compared to other genetic conditions.

Extrachromosomal nucleic acids in bovine babesia

Two kinds of small extrachromosomal nucleic acid elements were found in the bovine babesias, Babesia bovis and B. bigemina. One element with an apparent size of 5.5 kilobase pairs (kbp) is a double stranded RNA related to virus like particles. Another molecule is a double stranded DNA with a molecular size of about 6.2 kbp. Southern blot comparison of restriction DNA fragments of the latter molecule, which is present in both B. bovis and B. bigemina is described.

Repeated unidirectional introgression towards Populus balsamifera in contact zones of exotic and native poplars.

As the evolutionary significance of hybridization is largely dictated by its extent beyond the first generation, we broadly surveyed patterns of introgression across a sympatric zone of two native poplars (Populus balsamifera, Populus deltoides) in Quebec, Canada within which European exotic Populus nigra and its hybrids have been extensively planted since the 1800s. Single nucleotide polymorphisms (SNPs) that appeared fixed within each species were characterized by DNA-sequencing pools of pure individuals. Thirty-five of these diagnostic SNPs were employed in a high-throughput assay that genotyped 635 trees of different age classes, sampled from 15 sites with various degrees of anthropogenic disturbance. The degree of admixture within sampled trees was then assessed through Bayesian clustering of genotypes. Hybrids were present in seven of the populations, with 2.4% of all sampled trees showing spontaneous admixture. Sites with hybrids were significantly more disturbed than pure stands, while hybrids comprised both immature juveniles and trees of reproductive age. All three possible F1s were detected. Advanced-generation hybrids were consistently biased towards P. balsamifera regardless of whether hybridization had occurred with P. deltoides or P. nigra. Gene exchange between P. deltoides and P. nigra was not detected beyond the F1 generation; however, detection of a trihybrid demonstrates that even this apparent reproductive isolation does not necessarily result in an evolutionary dead end. Collectively, results demonstrate the natural fertility of hybrid poplars and suggest that introduced genes could potentially affect the genetic integrity of native trees, similar to that arising from introgression between natives.

The worldwide expansion of the Argentine ant

Aim The aim of this study was to determine the number of successful establishments of the invasive Argentine ant outside native range and to see whether introduced supercolonies have resulted from single or multiple introductions. We also compared the genetic diversity of native versus introduced supercolonies to assess the size of the propagules (i.e. the number of founding individuals) at the origin of the introduced supercolonies. Location Global. Methods We used mitochondrial DNA (mtDNA) markers and microsatellite loci to study 39 supercolonies of the Argentine ant Linepithema humile covering both the native (n = 25) and introduced range (n = 14). Results Data from three mitochondrial genes and 13 nuclear microsatellites suggest that the introduced supercolonies studied originated from at least seven founding events out of the native area in Argentina (primary introductions). The distribution of mtDNA haplotypes also suggests that supercolonies in the introduced range each derive from a single source supercolony and that one of these source supercolonies has been particularly successful, being the basis of many introduced populations spread across the world. Comparison of the genetic diversity of supercolonies based on the five most diverse loci also revealed that native and introduced supercolonies have greatly overlapping ranges of diversity, although the genetic diversity is on average less in introduced than in native supercolonies. Main conclusions Both primary introductions (from the native range) and secondary introductions (from sites with established invasive supercolonies) were important in the global expansion of the Argentine ant. In combination with the similar social organization of colonies in the native and introduced range, this indicates that invasiveness did not evolve recently as a unique and historically contingent event (e.g. reduction of genetic diversity) in this species. Rather, native L. humile supercolonies have characteristics that make them pre-adapted to invade new - and in particular disturbed - habitats when given the opportunity. These results have important implications with regard to possible strategies to be used to control invasive ants.

PIDD orchestrates translesion DNA synthesis in response to UV irradiation.

PIDD has been implicated in survival and apoptotic pathways in response to DNA damage, and a role for PIDD was recently identified in non-homologous end-joining (NHEJ) repair induced by γ-irradiation. Here, we present an interaction of PIDD with PCNA, first identified in a proteomics screen. PCNA has essential functions in DNA replication and repair following UV irradiation. Translesion synthesis (TLS) is a process that prevents UV irradiation-induced replication blockage and is characterized by PCNA monoubiquitination and interaction with the TLS polymerase eta (polη). Both of these processes are inhibited by p21. We report that PIDD modulates p21-PCNA dissociation, and promotes PCNA monoubiquitination and interaction with polη in response to UV irradiation. Furthermore, PIDD deficiency leads to a defect in TLS that is associated, both in vitro and in vivo, with cellular sensitization to UV-induced apoptosis. Thus, PIDD performs key functions upon UV irradiation, including TLS, NHEJ, NF-κB activation and cell death.

Species delimitation: A case study in a problematic ant taxon

Species delimitation has been invigorated as a discipline in systematics by an influx of new character sets, analytical methods, and conceptual advances. We use genetic data from 68 markers, combined with distributional, bioclimatic, and coloration information, to hypothesize boundaries of evolutionarily independent lineages (species) within the widespread and highly variable nominal fire ant species Solenopsis saevissima, a member of a species group containing invasive pests as well as species that are models for ecological and evolutionary research. Our integrated approach uses diverse methods of analysis to sequentially test whether populations meet specific operational criteria (contingent properties) for candidacy as morphologically cryptic species, including genetic clustering, monophyly, reproductive isolation, and occupation of distinctive niche space. We hypothesize that nominal S. saevissima comprises at least 4-6 previously unrecognized species, including several pairs whose parapatric distributions implicate the development of intrinsic premating or postmating barriers to gene flow. Our genetic data further suggest that regional genetic differentiation in S. saevissima has been influenced by hybridization with other nominal species occurring in sympatry or parapatry, including the quite distantly related Solenopsis geminata. The results of this study illustrate the importance of employing different classes of genetic data (coding and noncoding regions and nuclear and mitochondrial DNA [mtDNA] markers), different methods of genetic data analysis (tree-based and non-tree based methods), and different sources of data (genetic, morphological, and ecological data) to explicitly test various operational criteria for species boundaries in clades of recently diverged lineages, while warning against over reliance on any single data type (e.g., mtDNA sequence variation) when drawing inferences.

Genetic identity of the critically endangered Wimmer's shrew Crocidura wimmeri

Coastal primary rainforests have suffered damage in Côte d'Ivoire as a result of a lack of protection and urban pressures. Consequently, the highly endemic and critically endangered Wimmer's shrew, Crocidura wimmeri, known only from its type locality, Adiopodoumé, near Abidjan, was considered to have been extinct since 1976. Shrew species assignment is often problematic because of strong phenotypic similarities among many species. The phylogenetic position of C. wimmeri within the African Crocidura species should thus be clarified. In light of its recent rediscovery in the nearby small Banco National Park (34 km2), we investigated the genetic identity of seven specimens of C. wimmeri, based on 1020 bp of the mitochondrial DNA cytochrome b gene compared to other species sampled in the same region and published sequences from GenBank. Crocidura wimmeri formed a well-defined clade, the closest-related species being Crocidura sp., with a distance of 9.3%, a yet unknown species from Taï and Ziama forests. These results thus confirmed the validity of this species. This genetic characterization not only contributes to our knowledge of the evolution of West African shrews, but also may help in the discovery of additional populations of this critically endangered species.

Genetic variability and identification of the intermediate snail hosts of Schistosoma mansoni

Studies based on shell or reproductive organ morphology and genetic considerations suggest extensive intraspecific variation in Biomphalaria snails. The high variability at the morphological and genetic levels, as well as the small size of some specimens and similarities between species complicate the correct identification of these snails. Here we review our work using methods based on polymerase chain reaction (PCR) amplification for analysis of genetic variation and identification of Biomphalaria snails from Brazil, Argentina, Uruguay and Paraguay. Arbitrarily primed-PCR revealed that the genome of B. glabrata exihibits a remarkable degree of intraespecific polymorphism. Low stringency-PCR using primers for 18S rRNA permited the identification of B. glabrata, B. tenagophila and B. occidentalis. The study of individuals obtained from geographically distinct populations exhibits significant intraspecific DNA polymorphism, however specimens from the same species, exhibit some species specific LSPs. We also showed that PCR-restriction fragment of length polymorphism of the internal transcribed spacer region of Biomphalaria rDNA, using DdeI permits the differentiation of the three intermediate hosts of Schistosoma mansoni. The molecular biological techniques used in our studies are very useful for the generation of new knowledge concerning the systematics and population genetics of Biomphalaria snails.