Genetic diversity in widespread species is not congruent with species richness in alpine plant communities.

The Convention on Biological Diversity (CBD) aims at the conservation of all three levels of biodiversity, that is, ecosystems, species and genes. Genetic diversity represents evolutionary potential and is important for ecosystem functioning. Unfortunately, genetic diversity in natural populations is hardly considered in conservation strategies because it is difficult to measure and has been hypothesised to co-vary with species richness. This means that species richness is taken as a surrogate of genetic diversity in conservation planning, though their relationship has not been properly evaluated. We tested whether the genetic and species levels of biodiversity co-vary, using a large-scale and multi-species approach. We chose the high-mountain flora of the Alps and the Carpathians as study systems and demonstrate that species richness and genetic diversity are not correlated. Species richness thus cannot act as a surrogate for genetic diversity. Our results have important consequences for implementing the CBD when designing conservation strategies.

Wood ants protect their brood with tree resin

Social insects use multiple lines of collective defences to combat pathogens. One example of a behav- ioural group defence is the use of antimicrobial plant compounds to disinfect the nest. Indeed, wood ants collect coniferous tree resin, and the presence of resin in their nest protects them against fungal and bacterial pathogens. Many questions remain on the mechanisms of resin use, including which factors elicit resin collection and placement within nests. Here, we investigated whether the presence of brood induces Formica paralugubris workers to collect more resin, and whether the workers preferentially place resin near the brood. We also tested whether the collection and placement of resin depends on the presence of the fungal entomopathogen Beauveria bassiana. Workers brought more resin to their nest when brood was present, and preferentially placed the resin near the brood. In contrast, workers did not increase resin collection in response to exposure to B. bassiana, nor did they place resin closer to contaminated brood or contaminated areas of the nest. This lack of response may be explained by a limited effect of resin against the germination and growth of B. bassiana in vitro. Overall, our main result is that woods ants actively position resin near the brood, which probably confers prophylactic protection against other detrimental microorganisms.

On landslide risk, resilience and vulnerability of mountain communities in Central-Eastern Nepal

Executive SummaryIn Nepal, landslides are one of the major natural hazards after epidemics, killing over 100 persons per year. However, this figure is an underreported reflection of the actual impact that landslides have on livelihoods and food security in rural Nepal. With predictions of more intense rainfall patterns, landslide occurrence in the Himalayas is likely to increase and continue to be one of the major impediments to development. Due to the remoteness of many localities and lack of resources, responsibilities for disaster preparedness and response in mountain areas usually lie with the communities themselves. Everyday life is full of risk in mountains of Nepal. This is why mountain populations, as well as other populations living in harsh conditions have developed a number of coping strategies for dealing with adverse situations. Perhaps due to the dispersed and remote nature of landslides in Nepal, there have been few studies on vulnerability, coping- and mitigation strategies of landslide affected populations. There are also few recommendations available to guide authorities and populations how to reduce losses due to landslides in Nepal, and even less so, how to operationalize resilience and vulnerability.Many policy makers, international donors, NGOs and national authorities are currently asking what investments are needed to increase the so-called 'resilience' of mountain populations to deal with climate risks. However, mountain populations are already quite resilient to seasonal fluctuations, temperature variations, rainfall patterns and market prices. In spite of their resilience, they continue to live in places at risk due to high vulnerability caused by structural inequalities: access to land, resources, markets, education. This interdisciplinary thesis examines the concept of resilience by questioning its usefulness and validity as the current goal of international development and disaster risk reduction policies, its conceptual limitations and its possible scope of action. The goal of this study is two-fold: to better define and distinguish factors and relationships between resilience, vulnerability, capacities and risk; and to test and improve a participatory methodology for evaluating landslide risk that can serve as a guidance tool for improving community-based disaster risk reduction. The objective is to develop a simple methodology that can be used by NGOs, local authorities and communities to reduce losses from landslides.Through its six case studies in Central-Eastern Nepal, this study explores the relation between resilience, vulnerability and landslide risk based on interdisciplinary methods, including geological assessments of landslides, semi-structured interviews, focus groups and participatory risk mapping. For comparison, the study sites were chosen in Tehrathum, Sunsari and Dolakha Districts of Central/Eastern Nepal, to reflect a variety of landslide types, from chronic to acute, and a variety of communities, from very marginalized to very high status. The study uses the Sustainable Livelihoods Approach as its conceptual basis, which is based on the notion that access and rights to resources (natural, human/institutional, economic, environmental, physical) are the basis for coping with adversity, such as landslides. The study is also intended as a contribution to the growing literature and practices on Community Based Disaster Risk Reduction specifically adapted to landslide- prone areas.In addition to the six case studies, results include an indicator based methodology for assessing and measuring vulnerability and resilience, a composite risk assessment methodology, a typology of coping strategies and risk perceptions and a thorough analysis of the relation between risk, vulnerability and resilience. The methodology forassessing vulnerability, resilience and risk is relatively cost-effective and replicable in a low-data environment. Perhaps the major finding is that resilience is a process that defines a community's (or system's) capacity to rebound following adversity but it does not necessarily reduce vulnerability or risk, which requires addressing more structural issues related to poverty. Therefore, conclusions include a critical view of resilience as a main goal of international development and disaster risk reduction policies. It is a useful concept in the context of recovery after a disaster but it needs to be addressed in parallel with vulnerability and risk.This research was funded by an interdisciplinary grant (#26083591) from the Swiss National Science Foundation for the period 2009-2011 and a seed grant from the Faculty of Geosciences and Environment at the University of Lausanne in 2008.Résumé en françaisAu Népal, les glissements de terrain sont un des aléas les plus dévastateurs après les épidémies, causant 100 morts par an. Pourtant, ce chiffre est une sous-estimation de l'impact réel de l'effet des glissements sur les moyens de subsistance et la sécurité alimentaire au Népal. Avec des prévisions de pluies plus intenses, l'occurrence des glissements dans les Himalayas augmente et présente un obstacle au développement. Du fait de l'éloignement et du manque de ressources dans les montagnes au Népal, la responsabilité de la préparation et la réponse aux catastrophes se trouve chez les communautés elles-mêmes. Le risque fait partie de la vie quotidienne dans les montagnes du Népal. C'est pourquoi les populations montagnardes, comme d'autres populations vivant dans des milieux contraignants, ont développé des stratégies pour faire face aux situations défavorables. Peu d'études existent sur la vulnérabilité, ceci étant probablement dû à l'éloignement et pourtant, les stratégies d'adaptation et de mitigation des populations touchées par des glissements au Népal existent.Beaucoup de décideurs politiques, bailleurs de fonds, ONG et autorités nationales se demandent quels investissements sont nécessaires afin d'augmenter la 'resilience' des populations de montagne pour faire face aux changements climatiques. Pourtant, ces populations sont déjà résilientes aux fluctuations des saisons, des variations de température, des pluies et des prix des marchés. En dépit de leur résilience, ils continuent de vivre dans des endroits à fort risque à cause des vulnérabilités créées par les inégalités structurelles : l'accès à la terre, aux ressources, aux marchés et à l'éducation. Cette thèse interdisciplinaire examine le concept de la résilience en mettant en cause son utilité et sa validité en tant que but actuel des politiques internationales de développement et de réduction des risques, ainsi que ses limitations conceptuelles et ses possibles champs d'action. Le but de cette étude est double : mieux définir et distinguer les facteurs et relations entre la résilience, la vulnérabilité, les capacités et le risque ; Et tester et améliorer une méthode participative pour évaluer le risque des glissements qui peut servir en tant qu'outil indicatif pour améliorer la réduction des risques des communautés. Le but est de développer une méthodologie simple qui peut être utilisée par des ONG, autorités locales et communautés pour réduire les pertes dues aux glissements.A travers les études de cas au centre-est du Népal, cette étude explore le rapport entre la résilience, la vulnérabilité et les glissements basée sur des méthodes interdisciplinaires ; Y sont inclus des évaluations géologiques des glissements, des entretiens semi-dirigés, des discussions de groupes et des cartes de risques participatives. Pour la comparaison, les zones d'études ont été sélectionnées dans les districts de Tehrathum, Sunsari et Dolakha dans le centre-est du Népal, afin de refléter différents types de glissements, de chroniques à urgents, ainsi que différentes communautés, variant de très marginalisées à très haut statut. Pour son cadre conceptuel, cette étude s'appuie sur l'approche de moyens de subsistance durable, qui est basée sur les notions d'accès et de droit aux ressources (naturelles, humaines/institutionnelles, économiques, environnementales, physiques) et qui sont le minimum pour faire face à des situations difficiles, comme des glissements. Cette étude se veut aussi une contribution à la littérature et aux pratiques en croissantes sur la réduction des risques communautaires, spécifiquement adaptées aux zones affectées par des glissements.En plus des six études de cas, les résultats incluent une méthodologie basée sur des indicateurs pour évaluer et mesurer la vulnérabilité et la résilience, une méthodologie sur le risque composé, une typologie de stratégies d'adaptation et perceptions des risques ainsi qu'une analyse fondamentale de la relation entre risque, vulnérabilité et résilience. Les méthodologies pour l'évaluation de la vulnérabilité, de la résilience et du risque sont relativement peu coûteuses et reproductibles dans des endroits avec peu de données disponibles. Le résultat probablement le plus pertinent est que la résilience est un processus qui définit la capacité d'une communauté (ou d'un système) à rebondir suite à une situation défavorable, mais qui ne réduit pas forcement la vulnérabilité ou le risque, et qui requiert une approche plus fondamentale s'adressant aux questions de pauvreté. Les conclusions incluent une vue critique de la résilience comme but principal des politiques internationales de développement et de réduction des risques. C'est un concept utile dans le contexte de la récupération après une catastrophe mais il doit être pris en compte au même titre que la vulnérabilité et le risque.Cette recherche a été financée par un fonds interdisciplinaire (#26083591) du Fonds National Suisse pour la période 2009-2011 et un fonds de préparation de recherches par la Faculté des Géosciences et Environnement à l'Université de Lausanne en 2008.

The role of phylogeny in the spatial distributions and assembly of mountain plant communities.

A major challenge in community ecology is a thorough understanding of the processes that govern the assembly and composition of communities in time and space. The growing threat of climate change to the vascular plant biodiversity of fragile ecosystems such as mountains has made it equally imperative to develop comprehensive methodologies to provide insights into how communities are assembled. In this perspective, the primary objective of this PhD thesis is to contribute to the theoretical and methodological development of community ecology, by proposing new solutions to better detect the ecological and evolutionary processes that govern community assembly. As phylogenetic trees provide by far, the most advanced tools to integrate the spatial, ecological and evolutionary dynamics of plant communities, they represent the cornerstone on which this work was based. In this thesis, I proposed new solutions to: (i) reveal trends in community assembly on phylogenies, depicted by the transition of signals at the nodes of the different species and lineages responsible for community assembly, (ii) contribute to evidence the importance of evolutionarily labile traits in the distribution of mountain plant species. More precisely, I demonstrated that phylogenetic and functional compositional turnover in plant communities was driven by climate and human land use gradients mostly influenced by evolutionarily labile traits, (iii) predict and spatially project the phylogenetic structure of communities using species distribution models, to identify the potential distribution of phylogenetic diversity, as well as areas of high evolutionary potential along elevation. The altitudinal setting of the Diablerets mountains (Switzerland) provided an appropriate model for this study. The elevation gradient served as a compression of large latitudinal variations similar to a collection of islands within a single area, and allowed investigations on a large number of plant communities. Overall, this thesis highlights that stochastic and deterministic environmental filtering processes mainly influence the phylogenetic structure of plant communities in mountainous areas. Negative density-dependent processes implied through patterns of phylogenetic overdispersion were only detected at the local scale, whereas environmental filtering implied through phylogenetic clustering was observed at both the regional and local scale. Finally, the integration of indices of phylogenetic community ecology with species distribution models revealed the prospects of providing novel and insightful explanations on the potential distribution of phylogenetic biodiversity in high mountain areas. These results generally demonstrate the usefulness of phylogenies in inferring assembly processes, and are worth considering in the theoretical and methodological development of tools to better understand phylogenetic community structure.

Venous cannula performance assessment in a realistic caval tree model.

OBJECTIVES: A new caval tree system was designed for realistic in vitro simulation. The objective of our study was to assess cannula performance for virtually wall-less versus standard percutaneous thin-walled venous cannulas in a setting of venous collapse in case of negative pressure. METHODS: For a collapsible caval model, a very flexible plastic material was selected, and a model with nine afferent veins was designed according to the anatomy of the vena cava. A flow bench was built including a lower reservoir holding the caval tree, built by taking into account the main afferent vessels and their flow provided by a reservoir 6 cm above. A cannula was inserted in this caval tree and connected to a centrifugal pump that, in turn, was connected to a reservoir positioned 83 cm above the second lower reservoir (after-load = 60 mmHg). Using the same pre-load, the simulated venous drainage for cardiopulmonary bypass was realized using a 24 F wall-less cannula (Smartcanula) and 25 F percutaneous cannula (Biomedicus), and stepwise increased augmentation (1500 RPM, 2000 and 2500 RPM) of venous drainage. RESULTS: For the thin wall and the wall-less cannulas, 36 pairs of flow and pressure measurements were realized for three different RPM values. The mean Q-values at 1500, 2000 and 2500 RPM were: 3.98 ± 0.01, 6.27 ± 0.02 and 9.81 ± 0.02 l/min for the wall-less cannula (P <0.0001), versus 2.74 ± 0.02, 3.06 ± 0.05, 6.78 ± 0.02 l/min for the thin-wall cannula (P <0.0001). The corresponding inlet pressure values were: -8.88 ± 0.01, -23.69 ± 0.81 and -70.22 ± 0.18 mmHg for the wall-less cannula (P <0.0001), versus -36.69 ± 1.88, -80.85 ± 1.71 and -101.83 ± 0.45 mmHg for the thin-wall cannula (P <0.0001). The thin-wall cannula showed mean Q-values 37% less and mean P values 26% more when compared with the wall-less cannula (P <0.0001). CONCLUSIONS: Our in vitro water test was able to mimic a negative pressure situation, where the wall-less cannula design performs better compared with the traditional thin-wall cannula.

Multiscale determinants of tree frog (Hyla arborea L.) calling ponds in western Switzerland

A tree frog (Hyla arborea L., 1758) metapopulation in western Switzerland was studied during spring 2001. All potential calling ponds in an area of 350 km(2) were searched for tree frog calling males. Twenty-nine out of 111 ponds sheltered between 1 and 250 callers. Most ponds were occupied by less than 12 males. Pond parameters were measured at three different levels using field analysis and a Geographical Information System (GIS). The first level was water chemistry and pond-associated measures. The second level was the surrounding land use in a 30 m buffer around the pond. The third level consisted of landscape indices on a broader scale (up to 2 km). Logistic regression was applied to identify parameters that can predict the presence of calling males in a pond. Response variable was the presence or absence of callers. Four significant parameters allowed us to explain about 40% of the total deviance of the observed occupational pattern. Urbanization around the pond had a highly negative impact on the probability of presence of calling males. Hours of direct sunlight on the pond was positively correlated with callers. Higher water conductivity was associated with a lesser probability of species presence. Finally, the further the closest two-lane road, the higher the probability of callers presence. Our results show that presence or absence of callers is influenced by factors acting at various geographical scales.

A sex-specific marker reveals male heterogamety in European tree frogs.

Most amphibians examined so far show undifferentiated sex chromosomes. The heterogametic sex's identity, usually revealed through indirect means, often varies among closely related species or even populations (as do sex-linkage groups), suggesting great evolutionary instability of the sex-determining genes. Here we take advantage of a sex-specific marker that amplifies in several related species of European tree frogs (Hyla arborea group) to disclose a homogeneous pattern of male heterogamety. Besides relevance for evolutionary studies of sex determination in amphibians, our results have potential for addressing practical issues in conservation biology because sex reversal by anthropogenic endocrine disruptors is considered one possible cause of amphibian decline.

Ecological assembly rules in plant communities--approaches, patterns and prospects.

Understanding how communities of living organisms assemble has been a central question in ecology since the early days of the discipline. Disentangling the different processes involved in community assembly is not only interesting in itself but also crucial for an understanding of how communities will behave under future environmental scenarios. The traditional concept of assembly rules reflects the notion that species do not co-occur randomly but are restricted in their co-occurrence by interspecific competition. This concept can be redefined in a more general framework where the co-occurrence of species is a product of chance, historical patterns of speciation and migration, dispersal, abiotic environmental factors, and biotic interactions, with none of these processes being mutually exclusive. Here we present a survey and meta-analyses of 59 papers that compare observed patterns in plant communities with null models simulating random patterns of species assembly. According to the type of data under study and the different methods that are applied to detect community assembly, we distinguish four main types of approach in the published literature: species co-occurrence, niche limitation, guild proportionality and limiting similarity. Results from our meta-analyses suggest that non-random co-occurrence of plant species is not a widespread phenomenon. However, whether this finding reflects the individualistic nature of plant communities or is caused by methodological shortcomings associated with the studies considered cannot be discerned from the available metadata. We advocate that more thorough surveys be conducted using a set of standardized methods to test for the existence of assembly rules in data sets spanning larger biological and geographical scales than have been considered until now. We underpin this general advice with guidelines that should be considered in future assembly rules research. This will enable us to draw more accurate and general conclusions about the non-random aspect of assembly in plant communities.

Pollution stress on aquatic microbial communities

Thousands of chemical compounds enter the natural environment but many have unknown effects and consequences, in particular at low concentrations. This thesis work contributes to our understanding of pollution effects by using bacteria as test organisms. Bacteria are important for this question because some of them degrade and transform pollutants into less harmful compounds, but secondly because they themselves can be inhibited in their reproduction by exposure to toxic compounds. When inhibitory effects occur this may change the composition of the microbial com¬munity in the long run, leading to altered or diminished ecosystem services by those communities. As a result chemicals of anthropogenic origin may accumulate and per¬sist in the environment, and finally, affect higher organisms as well. In addition to acquiring basic understanding of pollutant effects at low concentrations on bacterial communities an applied goal of this thesis work was to develop bacteria-based tests to screen new organic chemicals for toxicity and biodégradation. In the first part of this work we developed a flow cytometry-based assay on SYT09 plus ethidium-bromide or propidium-iodide stained cells of Pseudomonas ûuorescens exposed or not to a variety of pollutants under oligotrophic growth conditions. Flow cytometry (FC) allows fast and accurate counting of bacterial cells under simul¬taneous assessment of their physiological state, in particular in combination with different fluorescent dyes. Here we employed FC and fluorescent dyes to monitor the effect that pollutants may exert on Pseudomonas ûuorescens SV3. First we designed an oligotrophic growth test, which enabled us to follow population growth at low densities (104 - 10 7 cells per ml) using 0.1 mM sodium acetate as carbon source. Cells in the oligotrophic milieu were then exposed or not to a variety of common pollutants, such as 2-chlorobiphenyl (2CBP), naphthalene (NAH), 4-chlorophenol (4CP), tetradecane (TD), mercury chloride (HgCl2) or benzene, in different dosages. Exposed culture samples were stained with SYT09 (green fluorescent dye binding nucleic acids, generally staining all cells) in combination with propidium iodide (PI) or ethidium bromide (EB), both dyes being membrane integrity indicators. We ob- served that most of the tested compounds decreased population growth in a dosage- dependent manner. SYT09/PI or SYT09/EB staining then revealed that chemical exposure led to arisal of subpopulations of live and injured or dead cells. By modeling population growth on the total cell numbers in population or only the subpopulation of live cells we inferred that even in stressed populations live cells multiply at rates no different to unexposed controls. The net decrease in population growth would thus be a consequence of more and more cells being not able to multiply at all, rather than all cells multiplying at slower rates. In addition, the proportion of injured cells correlated to the compound dosage. We concluded that the oligotrophic test may be useful to asses toxicity of unknown chemicals on a variety of model bacteria. Mul¬tiple tests can be run in parallel and effects are rapidly measured within a period of 8 hours. Interestingly, in the same exposure tests with P. fluorescens SV3 we observed that some chemicals which did not lead to a reduction of net population growth rates did cause measurable effects on live cells. This was mainly observed in cells within the live subpopulation as an increase of the EB fluorescence signal. We showed that SYT09/EB is a more useful combination of dyes than SYT09/PI because PI fluorescence tend to increase only when cells are effectively dead, but not so much in live cells (less then twofold). In contrast, EB geometric mean fluorescence in live cells increased up to eightfold after exposure to toxic compounds. All compounds even at the lowest concentration caused a measurable increase in EB geometric mean fluorescence especially after 2 h incubation time. This effect was found to be transient for cells exposed to 2CBP and 4CP, but chronic for cells incubated with TD and NAH (ultimately leading to cell death). In order to understand the mechanism underlying the observed effects we used known membrane or energy uncouplers. The pattern of EB signal increase in chemical-exposed populations resembled mostly that of EDTA, although EB fluorescence in EDTA-treated or pasteurized cells was even higher than after exposure to the four test chemicals. We conclude that the ability of cells to efflux EB under equilibrium conditions is an appropriate measure for the potential of a chemical to exert toxicity. Since most bacterial species possess efflux systems for EB that all require cellular energy, our test should be more widely relevant to infer toxicity effects of chemical exposure on the physiological status of the bacterial cell. To better understand the effect of toxicant exposure on efflux defense systems, we studied 2-hydroxybiphenyl toxicity to Pseudomonas azeiaica HBP1. We showed that 2-HBP exerts toxicity even to P. azelaica HBP1, but only at concentrations higher than 0.5 mM. Above this concentration transient loss of membrane polarization and integrity occurred, which we conclude from staining of growing cells with fluorescent dyes. Cells finally recover and resume growth on 2HBP. The high resistance of P. azelaica HBP1 to 2-HBP was found to be the result of an efficient MexABOprM- type efflux pump system counteracting passive influx of this compound into the membrane and cellular interior. Mutants with disrupted mexA, mexB and oprM genes did no longer grow on 2-HBP at concentrations above 100 μΜ, whereas below this concentration we found 2-HBP-concentration dependent decrease of growth rate. The MexAB-OprM system in P. azeiaica HBP1 is indeed an efflux pump for ethidium bromide as well. By introducing gfp reporter fusions responsive to intracellular 2- HBP concentrations into HBP1 wild-type or the mutants we demonstrated that 2HBP enters into the cells in a similar way. In contrast, the reporter system in the wild-type cells does not react to 2-HBP at an outside concentration of 2.4 μΜ, whereas in mutant cells it does. This suggests that wild-type cells pump 2-HBP to the outside very effectively preventing accumulation of 2-HBP. 2HBP metabolism, therefore, is not efficient enough to lower the intracellular concentration and prevent toxicity. We conclude that P. azelaica HBP1 resistance to 2-HBP is mainly due to an efficient efflux system and that 2HBP in high concentrations exerts narcotic effects on the bacterial membrane. In the part of this thesis, we investigated the possibilities of bacteria to degrade pollutants at low concentrations (1 mg per L and below). As test components we used 2-hydroxybiphenyl, antibiotics and a variety of fragrances, many of which are known to be difficult to biodegrade. By using accurate counting of low numbers of bacterial cells we could demonstrate that specific growth on these compounds is possible. We demonstrated the accuracy of FC counting at low cell numbers (down to 103 bacterial cells per ml). Then we tested whether bacterial population growth could be specifically monitored at the expense of low substrate concentrations, us¬ing P. azelaica HBP1. A perfect relationship was found between growth rate, yield and 2-HBP concentrations in the range of 0.1 up to 5 mg per L. Mixing P. azelaica within sludge, however, suggested that growth yields in a mixed community can be much lower than in pure culture, perhaps because of loss of metabolic intermediates. We then isolated new strains from activated sludge using 2-HBP or antibiotics (Nal, AMP, SMX) at low concentrations (0.1-1 mg per L) as sole carbon and energy sub¬strate and PAO microdishes. The purified strains were then examined for growth on their respective substrate, which interestingly, showed that all strains can not with¬stand higher than 1 or 10 mg per L concentrations of target substrate. Thus, bacteria must exist that contribute to compound degradation at low pollutant concentrations but are inhibited at higher concentrations. Finally we tested whether specific biomass growth (in number of cells) at the expense of pollutants can also be detected with communities as starting material. Hereto, we focused on a number of fragrance chemicals and measured community biomass increase by flow cytometry cell counting on two distinct starter communities: (i) diluted Lake Geneva water, and dilute activated sludge from a wastewater treatment plant. We observed that most of the test compounds indeed resulted in significant biomass increase in the starter community compared to a no-carbon added control, but activated sludge and lake Geneva water strongly differed (almost mutually ex¬clusive) in their capacity to degrade the test chemicals. In two cases for activated sludge the same type of microbial community developed upon compound exposure, as concluded from transcription fragment length polymorphism analysis on community purified and PCR amplified 16S rRNA gene fragments. To properly test compound biodegradability it is thus important to use starter communities of different origin. We conclude that FC counting can be a valuable tool to screen chemicals for their biodegradability and toxicity. - Des milliers de produits chimiques sont libérés dans l'environnement mais beaucoup ont des effets inconnus, en particulier à basses concentrations. Ce travail de thèse contribue à notre comprehension des effets de la pollution en utilisant des bacteries comme des organismes-tests. Les bacteries sont importantes pour etudier cette ques¬tion car certaines d'entre elles peuvent degrader ou transformer les polluants, mais également parce qu'elles-mmes peuvent tre inhibees dans leur reproduction après avoit ete exposees à ces composes toxiques. Quand des effets inhibiteurs ont lieu, la composition de la communauté microbienne peut tre changee à long terme, ce qui mène à une reduction du service d'ecosystème offert par ces communautés. En consequence, après leur liberation dans l'environnement, les produits chimiques d'origine anthropogenique peuvent soit s'y accumuler et per¬sister, exerant ainsi des effets encore inconnus sur les organismes vivants. En plus d'acquérir des connaissances de base sur les effets des polluants à basses concentra¬tions sur les communautés microbiennes, un but applique de cette thèse était de développer des tests bases sur les bacteries afin d'identifier de nouveau composes pour leur toxicité ou leur biodégradation. Dans la première partie de ce travail, nous avons developpe un test base sur la cytometrie de flux (FC) sur des cellules de Pseudomonas fluorescens colorees par du bromure d'ethidium ou de l'iodure de propidium et exposees ou non à une palette de polluants sous des conditions de croissance oligotrophique. La cytometrie de flux est une technique qui connaît de nombreuses applications dans la microbiologie environ¬nementale. Cela est principalement du au fait qu'elle permet un comptage rapide et precis ainsi que l'évaluation de l'état physiologique, en particulier lorsqu'elle est combinée h des colorations fluorescentes. Ici, nous avons utilise la technique FC et des colorants fluorescents afin de mesurer l'effet que peuvent exercer certains pollu¬ants sur Pseudomonas ûuorescens SV3 . D'abord nous avons conu des tests oligo- trophiques qui nous permettent de suivre la croissance complète de cellules en culture h des densites faibles (104 -10 7 cellules par ml), sur de l'acetate de sodium à 0.1 mM, en presence ou absence de produits chimiques (2-chlorobiphenyl (2CBP), naphthalène (NAH), 4-chlorophenol (4CP), tetradecane (TD), chlorure de mercure(II) (HgCl2)) à différentes concentrations. Afin de montrer le devenir des bacteries tant au niveau de la cellule individuelle que celui de la population globale, après exposition à des series de composes chimiques, nous avons compte les cellules colorees avec du SYT09 (col¬orant fluorescent vert des acides nucléiques pour la discrimination des cellules par rapport au bruit de fond) en combinaison avec l'iodure de propidium (PI) ou le bromure d'ethidium (EB), indicateurs de l'intégrité de la membrane cellulaire avec FC. Nous avons observe que de nombreux composes testes avaient un effet sur la croissance bacterienne, resultant en une baisse du taux de reproduction de la pop¬ulation. En outre, la double coloration que nous avons utilisee dans cette etude SYT09/PI ou SYT09/EB a montre que les produits chimiques testes induisaient une reponse heterogène des cellules dans la population, divisant celle-ci en sous- populations "saine", "endommagee" ou "morte". Les nombres de cellules à partir du comptage et de la proportion de celles "saines" et "endommagees/mortes" ont ensuite ete utilises pour modeliser la croissance de P. ûuorescens SV3 exposee aux produits chimiques. La reduction nette dans la croissance de population est une consequence du fait que de plus en plus de cellules sont incapables de se reproduire, plutt que du fait d'une croissance plus lente de l'ensemble de la population. De plus, la proportion de cellules endommagees est correllee au dosage du compose chimique. Les résultats obtenus nous ont permis de conclure que le test oligotrophique que nous avons developpe peut tre utilise pour l'évaluation de la toxicité de produits chimiques sur différents modèles bacteriens. Des tests multiples peuvent tre lances en parallèle et les effets sont mesures en l'espace de huit heures. Par ailleurs, nous en déduisons que les produits chimiques exercént un effet sur la croissance des cellules de P. ûuorescens SV3, qui est heterogène parmi les cellules dans la population et depend du produit chimique. Il est intéressant de noter que dans les mmes tests d'exposition avec P. ûuorescens SV3, nous avons observe que certains composes qui n'ont pas conduit à une reduction du taux de la croissance nette de la population, ont cause des effets mesurables sur les cellule saines. Ceci a ete essentiellement observe dans la portion "saine" des cellules en tant qu'augmentation du signal de la fluorescence de 1ΈΒ. D'abord nous avons montre que SYT09/EB était une com¬binaison de colorants plus utile que celle de SYT09/PI parce que la fluorescence du PI a tendance à augmenter uniquement lorsque les cellules sont effectivement mortes, et non pas dans les cellules saines (moins de deux fois plus). Par opposi¬tion, la fluorescence moyenne de l'EB dans les cellules saines augmente jusqu'à huit fois plus après exposition aux composes toxiques. Tous les composes, mme aux plus basses concentrations, induisent une augmentation mesurable de la fluorescence moy¬enne de 1ΈΒ, plus particulièrement après deux heures d'incubation. Cet effet s'est revele tre transitoire pour les cellules exposees aux 2CNP et 4CP, mais est chro¬nique pour les cellules incubees avec le TD et le NAH (entranant la mort cellulaire). Afin de comprendre les mécanismes qui sous-tendent les effets observes, nous avons utilise des decoupleurs d'energie ou de membrane. L'augmentation du signal EB dans les populations causee par des produits chimiques ressemblait à celle exerce par le chelateur des ions divalents EDTA. Cependant, les intensités du signal EB des cellules exposees aux produits chimiques testees n'ont jamais atteint les valeurs des cellules traitees avec l'EDTA ou pasteurises. Nous en concluons que le test oli- gotrophique utilisant la coloration (SYT09/)EB des cellules exposees ou non à un produit chimique est utile afin d'evaluer l'effet toxique exerce par les polluants sur la physiologie bacterienne. Afin de mieux comprendre la reaction d'un système de defense par pompe à efflux après exposition à une toxine, nous avons étudié la toxicité du 2-hydroxybiphenyl (2-HBP) sur Pseudomonas azeiaica HBP1. Nous avons montre que le 2-HBP exerce une toxicité mme sur HBP1, mais uniquement à des concentrations supérieures à 0.5 mM. Au-dessus de cette concentration, des pertes transitoires d'intégrité et de polarization membranaire ont lieu, comme cela nous a ete montre par coloration des cellules en croissance. Les cellules sont finalement capables de se rétablir et de reprendre leur croissance sur 2-HBP. La forte resistance de P. azeiaica HBP1 h 2-HBP physiologie bacterienne s'est revele tre le résultat d'un système de pompe h efflux de type MexABOprM qui contre-balance l'influx passif de ce compose h travers la membrane. Nous avons montre, en construisant des mutants avec des insertions dans les gènes mexA, mexB and oprM et des fusions avec le gène rapporteur gfp, que l'altération de n'importe quelle partie du système d'efflux conduisait à accroître l'accumulation de 2-HBP dans la cellule, en comparaison avec la souche sauvage HBP1, provoquant une diminution de la resistance au 2-HBP ainsi qu'une baisse du taux de reproduction des cellules. Des systèmes d'efflux similaires sont répandus chez de nombreuses espèces bactériennes. Ils seraient responsables de la resistance aux produits chimiques tels que les colorants fluorescents (bromure d'ethidium) et des antibiotiques. Nous concluons que la resistance de P. azelaica HBP1 à 2-HBP est principalement due à un système d'efflux efficace et que 2-HBP, à des concentrations elevees, exerce un effet deletère sur la membrane bacterienne. En se basant sur le comptage des cellules avec la FC, nous avons developpe ensuite une methode pour evaluer la biodegradabilite de polluants tels que le 2-HBP ainsi que les antibiotiques (acide nalidixique (Nal), ampicilline (AMP) ou sulfamethoxazole (SMX)) à de faibles concentrations lmg par L et moins), par le suivi de la croissance spécifique sur le compose de cultures microbiennes pures et mixtes. En utilisant un comptage precis de faibles quantités de cellules nous avons pu demontrer que la croissance spécifique sur ces composes est possible. Nous avons pu illustrer la precision du comptage par cytometrie de flux à faible quantité de cellules (jusqu'à 10 3 cellules par ml). Ensuite, nous avons teste s'il était possible de suivre dynamiquement la croissance de la population de cellules sur faibles concentrations de substrats, en utilisant P. azelaica HBP1. Une relation parfaite a ete trouvee entre le taux de croissance, le rendement et les concentrations de 2-HBP (entre 0.1 et 5 mg par L). En mélangeant HBP1 à de la boue active, nous avons pu montrer que le rendement en communauté mixtes pouvait tre bien inférieur qu'en culture pure. Ceci étant peut tre le résultat d'une perte d'intermédiaires métaboliques. Nous avons ensuite isole de nouvelles souches à partir de la boue active en utilisant le 2-HBP ou des antibiotiques (Nal, AMP, SMX) h basses concentrations (0.1-1 mg par L) comme seules sources de carbone et d'energie. En combinaison avec ceci, nous avons également utilise des microplaques PAO. Les souches purifiees ont ensuite ete examinees pour leurs croissances sur leurs substrats respectifs. De faon intéressante, toutes ces souches ont montre qu'elles ne pouvaient pas survivre à des concentrations de substrats supérieures à 1 ou 10 mg par L. Ainsi, il existe des bacteries qui contribuent à la degradation de composes à basses concentrations de polluant mais sont inhibes lorsque ces concentrations deviennent plus hautes. Finalement, nous avons cherche à savoir s'il est possible de detecter une croissance spécifique à une biomasse au depend d'un polluant, en partant d'une communauté microbienne. Ainsi, nous nous sommes concentre sur certains composes et avons mesure l'augmentation de la biomasse d'une communauté grce à la cytometrie de flux. Nous avons compte deux communautés de depart distinctes: (i) une dilution d'eau du Lac Léman, et une dilution de boue active d'une station d'épuration. Nous avons observe que la plupart des composes testes ont entrane une augmentation de la biomasse de depart par rapport au control sans addition de source de carbone. Néanmoins, les échantillons du lac Léman et de la station d'épuration différaient largement (s'excluant mutuellement l'un l'autre) dans leur capacité à degrader les composes chimiques. Dans deux cas provenant de la station d'épuration, le mme type de communauté microbienne s'est developpe après exposition aux composes, comme l'a démontré l'analyse TRFLP sur les fragments d'ARN 16S purifie de la communauté et amplifie par PCR. Afin de tester correctement la biodegradabilite d'un compose, il est donc important d'utiliser des communautés de depart de différentes origines Nous en concluons que le comptage par cytometrie de flux peut tre un outil de grande utilité pour mettre en valeur la biodegradabillite et la toxicité des composes chimiques.

Tree of Sex: A database of sexual systems

The vast majority of eukaryotic organisms reproduce sexually, yet the nature of the sexual system and the mechanism of sex determination often vary remarkably, even among closely related species. Some species of animals and plants change sex across their lifespan, some contain hermaphrodites as well as males and females, some determine sex with highly differentiated chromosomes, while others determine sex according to their environment. Testing evolutionary hypotheses regarding the causes and consequences of this diversity requires interspecific data placed in a phylogenetic context. Such comparative studies have been hampered by the lack of accessible data listing sexual systems and sex determination mechanisms across the eukaryotic tree of life. Here, we describe a database developed to facilitate access to sexual system and sex chromosome information, with data on sexual systems from 11,038 plant, 705 fish, 173 amphibian, 593 non-avian reptilian, 195 avian, 479 mammalian, and 11,556 invertebrate species.

Consequences of genetic erosion on fitness and phenotypic plasticity in European tree frog populations (Hyla arborea).

The detrimental effects of genetic erosion on small isolated populations are widely recognized contrary to their interactions with environmental changes. The ability of genotypes to plastically respond to variability is probably essential for the persistence of these populations. Genetic erosion impact may be exacerbated if inbreeding affects plastic responses or if their maintenance were at higher phenotypic costs. To understand the interplay 'genetic erosion-fitness-phenotypic plasticity', we experimentally compared, in different environments, the larval performances and plastic responses to predation of European tree frogs (Hyla arborea) from isolated and connected populations. Tadpoles from isolated populations were less performant, but the traits affected were environmental dependant. Heterosis observed in crosses between isolated populations allowed attributing their low fitness to inbreeding. Phenotypic plasticity can be maintained in the face of genetic erosion as inducible defences in response to predator were identical in all populations. However, the higher survival and developmental costs for isolated populations in harsh conditions may lead to an additional fitness loss for isolated populations.

Maintenance of ancestral sex chromosomes in Palearctic tree frogs: direct evidence from Hyla orientalis.

Contrasting with the situation found in birds and mammals, sex chromosomes are generally homomorphic in poikilothermic vertebrates. This homomorphy was recently shown to result from occasional X-Y recombinations (not from turnovers) in several European species of tree frogs (Hyla arborea, H. intermedia and H. molleri). Because of recombination, however, alleles at sex-linked loci were rarely diagnostic at the population level; support for sex linkage had to rely on multilocus associations, combined with occasional sex differences in allelic frequencies. Here, we use direct evidence, obtained from anatomical and histological analyses of offspring with known pedigrees, to show that the Eastern tree frog (H. orientalis) shares the same pair of sex chromosomes, with identical patterns of male heterogamety and complete absence of X-Y recombination in males. Conservation of an ancestral pair of sex chromosomes, regularly rejuvenated via occasional X-Y recombination, seems thus a widespread pattern among Hyla species. Sibship analyses also identified discrepancies between genotypic and phenotypic sex among offspring, associated with abnormal gonadal development, suggesting a role for sexually antagonistic genes on the sex chromosomes.

Molecular Proxies for Climate Maladaptation in a Long-Lived Tree (Pinus pinaster Aiton, Pinaceae).

Understanding adaptive genetic responses to climate change is a main challenge for preserving biological diversity. Successful predictive models for climate-driven range shifts of species depend on the integration of information on adaptation, including that derived from genomic studies. Long-lived forest trees can experience substantial environmental change across generations, which results in a much more prominent adaptation lag than in annual species. Here, we show that candidate-gene SNPs (single nucleotide polymorphisms) can be used as predictors of maladaptation to climate in maritime pine (Pinus pinaster Aiton), an outcrossing long-lived keystone tree. A set of 18 SNPs potentially associated with climate, 5 of them involving amino acid-changing variants, were retained after performing logistic regression, latent factor mixed models, and Bayesian analyses of SNP-climate correlations. These relationships identified temperature as an important adaptive driver in maritime pine and highlighted that selective forces are operating differentially in geographically discrete gene pools. The frequency of the locally advantageous alleles at these selected loci was strongly correlated with survival in a common garden under extreme (hot and dry) climate conditions, which suggests that candidate-gene SNPs can be used to forecast the likely destiny of natural forest ecosystems under climate change scenarios. Differential levels of forest decline are anticipated for distinct maritime pine gene pools. Geographically defined molecular proxies for climate adaptation will thus critically enhance the predictive power of range-shift models and help establish mitigation measures for long-lived keystone forest trees in the face of impending climate change.