Comparative phylogeography in a specific and obligate pollination antagonism.

In specific and obligate interactions the nature and abundance of a given species can have important effects on the survival and population dynamics of associated organisms. In a phylogeographic framework, we therefore expect that the fates of organisms interacting specifically are also tightly interrelated. Here we investigate such a scenario by analyzing the genetic structures of species interacting in an obligate plant-insect pollination lure-and-trap antagonism, involving Arum maculatum (Araceae) and its specific psychodid (Diptera) visitors Psychoda phalaenoides and Psycha grisescens. Because the interaction is asymmetric (i.e., only the plant depends on the insect), we expect the genetic structure of the plant to be related with the historical pollinator availability, yielding incongruent phylogeographic patterns between the interacting organisms.Using insect mtDNA sequences and plant AFLP genome fingerprinting, we inferred the large-scale phylogeographies of each species and the distribution of genetic diversities throughout the sampled range, and evaluated the congruence in their respective genetic structures using hierarchical analyses of molecular variances (AMOVA). Because the composition of pollinator species varies in Europe, we also examined its association with the spatial genetic structure of the plant.Our findings indicate that while the plant presents a spatially well-defined genetic structure, this is not the case in the insects. Patterns of genetic diversities also show dissimilar distributions among the three interacting species. Phylogeographic histories of the plant and its pollinating insects are thus not congruent, a result that would indicate that plant and insect lineages do not share the same glacial and postglacial histories. However, the genetic structure of the plant can, at least partially, be explained by the type of pollinators available at a regional scale. Differences in life-history traits of available pollinators might therefore have influenced the genetic structure of the plant, the dependent organism, in this antagonistic interaction.

Evolutionary and dispersal history of Eurasian house mice Mus musculus clarified by more extensive geographic sampling of mitochondrial DNA.

We examined the sequence variation of mitochondrial DNA control region and cytochrome b gene of the house mouse (Mus musculus sensu lato) drawn from ca. 200 localities, with 286 new samples drawn primarily from previously unsampled portions of their Eurasian distribution and with the objective of further clarifying evolutionary episodes of this species before and after the onset of human-mediated long-distance dispersals. Phylogenetic analysis of the expanded data detected five equally distinct clades, with geographic ranges of northern Eurasia (musculus, MUS), India and Southeast Asia (castaneus, CAS), Nepal (unspecified, NEP), western Europe (domesticus, DOM) and Yemen (gentilulus). Our results confirm previous suggestions of Southwestern Asia as the likely place of origin of M. musculus and the region of Iran, Afghanistan, Pakistan, and northern India, specifically as the ancestral homeland of CAS. The divergence of the subspecies lineages and of internal sublineage differentiation within CAS were estimated to be 0.37-0.47 and 0.14-0.23 million years ago (mya), respectively, assuming a split of M. musculus and Mus spretus at 1.7 mya. Of the four CAS sublineages detected, only one extends to eastern parts of India, Southeast Asia, Indonesia, Philippines, South China, Northeast China, Primorye, Sakhalin and Japan, implying a dramatic range expansion of CAS out of its homeland during an evolutionary short time, perhaps associated with the spread of agricultural practices. Multiple and non-coincident eastward dispersal events of MUS sublineages to distant geographic areas, such as northern China, Russia and Korea, are inferred, with the possibility of several different routes.

The genomic distribution of intraspecific and interspecific sequence divergence of human segmental duplications relative to human/chimpanzee chromosomal rearrangements

Background: It has been suggested that chromosomal rearrangements harbor the molecular footprint of the biological phenomena which they induce, in the form, for instance, of changes in the sequence divergence rates of linked genes. So far, all the studies of these potential associations have focused on the relationship between structural changes and the rates of evolution of single-copy DNA and have tried to exclude segmental duplications (SDs). This is paradoxical, since SDs are one of the primary forces driving the evolution of structure and function in our genomes and have been linked not only with novel genes acquiring new functions, but also with overall higher DNA sequence divergence and major chromosomal rearrangements.Results: Here we take the opposite view and focus on SDs. We analyze several of the features of SDs, including the rates of intraspecific divergence between paralogous copies of human SDs and of interspecific divergence between human SDs and chimpanzee DNA. We study how divergence measures relate to chromosomal rearrangements, while considering other factors that affect evolutionary rates in single copy DNA. Conclusion: We find that interspecific SD divergence behaves similarly to divergence of single-copy DNA. In contrast, old and recent paralogous copies of SDs do present different patterns of intraspecific divergence. Also, we show that some relatively recent SDs accumulate in regions that carry inversions in sister lineages.

Transcriptome analyses of primitively eusocial wasps reveal novel insights into the evolution of sociality and the origin of alternative phenotypes

BACKGROUND: Understanding how alternative phenotypes arise from the same genome is a major challenge in modern biology. Eusociality in insects requires the evolution of two alternative phenotypes - workers, who sacrifice personal reproduction, and queens, who realize that reproduction. Extensive work on honeybees and ants has revealed the molecular basis of derived queen and worker phenotypes in highly eusocial lineages, but we lack equivalent deep-level analyses of wasps and of primitively eusocial species, the latter of which can reveal how phenotypic decoupling first occurs in the early stages of eusocial evolution. RESULTS: We sequenced 20 Gbp of transcriptomes derived from brains of different behavioral castes of the primitively eusocial tropical paper wasp Polistes canadensis. Surprisingly, 75% of the 2,442 genes differentially expressed between phenotypes were novel, having no significant homology with described sequences. Moreover, 90% of these novel genes were significantly upregulated in workers relative to queens. Differential expression of novel genes in the early stages of sociality may be important in facilitating the evolution of worker behavioral complexity in eusocial evolution. We also found surprisingly low correlation in the identity and direction of expression of differentially expressed genes across similar phenotypes in different social lineages, supporting the idea that social evolution in different lineages requires substantial de novo rewiring of molecular pathways. CONCLUSIONS: These genomic resources for aculeate wasps and first transcriptome-wide insights into the origin of castes bring us closer to a more general understanding of eusocial evolution and how phenotypic diversity arises from the same genome.

Differential miRNA and IncRNA expression in embryonic stem cells lacking the Notch1 receptor

Embryonic stem (ES) cells-derived cardiomyocytes represent an attractive source of cells in cell replacement therapies for heart disease. However, controlled cardiogenic differentiation of ES cells requires a complete understanding of the complex molecular mechanisms regulating the differentiation process. We have previously shown that differentiation of ES cells into cardiomyocytes is favored by inactivation of the Notch 1 receptor pathway. In the present study, we therefore compared two ES cell lines, one with normal Notchl expression and one carrying deleted Notchl receptor alleles (Notchl-deleted ES cells) in order to identify genes responsible for the increased propensity of Notchl-deleted ES cells to produce cardiomyocytes. Using RNA-sequencing, we found approximately 300 coding and noncoding transcripts, which are differently expressed in undifferentiated Notchl-deleted ES cells. Since accumulating evidences indicate that long noncoding RNAs (IncRNAs) play important roles in ES cell pluripotency and differentiation, we focused our analysis on modulated IncRNAs. In particular, two IncRNAs, named here lnc 1230 and lnc 1335, are highly induced in the absence of Notchl receptor expression. These represent therefore prime candidates that could favor cardiogenic commitment in undifferentiated ES cells. Indeed, we demonstrate that forced expression of these two IncRNAs in wild-type ES cells result in a significant increase of the number of cardiac progenitor cells and cardiomyocytes in the differentiated progeny of these ES cells. Furthermore, we also identify several microRNAs that are differentially modulated in absence of Notchl expression. Among these are miR-142-5p and miR- 381-3p. Interestingly, both lncl230 and lncl335 are targets of these two microRNAs. Altogether, these data suggest that Notchl-dependent noncoding gene networks, implicating microRNAs and IncRNAs, control embryonic stem cell commitment into the mesodermal and cardiac lineages already at the undifferentiated state. - Les cardiomyocytes issus cellules souches embryonnaires sont une source très prometteuse pour les thérapies cellulaire de remplacement dans le cadre des maladies cardiaques. Cependant, l'utilisation de telles cellules requiert une compréhension poussée des mécanismes moléculaire régulant la différenciation. Nous avons par le passé démontré que la différenciation des cellules souches embryonnaires en cardiomyocytes est favorisée par l'inactivation de la voie d'activation intracellulaire dépendante du récepteur Notch 1. Nous avons donc comparé deux lignées de cellules souches embryonnaires, une présentant une voie d'activation Notchl normale et une chez laquelle les allèles codant pour le récepteur Notchl avaient été invalidés, de façon à identifier les gènes impliqués dans la capacité augmentée des cellules déficientes à produire des cardiomyocytes. En utilisant du séquençage d'ARN à haut débit, nous avons trouvé environ 300 gènes différemment exprimés dans les cellules déficientes pour Notchl. Par ailleurs, des évidences de plus en plus nombreuses suggèrent qu'une nouvelle classe de molécules appelée « long noncoding RNAs » joue un rôle prépondérant dans la maintenance de l'état non différencié et de la capacité de différenciation des cellules souches embryonnaires. Nous avons trouvé que plusieurs « long noncoding RNAs » étaient modulés en l'absence de Notchl, et en particulier deux molécules que nous avons appelées lncl230 et lncl335. Ces derniers représentent des candidats potentiels devant permettre de favoriser la production de cardiomyocytes. Nous avons en effet démontré que la surexpression de ces deux candidats dans des cellules souches embryonnaires résultait en une surproduction de cardiomyocytes. De plus, nous avons également identifié plusieurs microRNAs dont l'expression était modulée dans les cellules souches embryonnaires déficientes dans la voie Notchl. De façon intéressante, parmi ces microRNAs, le miR-142-5p et le miR-381-3p sont capables de cibler lncl230 and lncl335. Dans l'ensemble, ces résultats indiquent donc que des réseaux d'interaction dépendant de la voie d'activation Notch 1 et impliquant des ARNs non codant existent dans les cellules souches embryonnaires pour réguler leur différenciation en différent types cellulaires spécifiques.

Multilocus phylogeography of the common lizard Zootoca vivipara at the Ibero-Pyrenean suture zone reveals lowland barriers and high-elevation introgression.

BACKGROUND: The geographic distribution of evolutionary lineages and the patterns of gene flow upon secondary contact provide insight into the process of divergence and speciation. We explore the evolutionary history of the common lizard Zootoca vivipara (= Lacerta vivipara) in the Iberian Peninsula and test the role of the Pyrenees and the Cantabrian Mountains in restricting gene flow and driving lineage isolation and divergence. We also assess patterns of introgression among lineages upon secondary contact, and test for the role of high-elevation trans-mountain colonisations in explaining spatial patterns of genetic diversity. We use mtDNA sequence data and genome-wide AFLP loci to reconstruct phylogenetic relationships among lineages, and measure genetic structure RESULTS: The main genetic split in mtDNA corresponds generally to the French and Spanish sides of the Pyrenees as previously reported, in contrast to genome-wide AFLP data, which show a major division between NW Spain and the rest. Both types of markers support the existence of four distinct and geographically congruent genetic groups, which are consistent with major topographic barriers. Both datasets reveal the presence of three independent contact zones between lineages in the Pyrenean region, one in the Basque lowlands, one in the low-elevation mountains of the western Pyrenees, and one in the French side of the central Pyrenees. The latter shows genetic evidence of a recent, high-altitude trans-Pyrenean incursion from Spain into France. CONCLUSIONS: The distribution and age of major lineages is consistent with a Pleistocene origin and a role for both the Pyrenees and the Cantabrian Mountains in driving isolation and differentiation of Z. vivipara lineages at large geographic scales. However, mountain ranges are not always effective barriers to dispersal, and have not prevented a recent high-elevation trans-Pyrenean incursion that has led to asymmetrical introgression among divergent lineages. Cytonuclear discordance in patterns of genetic structure and introgression at contact zones suggests selection may be involved at various scales. Suture zones are important areas for the study of lineage formation and speciation, and our results show that biogeographic barriers can yield markedly different phylogeographic patterns in different vertebrate and invertebrate taxa.

Hes1 is a critical but context-dependent mediator of canonical Notch signaling in lymphocyte development and transformation.

Although canonical Notch signaling regulates multiple hematopoietic lineage decisions including T cell and marginal zone B cell fate specification, the downstream molecular mediators of Notch function are largely unknown. We showed here that conditional inactivation of Hes1, a well-characterized Notch target gene, in adult murine bone marrow (BM) cells severely impaired T cell development without affecting other Notch-dependent hematopoietic lineages such as marginal zone B cells. Competitive mixed BM chimeras, intrathymic transfer experiments, and in vitro culture of BM progenitors on Delta-like-expressing stromal cells further demonstrated that Hes1 is required for T cell lineage commitment, but dispensable for Notch-dependent thymocyte maturation through and beyond the beta selection checkpoint. Furthermore, our data strongly suggest that Hes1 is essential for the development and maintenance of Notch-induced T cell acute lymphoblastic leukemia. Collectively, our studies identify Hes1 as a critical but context-dependent mediator of canonical Notch signaling in the hematopoietic system.

Phylogeography and dispersal in the velvet gecko (Oedura lesueurii), and potential implications for conservation of an endangered snake (Hoplocephalus bungaroides).

ABSTRACT: BACKGROUND: To conserve critically endangered predators, we also need to conserve the prey species upon which they depend. Velvet geckos (Oedura lesueurii) are a primary prey for the endangered broad-headed snake (Hoplocephalus bungaroides), which is restricted to sandstone habitats in southeastern Australia. We sequenced the ND2 gene from 179 velvet geckos, to clarify the lizards' phylogeographic history and landscape genetics. We also analysed 260 records from a longterm (3-year) capture-mark-recapture program at three sites, to evaluate dispersal rates of geckos as a function of locality, sex and body size. RESULTS: The genetic analyses revealed three ancient lineages in the north, south and centre of the species' current range. Estimates of gene flow suggest low dispersal rates, constrained by the availability of contiguous rocky habitat. Mark-recapture records confirm that these lizards are highly sedentary, with most animals moving < 30 m from their original capture site even over multi-year periods. CONCLUSION: The low vagility of these lizards suggests that they will be slow to colonise vacant habitat patches; and hence, efforts to restore degraded habitats for broad-headed snakes may need to include translocation of lizards.

Contributions of the peroxisome and β-oxidation cycle to biotin synthesis in fungi.

The first step in the synthesis of the bicyclic rings of D-biotin is mediated by 8-amino-7-oxononanoate (AON) synthase, which catalyzes the decarboxylative condensation of l-alanine and pimelate thioester. We found that the Aspergillus nidulans AON synthase, encoded by the bioF gene, is a peroxisomal enzyme with a type 1 peroxisomal targeting sequence (PTS1). Localization of AON to the peroxisome was essential for biotin synthesis because expression of a cytosolic AON variant or deletion of pexE, encoding the PTS1 receptor, rendered A. nidulans a biotin auxotroph. AON synthases with PTS1 are found throughout the fungal kingdom, in ascomycetes, basidiomycetes, and members of basal fungal lineages but not in representatives of the Saccharomyces species complex, including Saccharomyces cerevisiae. A. nidulans mutants defective in the peroxisomal acyl-CoA oxidase AoxA or the multifunctional protein FoxA showed a strong decrease in colonial growth rate in biotin-deficient medium, whereas partial growth recovery occurred with pimelic acid supplementation. These results indicate that pimeloyl-CoA is the in vivo substrate of AON synthase and that it is generated in the peroxisome via the β-oxidation cycle in A. nidulans and probably in a broad range of fungi. However, the β-oxidation cycle is not essential for biotin synthesis in S. cerevisiae or Escherichia coli. These results suggest that alternative pathways for synthesis of the pimelate intermediate exist in bacteria and eukaryotes and that Saccharomyces species use a pathway different from that used by the majority of fungi.

Control of gene expression in melanocytes and RPE by conserved distal regulatory elements

Résumé Durant le développement embryonnaire, les cellules pigmentaires des mammifères se développent à partir de deux origines différentes : les melanocytes se développent à partir de la crête neurale alors que les cellules de la rétine pigmentaire (RP) ont une origine neuronale. Un grand nombre de gènes sont impliqués dans la pigmentation dont les gènes de la famille tyrosinase à savoir Tyr, Tyrp1 et Dct. Certaines études ont suggéré que les gènes de la pigmentation sont régulés de manière différentielle dans les mélanocytes et dans la RP. Dans ce travail, les gènes de la famille tyrosinase ont été étudiés comme modèle de la régulation des gènes de la pigmentation par des éléments régulateurs agissant à distance. II a été montré que le promoteur du gène Tyrp1pouvait induire l'expression d'un transgène uniquement dans la RP alors que ce gène est aussi exprimé dans les mélanocytes comme le montre le phénotype des souris mutantes pour Tyrp1. Ce résultat suggère que les éléments régulateurs du promoteur sont suffisants pour l'expression dans la RP mais pas pour l'expression dans les mélanocytes. J'ai donc cherché à identifier la séquence qui régule l'expression dans les mélanocytes. Un chromosome artificiel bactérien (CAB) contenant le gène Tyrp1 s'est avéré suffisant pour induire l'expression dans les mélanocytes, comme démontré par la correction du phénotype mutant. La séquence de ce CAB contient plusieurs régions très conservées qui pourraient représenter de nouveaux éléments régulateurs. Par la suite, j'ai focalisé mon analyse sur une séquence située à -I5 kb qui s'est révélée être un amplificateur spécifique aux mélanocytes comme démontré par des expériences de cultures cellulaire et de transgenèse. De plus, une analyse poussée de cet élément a révélé que le facteur de transcription Sox 10 représentait un transactivateur de cet amplificateur. Comme pour Tyrp1, la régulation du gène tyrosinase est contrôlée par différents éléments régulateurs dans les mélanocytes et la RP. Il a été montré que le promoteur de tyrosinase n'était pas suffisant pour une forte expression dans les mélanocytes et la RP. De plus, l'analyse de la région située en amont a révélé la présence d'un amplificateur nécessaire à l'expression dans les mélanocytes à la position -15 kb. Cet amplificateur n'est toutefois pas actif dans la RP mais agit comme un répresseur dans ces cellules. Ces résultats indiquent que certains éléments nécessaires à l'expression dans les deux types de cellules pigmentaires sont absents de ces constructions. Comme pour Tyrp1, j'ai en premier lieu démontré qu'un CAB était capable de corriger le phénotype albinique, puis ai inséré un gène reporter (lacZ) dans le CAB par recombinaison homologue et ai finalement analysé l'expression du reporter en transgenèse. Ces souris ont montré une expression forte du lacZ dans les mélanocytes et la RP, ce qui indique que le CAB contient les séquences régulatrices nécessaires à l'expression correcte de tyrosinase. Afin de localiser plus précisément les éléments régulateurs, j'ai ensuite généré des délétions dans le CAB et analysé l'expression du lacZ en transgenèse. La comparaison de séquences génomiques provenant de différentes espèces a permis par la suite d'identifier des régions représentant de nouveaux éléments régulateurs potentiels. En utilisant cette approche, j'ai identifié une région qui se comporte comme un amplificateur dans la RP et qui est nécessaire à l'expression de tyrosinase dans ce tissu. De plus, j'ai identifié les facteurs de transcription Mitf et Sox10 comme transactivateurs de l'amplificateur spécifique aux mélanocytes situé à -15 kb. L'identification et la caractérisation des ces éléments régulateurs des gènes tyrosinase et Tyrp1confirme donc que la régulation différentielle des gènes dans les mélanocytes et la RP est liée à des éléments régulateurs séparés. Summary Pigment cells of mammals originate from two different lineages: melanocytes arise from the neural crest, whereas cells of the retinal pigment epithelium (RPE) originate from the optic cup of the developing forebrain. A large set of genes are involved in pigmentation, including the members of the tyrosinase gene family, namely tyrosinase, Tyrp1 and Dct. Previous studies have suggested that pigmentation genes are differentially regulated in melanocytes and RPE. In this work, the tyrosinase gene family was used as a model for studying the involvement of distal regulatory elements in pigment cell-specific gene expression. The promoter of the Tyrp1 gene has been shown to drive detectable transgene expression only to the RPE, even though the gene is also expressed in melanocytes as evident from Tyrp1-mutant mice. This indicates that the regulatory elements responsible for Tyrp1 gene expression in the RPE are not sufficient for expression in melanocytes. I thus searched for a putative melanocyte-specific regulatory sequence and demonstrate that a bacterial artificial chromosome (BAC) containing the Tyrp1 gene and surrounding sequences is able to target transgenic expression to melanocytes and to rescue the Tyrp1 b (brown) phenotype. This BAC contains several highly conserved non-coding sequences that might represent novel regulatory elements. I further focused on a sequence located at -15 kb which I identified as amelanocyte-specific enhancer as shown by cell culture and transgenic mice. In addition, further functional analysis identified the transcription factor Sox10 as being able to bind and transactivate this enhancer. As for Tyrp1, tyrosinase gene regulation is mediated by different cis-regulatory elements in melanocytes and RPE. It was shown that the tyrosinase promoter was not sufficient to confer strong and specific expression in melanocytes and RPE. Moreover, analysis of tyrosinase upstream sequence, revealed the presence of a specific enhancer at position -15 kb which was necessary to confer strong expression in melanocytes. This enhancer element however failed to act as an enhancer in the RPE, but rather repressed expression. This indicates that some regulatory elements required for tyrosinase expression in both RPE and melanocytes are still missing from these constructs. As for Tyrp1, I first demonstrated that a BAC containing the Tyr gene is able to rescue the Tyr c (albino) phenotype in mice, then I inserted a lacZ reporter gene in the BAC by homologous recombination, and finally analysed the pattern of lacZ expression in transgenic mice. These mice showed strong lacZ expression in both RPE and melanocytes, indicating that the BAC contains the regulatory sequences required for proper tyrosinase expression. In order to localize more precisely these regulatory elements, I have then generated several deletions in the BAC and analysed lacZ expression in transgenic mice. Multi-species comparative genomic analysis then allowed identifying conserved sequences that potentially represent novel regulatory elements. Using this experimental approach, I identified a region that behaves as a RPE-specific enhancer and that is required for tyrosinase expression in the retina] pigment epithelium. In addition, I identified the transcription factors Mitf and Sox l0 as being transactivators of the melanocyte-specific enhancer located at -l5 kb. The identification and characterization of these tyrosinase and Tyrp1 distal regulatory element supports the idea that separate regulatory sequences mediate differential gene expression in melanocytes and RPE.

Origin and genome evolution of polyploid green toads in Central Asia: evidence from microsatellite markers.

Polyploidization, which is expected to trigger major genomic reorganizations, occurs much less commonly in animals than in plants, possibly because of constraints imposed by sex-determination systems. We investigated the origins and consequences of allopolyploidization in Palearctic green toads (Bufo viridis subgroup) from Central Asia, with three ploidy levels and different modes of genome transmission (sexual versus clonal), to (i) establish a topology for the reticulate phylogeny in a species-rich radiation involving several closely related lineages and (ii) explore processes of genomic reorganization that may follow polyploidization. Sibship analyses based on 30 cross-amplifying microsatellite markers substantiated the maternal origins and revealed the paternal origins and relationships of subgenomes in allopolyploids. Analyses of the synteny of linkage groups identified three markers affected by translocation events, which occurred only within the paternally inherited subgenomes of allopolyploid toads and exclusively affected the linkage group that determines sex in several diploid species of the green toad radiation. Recombination rates did not differ between diploid and polyploid toad species, and were overall much reduced in males, independent of linkage group and ploidy levels. Clonally transmitted subgenomes in allotriploid toads provided support for strong genetic drift, presumably resulting from recombination arrest. The Palearctic green toad radiation seems to offer unique opportunities to investigate the consequences of polyploidization and clonal transmission on the dynamics of genomes in vertebrates.

Sex-chromosome evolution of Palearctic tree frogs in space and time

Sexual reproduction is nearly universal in eukaryotes and genetic determination of sex prevails among animals. The astonishing diversity of sex-determining systems and sex chromosomes is yet bewildering. Some taxonomic groups possess conserved and dimorphic sex chromosomes, involving a functional copy (e.g. mammals' X, birds' Z) and a degenerated copy (mammals' Y, birds' W), implying that sex- chromosomes are expected to decay. In contrast, others like amphibians, reptiles and fishes yet maintained undifferentiated sex chromosomes. Why such different evolutionary trajectories? In this thesis, we empirically test and characterize the main hypotheses proposed to prevent the genetic decay of sex chromosomes, namely occasional X-Y recombination and frequent sex-chromosome transitions, using the Palearctic radiation of Hyla tree frogs as a model system. We take a phylogeographic and phylogenetic approach to relate sex-chromosome recombination, differentiation, and transitions in a spatial and temporal framework. By reconstructing the recent evolutionary history of the widespread European tree frog H. arborea, we showed that sex chromosomes can recombine in males, preventing their differentiation, a situation that potentially evolves rapidly. At the scale of the entire radiation, X-Y recombination combines with frequent transitions to prevent sex-chromosome degeneration in Hyla: we traced several turnovers of sex-determining system within the last 10My. These rapid changes seem less random than usually assumed: we gathered evidences that one chromosome pair is a sex expert, carrying genes with key role in animal sex determination, and which probably specialized through frequent reuse as a sex chromosome in Hyla and other amphibians. Finally, we took advantage of secondary contact zones between closely-related Hyla lineages to evaluate the consequences of sex chromosome homomorphy on the genetics of speciation. In comparison with other systems, the evolution of sex chromosomes in Hyla emphasized the existence of consistent evolutionary patterns within the chaotic diversity of flexibility of cold-blooded vertebrates' sex-determining systems, and provides insights into the evolution of recombination. Beyond sex-chromosome evolution, this work also significantly contributed to speciation, phylogeography and applied conservation research. -- La reproduction sexuée est quasi-universelle chez les eucaryotes et le sexe est le plus souvent déterminé génétiquement au sein du règne animal. L'incroyable diversité des systèmes de reproduction et des chromosomes sexuels est particulièrement étonnante. Certains groupes taxonomiques possèdent des chromosomes sexuels dimorphiques et très conservés, avec une copie entièrement fonctionnelle (ex : le X des mammifères, le Z des oiseaux) et une copie dégénérée (ex : le Y des mammifères, le W des oiseaux), suggérant que les chromosomes sexuels sont voués à se détériorer. Cependant les chromosomes sexuels d'autres groupes tels que les amphibiens, les reptiles et les poissons sont pour la plupart indifférenciés. Comment expliquer des trajectoires évolutives si différentes? Au cours de cette thèse, nous avons étudié empiriquement les processus évolutifs pouvant maintenir les chromosomes sexuels intacts, à savoir la recombinaison X-Y occasionnel ainsi que les substitutions fréquentes de chromosomes sexuels, en utilisant les rainettes Paléarctiques du genre Hyla comme modèle d'étude. Nous avons adopté une approche phylogéographique et phylogénétique pour appréhender les événements de recombinaison, de différenciation et de transitions de chromosomes sexuels dans un contexte spatio-temporel. En retraçant l'histoire évolutive récente de la rainette verte H. arborea, nous avons mis en évidence que les chromosomes sexuels pouvaient recombiner chez les mâles, empêchant ainsi leur différenciation, et que ce processus avait le potentiel d'évoluer très rapidement. A l'échelle plus globale de la radiation, il apparait que les phénomènes de recombinaison X-Y soient également accompagnés de substitutions de chromosomes sexuels, et participent de concert au maintien de chromosomes sexuels intacts dans les populations: le système de détermination du sexe des rainettes a changé plusieurs fois au cours des 10 derniers millions d'années. Ces transitions fréquentes ne semblent pas aléatoires: nous avons identifié une paire de chromosomes qui présente des caractéristiques présageant d'une spécialisation dans le déterminisme du sexe (notamment car elle possède des gènes importants pour cette fonction), et qui a été réutilisée plusieurs fois comme tel chez les rainettes ainsi que d'autres amphibiens. Enfin, nous avons étudié l'hybridation entre différentes espèces dans leurs zones de contact, afin d'évaluer si l'absence de différenciation entre X et Y jouaient un rôle dans les processus génétiques de spéciation. Outre son intérêt pour la compréhension de l'évolution des chromosomes sexuels, ce travail contribue de manière significative à d'autres domaines de recherche tels que la spéciation, la phylogéographie, ainsi que la biologie de la conservation.

Prevalence and diversity of Chlamydiales in Swiss ruminant farms.

Chlamydia and Chlamydia-related bacteria are known to infect various organisms and may cause a wide range of diseases, especially in ruminants. To gain insight into the prevalence of these bacteria in the ruminant environment, we applied a pan-Chlamydiales PCR followed by sequencing to 72 ruminant environmental samples from water, feed bunks and floors. Chlamydiales from four family-level lineages were detected indicating a high biodiversity of Chlamydiales in ruminant farms. Parachlamydiaceae were detected in all three types of environmental samples and was the most abundant family-level taxon (60%). In contrast, only one bacterium from each of the following family-level lineages was identified: Chlamydiaceae, Criblamydiaceae and Simkaniaceae. The observed high prevalence of Parachlamydiaceae in water samples may suggest water as the main source of contamination for ruminants as well as their environment due to spoilage. The absence of reported infections in the investigated ruminant farms might indicate that either detected Chlamydiales are of reduced pathogenicity or infective doses have not been reached.

Genes outside The Hla Region affecting Susceptibility to Type 1 Diabetes - The Role of Iddm2 and Iddm9 in the Finnish Population

Tyypin 1 diabeteksen perinnöllinen alttius Suomessa - HLA-alueen ulkopuolisten alttiuslokusten IDDM2 ja IDDM9 rooli taudin periytymisessä HLA-alue, joka sijaitsee kromosomissa 6p21.3, vastaa noin puolesta perinnöllisestä alttiudesta sairastua tyypin 1 diabetekseen. Myös HLA-alueen ulkopuolisten lokusten on todettu liittyvän sairausalttiuteen. Näistä kolmen lokuksen on varmistettu olevan todellisia alttiuslokuksia ja lisäksi useiden muiden, vielä varmistamattomien lokusten, on todettu liittyvän sairausalttiuteen. Tässä tutkimuksessa 12:n HLA-alueen ulkopuolisen alttiuslokuksen kytkentä tyypin 1 diabetekseen tutkittiin käyttäen 107:aa suomalaista multiplex-perhettä. Jatkotutkimuksessa analysoitiin IDDM9-alueen kytkentä ja assosiaatio sairauteen laajennetuissa perhemateriaaleissa sekä IDDM2-alueen mahdollinen interaktio HLA-alueen kanssa sairauden muodostumisessa. Lisäksi suoritettiin IDDM2-alueen suojaavien haplotyyppien alatyypitys tarkoituksena tutkia eri haplotyyppien käyttökelpoisuutta sairastumisriskin tarkempaa ennustamista varten. Ensimmäisessä kytkentätutkimuksessa ei löytynyt koko genomin tasolla merkitsevää tai viitteellistä kytkentää tutkituista HLA-alueen ulkopuolisista lokuksista. Voimakkain havaittu nimellisen merkitsevyyden tavoittava kytkentä nähtiin IDDM9-alueen markkerilla D3S3576 (MLS=1.05). Tutkimuksessa ei kyetty varmistamaan tai sulkemaan pois aiempia kytkentähavaintoja tutkituilla lokuksilla, mutta IDDM9-alueen jatkotutkimuksessa havaittu voimakas kytkentä (MLS=3.4) ja merkitsevä assosiaatio (TDT p=0.0002) viittaa vahvasti siihen, että 3q21-alueella sijaitsee todellinen tyypin 1 diabeteksen alttiusgeeni, jolloin alueen kattava assosiaatiotutkimus olisi perusteltu jatkotoimenpide. Sairauteen altistava IDDM2-alueen MspI-2221 genotyyppi CC oli nimellisesti yleisempi matalan tai kohtalaisen HLA-sairastumisriskin diabeetikoilla, verrattuna korkean HLA-riskin potilaisiin (p=0.05). Myös genotyyppijakauman vertailu osoitti merkitsevää eroa ryhmien välillä (p=0.01). VNTR-haplotyyppitutkimus osoitti, että IIIA/IIIA-homotsygootin sairaudelta suojaava vaikutus on merkitsevästi voimakkaampi kuin muiden luokka III:n genotyypeillä. Nämä tulokset viittaavat IDDM2-HLA -vuorovaikutukseen sekä siihen että IDDM2-alueen haplotyyppien välillä esiintyy etiologista heterogeniaa. Tämän johdosta IDDM2-alueen haplotyyppien tarkempi määrittäminen voisi tehostaa tyypin 1 diabeteksen riskiarviointia.

DNA barcoding of Corsican mayflies (Ephemeroptera) with implications on biogeography, systematics and biodiversity

Mayflies (Ephemeroptera) are known to generally present a high degree of insular endemism: half of the 28 species known from Corsica and Sardinia are considered as endemic. We sequenced the DNA barcode (a fragment of the mitochondrial COI gene) of 349 specimens from 50 localities in Corsica, Sardinia, continental Europe and North Africa. We reconstructed gene trees of eight genera or species groups representing the main mayfly families. Alternative topologies were built to test if our reconstructions suggested a single or multiple Corsican/Sardinian colonization event(s) in each genus or species group. A molecular clock calibrated with different evolution rates was used to try to link speciation processes with geological events. Our results confirm the high degree of endemism of Corsican and Sardinian mayflies and the close relationship between these two faunas. Moreover, we have evidence that the mayfly diversity of the two islands is highly underestimated as at least six new putative species occur on the two islands. We demonstrated that the Corsican and Sardinian mayfly fauna reveals a complex history mainly related to geological events. The Messinian Salinity Crisis, which is thought to have reduced marine barriers, thus facilitating gene flow between insular and continental populations, was detected as the most important event in the speciation of most lineages. Vicariance processes related to the split and rotation of the Corso-Sardinian microplate had a minor impact as they involved only two genera with limited dispersal and ecological range. Colonization events posterior to the Messinian Salinity Crisis had only marginal effects as we had indication of recent gene flow only in two clades. With very limited recent gene flow and a high degree of endemism, mayflies from Corsica and Sardinia present all the criteria for conservation prioritization.