Jean-Christophe Aymes

Edward Beall

Marc Jarry

Philippe Gaudin

Philippe Gaudin Brown

Brown trout life history and habitat use in the Kerguelen Islands as revealed by otolith microchemistry

Brown trout life history and habitat use in the Kerguelen Islands as revealed by otolith microchemistry

Résumé

Characterization of the life history traits of introduced brown trout (Salmo trutta L.) in the Kerguelen Islands is fundamental to understand the success of the islands invasion by this fish species. The capacity to use marine habitats (Anadromy) by brown trout is one of the most critical traits in the invasion process and its study is essential to understand how marine habitats contribute to dispersal in trout populations. Otoliths are calcified structure (aragonite) located in the inner ear of fishes; they record seasonal growth as scales but are also inert and able to trap trace element present in the fish environment (Campana and Thorrold, 2001). Strontium, particularly the Sr:Ca ratio, is a common indicator used to trace brackish/salt water migrations of salmonids (Limburg et al., 2001;Milton et al.,2008). We studied Sr:Ca ratios on otoliths of brown trout captured in specific habitats (lake, upstream and downstream zones of rivers, marine coastal waters) to characterize life history traits linked to freshwater or salt water migration (resident/migrant status, age at migration, homing, maternal origin). Results showed that the method is relevant because water and otolith present correlated chemical signatures across each selected Kerguelen habitat. A strong diversity of life histories appeared, ranging from pure residency in freshwater to permanent use of saltwater. Intermediate phenotypes were observed in downstream populations taking benefit of the highly productive estuarine habitats whereas specific migratory phenotypes could contribute to colonize new pristine aquatic systems.

Réferences

Campana, S.E., 1999. Chemistry and composition of fi sh otoliths: pathways, mechanisms and applications. Marine Ecology Progress Series 188, 263-297. Limburg, K.E., Landergren, P., Westin, L., Elfman, M., Kristiansson, P., 2001. Flexible modes of anadromy in Baltic sea trout: making the most of marginal spawning streams. Journal of Fish Biology 59 (3), 682-695. Milton, D.A., Chenery, Simon R, 2001. Sources and uptake of trace metals in otoliths of juvenile barramundi (Lates calcarifer). Journal of Experimental Marine Biology and Ecology 264 (1), 47e65.

J

  -C Aymes¹•², E. Beall¹•², M. Jarry²•¹, P. Gaudin¹•² ¹ INRA, UMR ECOBIOP, INRA/UPPA, Pôle d'Hydrobiologie de l'INRA, Quartier Ibarron 64310 Saint Pée sur Nivelle, France ² Univ Pau & Pays Adour, UMR ECOBIOP, INRA/UPPA, UFR Côte Basque, Allée du Parc Montaury, 64600 Anglet, France


Anne-Sophie Clémençon

Keywords: Urban forms, landscape, Canada, Quebec city

teaching and research institutions in France or abroad, or from public or private research centers.

The case study presented in Morisset's book refers to an important area of Quebec City whose name is Saint-Roch. The title of the book may be confusing. Quebec being a city and a province as well, we can guess that the study is about some other town. However, this confusion reveals something. One of the central subjects of the study is precisely the competition between Saint-Roch, the industrial, business, American looking district, with the administrative and European looking old part of the town, settled on the hill. The author traces the fascinating urban development of Saint-Roch from the 17th century to now and its different stages : a suburb cut from the city, the industrial and economic heart of the whole town, then a rejected and almost pulled down area and finally a rehabilitated part of the town center. By rediscovering the memory of this important part of Quebec City, on the one hand Morisset rehabilitates its image and, on the other hand, she gives us a completely new vision of the whole town.

History of urban forms is the methodological approach stated by Morisset and based on the current urban landscape and a majority of first-hand sources. It focuses on the network of streets, land divisions, volume built, and urban models used to plan the district, when this is the case, and the representations of this later. The different persons occuring in the making the town are identified as well as the traces they have left within the urban fabric. The complex connections between the economic, social and political history of Quebec city and the making of Saint-Roch district are well explained. The book contains 300 illustrations, well crossreferenced to the text, with long captions which provide a second level of comprehension :old and recent photographs, paintings, prints, typological models of dwellings…More than anything, the author gives us numerous maps and knows how to interpret them : maps from archives and the author's own maps summing up the increase of the network of the streets. The maps are sometimes too small and the page set-up is classical, but it is thorough and efficient for the reader. A general bibliography, sources list (texts and maps) and detailed index are also included.

There can be no doubt that this book represents a significant contribution to the understanding of the evolutionary process of urban forms. The scale of the case study chosen by the author, the district, is in my opinion, the best to analyse such processes. In fact, by studying a whole city, we usually focus on local council politics and main developments which tend then to hide the complexity of the making of the ordinary town. The district is the only scale where we can precisely observe the links between town and buildings. This study, made possible by an important and thorough work, is a very precise historical reconstitution. Divided in chapters following the chronological development, it gives an opportunity to identify numerous themes which come out from this history. The three chapters devoted to the 18th and 19th centuries, periods which are particularly stressed on, are especially interesting and new.

A detraction from the general excellence of the study is a general lack of synthesis. The reading is made difficult because Morisset tells us a story based on a lot of successive small events. I would say, a story from the inside. So, it is difficult for readers to locate themselfs within Saint-Roch and within the whole town if they don't know the place. If the urban processes are precisely described, the synthesis has not been made For example, numerous strong themes run through the book such as "identity and urban form" (French, British, North American and European influences), "actors making the town", "urban regulations", "connections (or not) between streets, plots and buildings"…but they are never identified for themselves. Except for the 20th century, there is no attempt to broaden the subject in the direction of other towns or other countries. This question aside, this is a valuable work which inclines a French reader to make comparisons. The most surprising is to realise how similar the evolutionary processes of urban forms are until the second world war, between a North American district and those studied at the same scale in Europe. Nevertheless, this is not the same continent, the same history nor the same culture. It is very interesting to observe this parallelism during a period where a strong centralised town planning doesn't exist yet, that is to say from the first settlement to the end of the 19th century. During the following period, until the second world war, the processes are close too, but they are better known. North American references to the main urban models are not far from European, even if the vocabulary is different. A comparative work between North American and European notions in different languages would be very useful (for example :"City beautiful", "haussmannisation", "embellissement des villes"…). After 1950, differences in town development between both continents increase, mainly because of a very different influence of cars. Comparative works in the history of urban forms have to be developed to bring to light the processes which are common in the making of towns. We strongly need studies made on the same scale and with the same accuracy as Morisset's book, this is why this latter is a valuable contribution towards a common knowlege.

Anne-Sophie Clémençon. Centre National de la Recherche Scientifique et Université Lumière Lyon 2. Institut des Sciences de l 'Homme,16 avenue Berthelot,69363 Lyon,

  Book review of The landscape's memory. The urban form history of a town centre : Saint-Roch, Quebec (La mémoire du paysage. Histoire de la forme urbaine d'un centre-ville : Saint-Roch, Québec", by Lucie K.Morisset Anne-Sophie A.S. Clémençon To cite this version: Anne-Sophie A.S. Clémençon. Book review of The landscape's memory. The urban form history of a town centre : Saint-Roch, Quebec (La mémoire du paysage. Histoire de la forme urbaine d'un centre-ville : Saint-Roch, Québec", by Lucie K.Morisset. Urban Morphology, 2003, 7 (2), pp.130. halshs-00280894


Gentsch Lydia

Sturm Patrick

Wingate Lisa

Ogée Jérôme

Siegwolf Rolf

Hammerle Albin

Plüss Peter

Baur Thomas

Barthel Matthias

Buchmann Nina

Knohl Alexander

NATURAL VARIATIONS OF THE 13 C/ 12 C SIGNATURE OF LEAF NOCTURNAL RESPIRATION IN FAGUS SYLVATICA BRANCHES RELATED TO PREVIOUS DAY PHOTOSYNTHESIS

Leaf respiration represents about a quarter of total plant carbon loss. Understanding the dynamics of this flux and identifying its substrates is therefore a prerequisite for an accurate description of the carbon budget of terrestrial ecosystems. The 13 C/ 12 C ratio of nocturnal leaf respiration (δ 13 C noc ) is one of the few signals that can provide clues on the dynamics of leaf non-structural carbohydrate pools in the field and their use for leaf respiration. However, the physiological mechanisms driving long-and short-term dynamics of δ 13 C noc are not straightforward, given likely impacts of post-photosynthetic fractionations, temporal dynamics in putative respiratory substrates and the modulation of the latter two by environmental drivers, such as temperature. Progress in laser spectrometry technology now enables high-frequency measurements of δ 13 C noc in the field and hence supports the pursuit for a better mechanistic understanding of nocturnal leaf respiration. Here, we present continuous, hourly measurements of nocturnal respiration (R noc ) and δ 13 C noc on leafy branches of three mature Fagus sylvatica trees from a temperate mixed-deciduous forest in Switzerland. These measurements were conducted with open branch chambers and a laser spectrometer for CO 2 isotopologue measurements (QCLAS-ISO, Aerodyne Research Inc.) in August and September during two consecutive years (in total 38 and 60 days). The data was then analysed for trends occurring over the course of individual nights and for trends from one night to the next. Night-to-night variation in flux-weighted means of δ 13 C noc was clearly linked to previous day photosynthetic 13 C discrimination, measured by the same chambers. This link was, however, modified by the strength of previous-day photosynthesis. For a large fraction of measured nights, δ 13 C noc decreased progressively over the course of the night (up to 0.7‰ hour -1 ). This trend was analysed with regard to concurrent dynamics of R noc , leaf temperature and previousday environmental conditions. Collectively, these results allowed us to draw a clearer picture of leaf respiratory processes of F. sylvatica branches in the field.


Nitrogen needs On line monitoring

In the future?

. A major challenge :

. to analyse complex criteria : typicity, aroma production … . using tests:

. mimicking industrial conditions, including variability . in standardized conditions

. New approaches :

.Genomic -post genomic -system biology approaches Relation between an ester and its precursor higher alcohol

  ability to grow and ferment without oxygen S2 De-aerated Chardonnay must S11 Julien et al., 2000

  effect than lipids Control of oxygenation -difficulties : Small amounts (several mg/l) During fermentation 'Useful oxygen' = oxygen transferred into the medium Oxygen additions : firstly to limit risks of stuck fermentations … About 10 mg/l Best moment : end of celle growth (about 20% of fermentation) … but also to modify wine aroma needs for additional studies needs for new tools , e.g., possibility of very slow addition (transfer) addition = f(time) or f(fermentation progress), e.g. 1 mg/l/

Sydney -July 15 , 2013

On line monitoring of fermentation

Sydney -July 15 , 2013 Higher alcohol and esters Markers of :

. aroma compounds : volatility . metabolism : key pathways of carbon and nitrogen metabolism Metabolic pathways well known but : . complex regulations . example : no direct relationship between must nitrogen composition and higher alcohol synthesis


Félix Massiot-Granier

Etienne Prevost

Gérald Chaput

Gordon Smith

Jonathan White

Ted Potter

Etienne Rivot

Demographic response of Atlantic salmon populations to multiple stressors: a Bayesian hierarchical modelling approach at broad ocean scale

Keywords: Atlantic salmon, Bayesian Hierarchical Model, life cycle, scale, climate change

de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

  Demographic response of Atlantic salmon populations to multiple stressors : a Bayesian hierarchical modelling approach at broad ocean scale Félix Massiot-Granier, Etienne Prévost, Gerald Chaput, Gordon Smith, Jonathan White, Ted Potter, Etienne Rivot To cite this version: Félix Massiot-Granier, Etienne Prévost, Gerald Chaput, Gordon Smith, Jonathan White, et al.. Demographic response of Atlantic salmon populations to multiple stressors : a Bayesian hierarchical modelling approach at broad ocean scale. ICES Annual Science Conference, International Council for the Exploration of the Sea (ICES). DNK., Sep 2013, Reykjavik, Iceland. 1 p. hal-01210279


domain: sdv sde

text:
Elena Orozco

Claire Ferret

Stéphanie Lawniczak

Isabelle Schalk

Guillaume Echevarria

Thibault Sterckeman

Valérie Geoffroy

New insight in fluorescence Pseudomonas iron acquisition in soil: pyoverdine plays a key role in iron oxydes weathering

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

♦ Pyoverdine has a high affinity for Fe (III) but is also able to chelate others metals (Al, Cd, Ni, Cu).

Iron oxides were prepared accorded to the methods of Schwertzmann and Cornell 1991. A goethite substituted with aluminium was prepared. Objectifs : Bacteria required iron for essential metabolic processes. However, ferric iron is poorly available in soil due to various parameters (pH, oxygen, cristallinity, specific area,…). To get access to this crucial element, bacteria produce in their environment siderophore, a high affinity Fe 3+ chelator. As a reference, pyoverdine was used as the main siderophore produced by fluorescent Pseudomonas. First, this research aims to understand the pyoverdine-mediated dissolution of different iron-oxides.

Electron micrographs of synthetic ferrihydrite 2-lines and goethite

Amorphous

We tested two iron-oxides, a well crystallized goethites and a poorly crystallized ferrihydrite. Secondly, the involvement of metabolizing siderophore-producing bacteria was investigated on iron-bearing minerals dissolution. In the presence of goethite, the fluorescence of pyoverdine decreased, meaning that a pyoverdine-Fe complex was formed. At 1 g. L -1 , the fluorescence was almost repressed.

Fe + Pvd-Fe Pvd

In the presence of pyoverdine, iron concentration increased in solution. The amorphous iron oxyde, ferrihydrite, was more easily weathered by pyoverdine than the well-crystallized oxyde, goethite.

+ Pvd (50 µM)

Time (h) Time (h)

Bacterial number was enumerated by plating on LB agar, incubation at 30°C/24H. In the presence of minerals, the bacterial growth was stimulated.

Alteration of iron oxides in the presence of pyoverdine-producing Pseudomonas

Pyoverdine concentration was measured by the absorbance at 400 nm.

In the presence of minerals containing a large amount of iron (11,2 mM), a decreased of 50% in the pyoverdine production was observed.

Iron determination was measured by ICP-AES after acidification of the supernatant. Iron was released in the supernatant of a 48h bacterial growth in an iron-deficient medium. With Al-goethite, both Fe and Al was detected in the supernatant.

Control in minimum medium

Control in minimum medium

In conclusion, the iron solubilisation from the iron oxides explained the growth stimulation and siderophore repression observed with Pseudomonas. The weathering of iron oxides by the siderophore itself and to a lesser extend by siderophore-producing bacteria, enhanced the release of substituted metal found in the crystals, that could be potentially toxic for the environment. Further investigations are in process to better understand the interaction between bacteria and minerals and to determine the mechanism involved in iron chelation.

1.♦

  Université de Strasbourg, Biotechnologie et signalisation cellulaire, UMR 7242, ESBS, 300 bd Brant BP 10413, 67412 Illkirch Cedex 2. Université de Strasbourg, Laboratoire d'Hydrologie et de Géochimie de Strasbourg, UMR7517,1 Rue BLESSIG, 67084 STRASBOURG 3. INRA, Université de Lorraine, Laboratoire Sols et Environnement, UMR 1120, BP 172, Vandoeuvre-lès-Nancy Cedex, F-54505, France New insight in fluorescence Pseudomonas iron acquisition in soil: pyoverdine plays a key role in iron oxydes weathering Bacteria: fluorescent Pseudomonas (P. aeruginosa ATCC 15692) ♦ Widespread bacteria in environmental niches (Soil, sediments, water,…) ♦ Siderophore produced: Pyoverdine type 1 (Pvd)

  Orozco Elena 1 , Ferret Claire 1* , Lawniczak Stéphanie 2 , Schalk Isabelle 1 , Echevarria Guillaume 3 , Sterckeman Thibault 3 and Geoffroy Valérie 1 .

Ferrihydrite


Introduction

A standpoint in systemic agronomy

Agronomy is envisaged here as a management science Agronomy is envisaged here as a management science, dealing with the management g g of agricultural vegetation covers

Classically:

Decisions, coordination of practices and Classically: , p organization of resources (workforce, land, machineries…) with the goal of:

Vegetation covers that are most directly productive: crops i) Insuring a desired production (nature, quantity, quality), productive: crops, grassland managed for fodder production and / ii) Insuring a coherent functioning of the entity of management (cropping system, f dd f f f ) or pasture.

fodder system, farm, group of farms, etc.) Malezieux, E., Crozat, Y., Dupraz, C., Laurans, M., Makowski, D., Ozier-Lafontaine, H., Rapidel, B., de Tourdonnet, S., Valantin-Morison, M., 2009

What processes to control to favour

The Ecosystem services depend on the functioning and resilience of agrol i l ecological systems

Resilience: Capacity of a system to b b h k ith i it absorb shocks with conserving its essential functions, its capacities of functioning

Walker, B., C. S. Holling, S. R. Carpenter and A. Kinzig, 2004. Resilience, adaptability and transformability in social-ecological systems. Ecology and Society 9. URL: http://www.ecologyandsociety.org/vol9/iss2/art5

Theories about sociotechnical transitions (e.g., Geels, 2010) Theories about resilience of socialecological systems (e.g., Gunderson and Holling, 2002) • aim at giving tools for not only identifying but also interpreting the features of evolution of social-ecological and socio-technical systems in terms of sustainability.

• share the idea of three key-features (among others) share the idea of three key features (among others) o path-dependency (including feed-back loops and cycles), o regime shifts, notably radical shift such as collapse or radical innovations o effects of "lock-in", "rigidity traps" 14 Geels, F.W., 2010. Ontologies, socio-technical transitions (to sustainability), and the multi-level perspective. Research policy 39, 495-510.

What kind of trajectories to describe and interprete from a farming management viewpoint? viewpoint?

«

  Processes at the origin of the service are observed Efficient service? Ecological processes are developing in such a way that farmer's decision making such a way that farmer s decision making can rely on 9 Inspiré de: Mérot, P.,Hubert-Moy, L., Gascuel-Odoux, C., Clément, B., Durand, P., Baudry, J., Thenail, C., 2006. A method for improving the management of controversial wetlands. Environmental Management 37, 258-270. Account for all kinds of practices dedicated to all kinds of landscape used in farm or sold e.g., sowing, bringing water to Thenail, C., Roche, B., Joannon, A., Le Coeur, D., in preparation. . Farmers' management of landscape resources: the case of field margins.cattle, collecting firewood. Explore farmers' decision making that relies or may rely on landscape Les services sont issus du fonctionnement d'un système et c'est la préservation du « Les services sont issus du fonctionnement d un système et c est la préservation du système qu'on veut atteindre » (Marie-Pierre 07/11/13).

  Proposals inspired from Godard, 2001 / framework of analysis of sustainability Medium term: Evolution of the tensions between: o practices of management of the production, o and practices of management of the landscape resources Medium term: Evolution of the tensions between: social, economic, ecological dimensions of farming practices (interd l h) disciplinary approach) Evolution of the tension between: o short -medium term management (production, maintenance, laying-out) o and long term patrimonial management 15 o and long term patrimonial management Godard, O., 2001. Le développement durable et la recherche scientifique, ou la difficile conciliation des logiques de l'action et de la connaissance. . In: Jollivet, M. (Ed.), Le développement durable, de l'utopie au concept. Nature Sciences Société -Elsevier, Paris, pp. 61

Evolution of the challenges in agriculture: debates in agronomy as well Minimize environmental impacts of agriculture Land sparing (ségrégation Minimize environmental impacts of agriculture while conserving even reinforce classical objectives of production, modes and objects of management (crops and grassland)

	dans l'espace entre nature
	et agriculture)
	management (

crops and grassland) Sustainability imposes to open agriculture to the management of natural resources… even of ecosystem services… which supposes to modify

		Land sharing
	significantly objectives, modes and objects of	( (partage de l'espace entre d l'
	management.	nature et agriculture)

1. From resources to ecosystem services at farm to landscape scales

	For enhancing	Ecosystem services of regulation
	ecosystem services	at a field scale:
	at field scales at field scales:	e g enhancement of organic e.g., enhancement of organic
	mixing plant species in cropping systems	matter, of favourable microclimate, control of weed flora development, control of flora development, control of
		pests, etc.

Hedgerows as resources associated to the land E l 20th Early 20th century

Early 21th Century

Late 20th century

The concerned owners and farmers may have changed several times


Patrick Von Aderkas

Thomas Boivin

Cindy Gidoin

Lionel Roques

Jean-Noël Candau

Steve Perlman

Ward Strong

Amber Paulson

Jan Hanneman

Ian Boyes

Jüergen Ehlting

Camille Péron

Marie-Anne Auger-Rozenberg

Alain Roques

Marie-Jo Hadrien

Julie Aurore

Marion

Emma Guillaume

for discussions and good times in Bamfield. Thank you

Introduction

Species living in seasonal and stochastic environments have to cope with future uncertainty.

There is therefore strong selection for life cycles to coincide with the spatial and temporal heterogeneity of abiotic and/or biotic environmental factors [START_REF] Tauber | Seasonal Adaptations of Insects[END_REF][START_REF] Danks | Insect Dormancy: an Ecological Perspective[END_REF]. In animal species, diapause is a kind of dormancy shown to have a key role in the evolution of life histories by providing an adaptive response to changing environments [START_REF] Tauber | Seasonal Adaptations of Insects[END_REF][START_REF] Roff | The evolution of life histories: Theory and analysis[END_REF]. Diapause has been described as an integrated response to the predictability of environmental conditions, allowing survival under inappropriate portions of the year for growth and reproduction [START_REF] Philippi | Hedging one's evolutionary bets, revisited[END_REF], but many species experiencing spatial or interannual fluctuations of their environment extend diapause over more than one year [START_REF] Waldbauer | Phenological adaptation and the polymodal emergence patterns of insects[END_REF][START_REF] Hanski | Four kinds of extra long diapause in insects: a review of theory and observations[END_REF][START_REF] Philippi | Bet-hedging germination of desert annuals: beyond the first year[END_REF]. Such a prolonged diapause is viewed as being an alternative strategy to dispersal [START_REF] Hanski | Four kinds of extra long diapause in insects: a review of theory and observations[END_REF][START_REF] Hairston | Zooplankton egg banks as biotic reservoirs in changing environments[END_REF], which, despite its associated metabolic, survival and reproductive costs [START_REF] Soula | Variability in diapause duration in the chestnut weevil: mixed ESS genetic polymorphism or bet-hedging?[END_REF], is generally thought to generate a population buffer against environmental constraints acting against non-diapausing stages [START_REF] Hairston | Zooplankton egg banks as biotic reservoirs in changing environments[END_REF][START_REF] Corley | Prolonged diapause and the stability of host-parasitoid interactions[END_REF][START_REF] Widenfalk | Slow response to strong disturbance in an insect population with a temporal refuge[END_REF][START_REF] Corley | Delayed emergence and the success of parasitoids in biological control[END_REF] In insects, emergence from prolonged diapause may be concentrated to a single year associated with the appearance of a periodically produced food resource [START_REF] Annila | Diapause and population fluctuations in Megastigmus specularis Walley and Megastigmus spermotrophus Wachtl. (Hymenoptera, Torymidae)[END_REF][START_REF] Roques | New results and some thinkings about significance and induction of prolonged diapause in cone insects with particular references to the larch cone fly (Lasiomma melania) and to the Douglas-fir seed chalcid (Megastigmus spermotrophus)[END_REF]. But in most cases environmental conditions are less predictable and emergences in a cohort are spread over several years, with a higher proportion of individuals emerging after the minimum diapause duration (obligatory diapause) and decreasing proportions of individuals emerging over the succeeding years [START_REF] Danks | Insect Dormancy: an Ecological Perspective[END_REF][START_REF] Hanski | Four kinds of extra long diapause in insects: a review of theory and observations[END_REF][START_REF] Menu | Strategies of emergence in the chestnut weevil Curculio elephas (Coleoptera: Curculionidae)[END_REF][START_REF] Boyce | The diapause phenomenon in insects, with special reference to Rhagoletis complete Cress.(Diptera: Trypetidae)[END_REF].

Variability in diapause duration and its evolutionary insights have been documented in various model systems such as insects [START_REF] Hanski | Four kinds of extra long diapause in insects: a review of theory and observations[END_REF][START_REF] Menu | Strategies of emergence in the chestnut weevil Curculio elephas (Coleoptera: Curculionidae)[END_REF][START_REF] Menu | Bet-hedging diapause strategies in stochastic environments[END_REF][START_REF] Soula | Variability in diapause duration in the chestnut weevil: mixed ESS genetic polymorphism or bet-hedging?[END_REF][START_REF] Gourbière | Adaptive dynamics of dormancy duration variability: evolutionary tradeoff and priority effect lead to suboptimal adaptation[END_REF] and crustaceans [START_REF] Ellner | Role of overlapping generations in maintaining genetic variation in a fluctuating environment[END_REF][START_REF] Hairston | Zooplankton egg banks as biotic reservoirs in changing environments[END_REF][START_REF] Caceres | Incidence of diapause varies among populations of Daphnia pulicari[END_REF]. Such variation in the diapause trait has been mainly attributed to diversified bet-hedging [START_REF] Hopper | Risk-spreading and bet-hedging in insect population biology[END_REF][START_REF] Menu | Bet-hedging diapause strategies in stochastic environments[END_REF][START_REF] Gourbière | Adaptive dynamics of dormancy duration variability: evolutionary tradeoff and priority effect lead to suboptimal adaptation[END_REF], a riskspreading strategy maximising the mean geometric fitness by minimising the fitness variance at the cost of a lower arithmetic mean fitness [START_REF] Philippi | Hedging one's evolutionary bets, revisited[END_REF][START_REF] Roff | The evolution of life histories: Theory and analysis[END_REF][START_REF] Hopper | Risk-spreading and bet-hedging in insect population biology[END_REF]. The role of prolonged diapause as a demographic refuge in stochastic environments has been demonstrated empirically and theoretically when variability of diapause duration decreases population extinction risks due to unpredictable catastrophic events such as sudden limitation in food supplies or enhanced predation or pathogenic risks [START_REF] Tuljapurkar | Environmental uncertainty and variable diapause[END_REF][START_REF] Menu | Coin-flipping plasticity and prolonged diapause in insects: example of the chestnut weevil Curculio elephas (Coleoptera: Curculionidae)[END_REF][START_REF] Menu | Bet-hedging diapause strategies in stochastic environments[END_REF]Menu and Deshouant, 2002;[START_REF] Widenfalk | Slow response to strong disturbance in an insect population with a temporal refuge[END_REF]. Indeed, prolonged diapause has been shown to increase the mean population growth rate in a stochastic environment, which may even facilitate the spreading phase of invasive populations when stochasticity is high [START_REF] Madjhoub | Prolonged diapause: A trait increasing invasion speed?[END_REF]. Moreover, prolonged diapause is likely to contribute to the stability and persistence of coupled host-parasitoid interactions [START_REF] Ringel | The evolution of diapause in a coupled host-parasitoid system[END_REF][START_REF] Corley | Prolonged diapause and the stability of host-parasitoid interactions[END_REF]. From a population genetics standpoint, it is generally assumed that prolonged diapause may help promoting the maintenance of genetic diversity in a diapausing population, as late-emerging individuals probably do not experience similar demographic or selection events than early-emerging ones [START_REF] Ellner | The role of fluctuating selection and long-term diapause in microevolution of diapause timing in a freshwater copepod[END_REF]. However, to our knowledge, there is still a lack of studies assessing specifically the impact of prolonged diapause on temporal gene flow within natural populations. In this study, we will aim at determining whether prolonged diapause may constitute a genetic refuge for natural populations of a seed-specialized chalcid wasp, Megastigmus schimitscheki (Novitzky), facing interannual fluctuations in food resource supplies by its conifer host.

Cones and seeds of conifers are exploited by approximately 400 species of phytophagous worldwide, among which most spermatophages, i.e., insects developing entirely within seeds, can prolong diapause in the last larval instar for 1 to 5 years [START_REF] Turgeon | Insect fauna of coniferous seed cones: Diversity, Host plant interactions, and Management[END_REF]. Spermatophagous species of the Megastigmus genus are known to extend diapause in response to the dramatic annual variation in seed production characterizing most conifer species [START_REF] Turgeon | Insect fauna of coniferous seed cones: Diversity, Host plant interactions, and Management[END_REF][START_REF] Roux | Effect of temperature and photoperiod on diapause development in Douglas fir seed chalcid, Megastimus spermotrophus[END_REF]. In many conifer species, large seed crops are indeed more or less periodical and regionally synchronized, a phenomenon referred to as masting [START_REF] Kelly | The evolutionary ecology of mast seeding[END_REF]. M. schimitscheki is an obligate predator of true cedar seeds (Cedrus spp.). It was introduced in Southeastern France in the early 90s from cedar seeds from Cyprus (Auger-Rozenberg et al., 2012, submitted). Its native range is the Eastern Mediterranean region, where it develops on C. libani in the Near East (Turkey, Syria and Lebanon) and C. brevifolia in Cyprus [START_REF] Fabre | Competition between exotic and native insects for seed resources in trees of a Mediterranean forest ecosystem[END_REF]. Although those two cedar species were introduced in many Mediterranean countries as ornamental trees, French invasive populations of M. schimitscheki were observed to date solely in Southeastern France. M. schimitscheki progressively invaded most of the planted C. atlantica stands (Lander et al., 2012, submitted), even showing a competitive advantage over its direct and closely related resident competitor M. pinsapinis [START_REF] Boivin | Differences in life history strategies between an invasive and a competing resident seed predator[END_REF]Auger-Rozenberg and Roques, 2012). Adult wasps emerge from mature cedar cones in May when the seeds in young cones begin to develop [START_REF] Boivin | Differences in life history strategies between an invasive and a competing resident seed predator[END_REF]. Females lay their eggs directly inside cedar ovules by inserting their ovipositor through the young cone scales and the larval instars develop within the seed by consuming the female gametophyte and the seed embryo [START_REF] Rouault | Oviposition strategies of conifer seed chalcids in relation to host phenology[END_REF]. As a univoltine species, M. schimitscheki produces one larval cohort per year. The main host of M. schimitscheki in France, C. atlantica, is a masting species [START_REF] Kelly | The evolutionary ecology of mast seeding[END_REF], displaying highly variable interannual seed productions [START_REF] Krouchi | Year and tree effect on reproductive organisation of Cedrus atlantica in a natural forest[END_REF]. In French C. atlantica stands, years of large seed crops alternate with years of low or null seed crops, suggesting an impact of such variations in resource availability on the demography of its specialized predator M. schimitscheki.

In this study we will address three questions:

-What is the pattern of prolonged diapause of M. schimitscheki in southeastern France?

-Is there a genetic signature of environmental stochasticity in M. schimitscheki populations? -Does prolonged diapause disrupt or maintain gene flow within natural French M. schimitscheki populations?

For this purpose, the pattern of adult emergence was characterized over four consecutive years in M. schimitscheki populations sampled throughout the current distribution of the species in France.

Using microsatellite markers, a temporal population genetic approach was conducted on a natural French population of M. schimitscheki sampled during 10 consecutive years. This allowed us to test for the occurrence of bottlenecks in the population history, and to estimate gene flow between successive cohorts in the population and between individuals emerging from obligatory diapause and those emerging from prolonged diapause.

Material and methods

Wasp sampling

Cohorts of M. schimitscheki were sampled as diapausing larvae within C. atlantica cones.

Twenty five cedar stands were sampled throughout southeastern France from 1999 to 2008. In each stand, 30 to 50 cones were collected in the autumn before seasonal natural cone disarticulation. In the laboratory, cones were individually disarticulated. Once extracted from cones, seeds were sorted by densitometry in 95% alcohol (separation of healthy seeds from infested and empty ones).

Infested seeds were further selected using numerical X-ray radiography (Faxitron ® , 15-20 kV, 0.3-3 mA), grouped according to year and site of collection and stored under natural conditions. Adults emerging in the spring were identified, sexed and stored in pure ethanol à -20°C. Because M. schimitscheki produces one larval cohort per year [START_REF] Fabre | Competition between exotic and native insects for seed resources in trees of a Mediterranean forest ecosystem[END_REF][START_REF] Boivin | Differences in life history strategies between an invasive and a competing resident seed predator[END_REF], each wasp sample corresponded to a cohort of a given site at a given year.

Pattern of prolonged diapause in M. schimitscheki

First adult emergences from a wasp cohort produced in spring of year n can only occur in spring of year n+2 because C. atlantica seeds are released from cones only in the autumn of n+1.

Adult emergences at n+2 thus related to obligatory diapause, and those occurring at further years related to prolonged diapause. Prolonged diapause was assessed in each of the 25 sampled populations by estimating proportions of adult emergence at n+2 and at further years until these proportions reached zero.

Genetic analyses DNA extraction and genotyping

Temporal gene flows among cohorts of M. schimitscheki were estimated within one of the 25 sampled natural populations, Ventouret 7 (44°08'29.41''N; 5°23'10.01''E, altitude: 1085 m), which was located near the introduction area of the wasp and which is thus one of the oldest French populations. Ten consecutive cohorts (1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008) were sampled at Ventouret 7 (Table 1) and a total of 491 wasp females were genotyped at 9 microsatellite loci (Table 2). For each cohort considered, we genotyped a sample of all individuals of the cohort, either individuals emerging in n+2, n+3, n+4 and n+5. For the DNA extraction each insect was placed in a 200µL tube with a solution of 10% chelex resin 100 and 6µL of 10mg/ml proteanase K [START_REF] Walsh | Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material[END_REF]. A steel ball was added to each tube and the insects were macerated in the tubes using a Qiagen TissueLyser, run twice for 10 seconds at 20 hz. The tubes with the macerated insects were then incubated at 56°C for 2 hours. After the incubation the tubes were heated twice to 100°C for 15 minutes to stop the enzymatic reaction and then centrifuged at 4000rpm to pellet the resin from the chelex resin 100. 50µL of the supernatant containing the DNA were removed. The tubes were heated to 100°C for thirty minute in a thermocycler, centrifuged and diluted in 30μL H20 for the PCR reactions. PCRs were performed for nine microsatellite markers distributed into two multiplex MKA and MKB (

Genetic diversity, population bottleneck and temporal genetic differentiation analyses

File format conversions were all performed using PGDspider [START_REF] Lischer | PGDSpider: An automated data conversion tool for connecting population genetics and genomics programs[END_REF].

Genetic diversity was quantified by the average number of allele for loci (na), the effective number of allele (Ne), the average (Rs) allelic richness, the observed heterozygoty (Ho), the Hardy-Weinberg expected heterozygoty (He) [START_REF] Nei | Estimation of average heterozygosity and genetic distance from a small number of individuals[END_REF]). Wright's F-statistics were calculated according to [START_REF] Weir | Estimating F-Statistics for analysis of population structure[END_REF] and significant deviations from Hardy-Weinberg expectations (FIS) in each cohort were tested at a significant level of α = 0.5 using FSTAT v. 2.9. 3.2 (Goudet, 2002).

Linkage disequilibrium (LD) describes the relationship between two alleles at two loci in a population and exists when the probability to observe a pair of alleles on a chromosome is not equal to the product of probabilities to observe these alleles individually. It was tested in each population for all pairs of loci with 10 000 permutations with a significant level of α = 0.1 using ARLEQUIN v. 3.5.1.2 (Excoffier and[START_REF] Excoffier | Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows[END_REF].

The existence of a genetic signature of environmental stochasticity in each cohort was tested with the program BOTTLENECK v. 1.2.02 [START_REF] Cornuet | Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data[END_REF]Piry et al., 1999). The theoretical prediction is that a population bottleneck generates a faster reduction in allelic diversity compared to heterozygosity, which in turn generates an excess of heterozygotes in the postbottleneck population. Because the number of our loci is lower than ten, we used the sign test in the software BOTTLENECK v 1.2.02. We used the two-phase mutation model (TPM) for microsatellite loci because it provides a better fit to observed allele frequency data than the infinite alleles model (IAM) [START_REF] Di Rienzo | Mutational processes of simple-sequence repeat loci in human populations[END_REF]. We defined 90% of mutations as following a stepwise mutation model and 10% as multistep with a variance (σ²g) = 12 for the geometric distribution of number of repeat units per multi-step [START_REF] Garza | Detection of reduction in population size using data from microsatellite loci[END_REF].

Gene flows were estimated using Wright's F-statistics according to [START_REF] Weir | Estimating F-Statistics for analysis of population structure[END_REF] tested for significant overall and pairwise genetic differentiation (Fst), using permutation tests (10 000 permutations with a significant level of α = 0.5) implemented in GENETIX v. 4.05.2 (Belkhir et al., 1996(Belkhir et al., -2004)).

We did a test to assign individuals to clusters based on their multilocus genotypes using a Bayesian inference method implemented in STRUCTURE v. 2.3.3 [START_REF] Pritchard | Inference of population structure using multilocus genotype data[END_REF] under admixture model. We used 50000 burn-in followed by 500000 MCMC simulation steps with a model allowing admixture. To assess the consistency of results, we performed 25 independent runs.

All value significances were corrected according to the Bonferroni correction with the program R v. 2.14.1 (R Development Core Team, 2011).

Results

Pattern of prolonged diapause in M. schimitscheki

Emergences of M. schimitscheki spread over a maximum of five years, i.e. adult emergences from cohorts produced at year n (collected at n+1) were recorded at n+2, n+3, n+4 and n+5, suggesting that a proportion of each cohort could prolong diapause over 3, 4 or 5 years. The pattern of adult emergence from n+2 to n+5 was relatively well conserved between the different collected cohorts, but showing a clear bimodal pattern as adults mostly emerged at n+2 and n+4 (80% +2.75, and 17% + 2.59, respectively), while much proportions of individuals emerged at both n+3 and n+5

(1.5% + 0.43 and 1.5% + 0.36, respectively)(Figure 1). Adult emergences at n+2 and n+4 could even reach similar proportions in some cohorts, such as in 2000 (52% + 7.4 and 46% + 7.2, respectively)

Genetic diversity

The population that was chosen for the temporal population genetic analysis, Ventouret 7, displayed the typical bimodal pattern of adult emergence, as mean emergence proportions reached 74% (SE= 5.6) and 19% (SE = 3.9) at n+2 and n+4 (respectively).

All loci were polymorphic in all sampled cohorts (1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008) The number of alleles per loci ranged from 2 (loci alpha and MS3-98) to 6 (loci MS2-162).

The effective number of alleles ranged from 1.66 (loci alpha) to 3.86 (loci MS1-110). The observed heterozygosities ranged from 0.29 (loci MW-34) to 0.92 (loci MS1-110 and MS1-43) with a mean equal to 0.61. The expected heterozygosities ranged from 0.40 (loci alpha) to 0.74 (loci MS1-110).

Allelic richness per locus ranged from 1.96 (loci alpha) to 4.07 (loci MS1-110). All microsatellite loci showed no deviation from Hardy-Weinberg equilibrium resulting in non-significant FIS values.

Four loci, MS2-162, MS3-105, MS3-91 and MS3-99 had one (MS3-99) to three (MS3-91) rare alleles (Annexe 2).

Linkage disequilibrium was significant (p = 0.01) for loci MS1-110 and MS3-91 in each cohort. The locus MS1-110 was thus removed from analyses to avoid bias associated with redundant genetic information between both markers.

Population bottleneck

There was statistical support for genetic bottlenecks in cohorts produced in 1999 (P=0.006), 2001 (P=0.007), 2003 (P=0.007) and 2006 (P=0.007).

Temporal genetic differentiation between cohorts produced in even years and in odd years

The clear bimodal pattern of adult emergence found in M. schimitscheki suggested that a very large proportion (>95%) of individuals of a cohort produced at year n will emerge only at year n+2 and n+4, while individuals issued form a cohort produced at n+1 will mainly emerge at n+3 and n+5. Consequently, in an even year, one could expect the frequency of individuals emerging from a cohort produced in an even year to be much higher than that of individuals produced in an odd year. Such a phenomenon could lead to gene flow disruption between cohorts produced in even years and those produced in odd years. We explored such a hypothesis by testing for genetic differentiation between pooled 'even' genetic data (from cohorts 2000, 2002, 2004, 2006 and 2008 cohorts) and pooled 'odd' genetic data (from 1999, 2001, 2003, 2005 and 2007 cohorts). There was a very low and non significant Fst value (0.2%) between these two groups, suggesting continuous gene flow between M. schimitscheki cohorts produced in even and odd years.

Temporal genetic differentiation between successive cohorts

The individuals that were genotyped in the cohort produced in 2000 were excluded from these analyses due to insufficient sample size (N=5). Cohorts produced in 2001, 2002, 2003, 2006 and 2007 showed low (FST 1.3%-2%) but significant differentiation from one or several other cohorts (Table 4). Cohorts produced in 1999 and 2004 showed low (FST 1.3%-2.3%) but significant differentiation from all other cohorts, in exception of those produced in 2005 and 2008, which were not statistically differentiated from all other cohorts (Table 3). In order to assess the potential for prolonged diapause to promote genetic diversity in a context of recurrent genetic population bottlenecks, we estimated levels of genetic differentiation between three groups of individuals:

-adult wasps emerging in 2008 from the cohort produced in 2006 (referred to as '2-year diapause wasps'),

-adult wasps emerging in 2008 from the cohort produced in 2004 (referred to as '4-year prolonged diapause wasps'),

-adult wasps emerging from the cohort produced in 2008 (referred to as 'progeny wasps')

One should thus note that both 2-year diapause and 4-year prolonged diapause wasps constitute the most likely parental generation of the progeny cohort, which allows estimating the potential contribution of wasps emerging from prolonged diapause to the genetic pool of the population in

2008.

There was statistical support for genetic differentiation between 2-year diapause wasps and 4-year prolonged diapause wasps (FST = 0.09, P=0.02), but not between 2-year diapause and progeny wasps (FST= 0.03, P=0.13) nor between 4-year prolonged diapause and progeny wasps progeny wasp (FST = 0.04, P=0.06).

Assignment tests were performed on individuals of these 3 groups considered above with STRUCTURE to test for the possible contribution of emigrants to the genetic diversity of progeny wasps. STRUCTURE revealed K = 2 genetically distinct clusters using the admixture model of structuration. However the three groups show high proportions of individuals assigned to both clusters, suggesting that progeny wasps mainly result from admixture between both 2-year diapause and 4-year prolonged diapause wasps rather than emigrants originating from peripherical populations.

Discussion

Prolonged diapause of M. schimitscheki

In most species in which diapause can be prolonged, the majority of individuals emerge during the first season, while frequencies of emergences during further years continuously decrease over time [START_REF] Danks | Insect Dormancy: an Ecological Perspective[END_REF]. For example, in the sawfly Gilpinia hercyniae, 50% of individuals generally emerge during the first year, 18% after four years and 0.04% after 6 years [START_REF] Prebble | The diapause and related phenomena in Gilpinia polytomum (Hartig), I-V. Can[END_REF]. Or in the chestnut weevil Curculio elephas, 61, 35 and 4% of individuals emerge after one, two and three winter diapauses, respectively [START_REF] Menu | Strategies of emergence in the chestnut weevil Curculio elephas (Coleoptera: Curculionidae)[END_REF]. In this study, we provide a particularly atypical example of prolonged diapause in insects, showing an unexpected bimodal pattern of adult emergence. Indeed, prolonged diapause in M. schimitscheki cohort spreads over five years with two significant emergence peaks occurring after 2 (80 %) and 4 years (17%) after egg-laying, while emergences after 3 and 5 years remain relate to an extremely small fraction of a cohort. In the seedspecialized Megastigmus species, prolonged diapause is viewed as one adaptation for coping with the spatial and temporal heterogeneity of food supplies and for reducing competition for a limited resource [START_REF] Turgeon | Insect fauna of coniferous seed cones: Diversity, Host plant interactions, and Management[END_REF][START_REF] Roux | Effect of temperature and photoperiod on diapause development in Douglas fir seed chalcid, Megastimus spermotrophus[END_REF]. This is particularly true for Megastigmus species exploiting coniferous hosts [START_REF] Roques | New results and some thinkings about significance and induction of prolonged diapause in cone insects with particular references to the larch cone fly (Lasiomma melania) and to the Douglas-fir seed chalcid (Megastigmus spermotrophus)[END_REF][START_REF] Turgeon | Insect fauna of coniferous seed cones: Diversity, Host plant interactions, and Management[END_REF], for which intervals between mast years may vary between 2 and 10 years, characterizing moderate, light or no cone crops [START_REF] Young | Seeds of Woody Plant in North America[END_REF]. Years of high levels of seed production is thought to generate a satiety phenomenon in predators, allowing greater proportion of seeds to escape predation. In contrast, during the years of low levels of seed production, predators cannot maintain high population densities, which may dramatically impact their demography [START_REF] Janzen | Seed Predation by Animals Annu[END_REF][START_REF] Kelly | Mast Seeding in Perennial Plants: Why, How, Where?[END_REF]. As considered as a dispersal factor in time, prolonged diapause in M. schimitscheki may limit the impact of C. atlantica masting on its demography and then may reduce the effectiveness of the predator satiation strategy.

Interestingly, such a bimodal emergence pattern and very low proportions of individuals emerging after 3 and 5 years of prolonged diapause (a total of 5%) did not result in any gene flow disruption between cohorts produced in even and odd years (FST = 0.002). This suggests that even such low proportions of 3-and 5-year individuals may contribute to the maintenance of sufficient gene flow gene fluxes between successive 'even' and 'odd' cohorts of M. schimitscheki. However, we cannot exclude the hypothesis of annual contributions of emigrants to the observed genetic diversity, which could counterbalance temporal genetic structuration in the Ventouret 7 population.

Additionally, one could also suggests that French populations of M. schimitscheki are too recent (Auger-Rozenberg et al., 2012, submitted) to show any significant within population genetic differentiation.

Evidence of recurrent population bottlenecks in M. schimitscheki

Bottlenecks in population size cause a loss of alleles at a faster rate than a loss of gene diversity, generating excesses in heterozygote or deficiencies in rare alleles [START_REF] Nei | The bottleneck effect and genetic variability in populations[END_REF].

Bottlenecks can be detected in a population with no known history of population reduction if sample is too small [START_REF] Leberg | Estimating allelic richness: Effects of sample size and bottlenecks[END_REF], or when a population is subjected to a strong reduction of its effective size, for example in exotic species during an introduction event [START_REF] Puillandre | Genetic bottleneck in invasive species: the potato tuber moth adds to the list[END_REF], but also in species facing significant variations in resource supplies. Population effective size of seed predators can be severely hampered in years of low seed production due to a drastic reduction in resource availability and to enhanced competition [START_REF] Brown | Life history evolution of seed-bank annuals in response to seed predation[END_REF]. The Ventouret 7 population of M. schimitscheki displayed four significant bottlenecks in population size (1999, 2001, 2003 and 2006), suggesting that its demography was significantly impacted by one or several environmental factors during the present 10-year study period. Although this was not formally tested in this study, one of the best environmental candidates may be the periodical low levels of seed production by C. atlantica [START_REF] Krouchi | Year and tree effect on reproductive organisation of Cedrus atlantica in a natural forest[END_REF], which is likely to periodically drive M. schimitscheki to particularly low population levels. But while population bottlenecks generate an erosion of genetic diversity that could lead to genetic differentiation [START_REF] Côté | Microsatellite DNA evidence for genetic drift and philopatry in Svalbard reindeer[END_REF], there was no clear evidence of such an impact in the studied M. schimitscheki population.

Prolonged diapause as a genetic refuge for M. schimitscheki populations

In the natural population of M. schimitscheki that was studied here, temporal genetic differentiation between successive cohorts was low (FST <0.05), despite significant recurrent bottlenecks in population size. Such a lack of clear temporal genetic differentiation suggests that this population did not experience significant genetic erosion during a period in which its demography may have been significantly affected by drastic reduction in food supplies. The guarantee of survival of the species vulnerable to changes in the environment, conversely, the loss of genetic variability could render population more vulnerable to extinction in case of habitat perturbation [START_REF] Allphin | Genetic diversity and gene flow in the endangered Dwarf Bear Poppy, Arctomecon Humilis (Papaveraceae)[END_REF]. However, one could also expect emigrants from neighboring populations to contribute to genetic diversity of the cohort produced in 2008. Structure assignment tests did not support such a hypothesis, but both small sample size in the cohort of 2008 (N=10) and the absence of genetic data obtained from surrounding populations may mitigate these results. But both the geographical position of the studied population within the distribution area of the species in France and the unidirectional pattern of dispersal of M. schimitscheki make the contribution of emigrants less plausible (Lander et al., 2012 submited). In this context, theoretical population genetic models manipulating both genetic diversity evolution and migration rates would constitute critical tools to formally test for the role of both prolonged diapause and emigration on the maintenance of genetic diversity in a stochastic environment.

Conclusion

Natural populations of M. schimitscheki are facing periodical variations in resource supplies that may lead to recurrent significant reductions in effective population size. Erosion of genetic diversity resulting from such recurrent bottlenecks may be compensated by prolonged diapause.

However the contribution of migrants cannot be completely excluded. This study showed that prolonged diapause may constitute a genetic refuge for insect populations living in stochastic environment.

Annexes

Annexe 1: Genetic population variability. N = cohort size; P = number of polymorphic loci; na = average number of allele; ne = average effective number of allele; Rs = average allelic richness; Ho = observed heterozygoty; He = expected heterozygoty; FIS = inbreeding coefficient. *p-value < 5%.

Abstract

Many species experiencing spatial or interannual fluctuations of their environment extend diapause, a kind of dormancy, over more than one year. Such a prolonged diapause has been shown to act as a demographic refuge in stochastic environments, but to date there is still lacking evidence of its potential role from a population genetic standpoint. In this study, we investigated how prolonged diapause may disrupt or promote gene flow within natural populations of an invasive seedspecialized chalcid wasp, Megastigmus schimitscheki which periodically face drastic fluctuations in food supplies by its obligate host Cedrus atlantica. We first characterized the pattern of prolonged diapause of M. schimitscheki by following adult emergence over five consecutive years from 25 field-collected larval cohorts. Prolonged diapause results in an atypical bimodal pattern of adult emergence, with two main emergence peaks after two (80%) and four (17%) years of diapause.

Using nine polymorphic microsatellite markers, estimations of temporal gene flow within a natural wasp population sampled for 10 consecutive years (1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008) suggested that such a bimodal pattern did not disrupt gene flow between cohorts produced even and odd years (FST = 0.002). However, there was significant evidence of recurrent bottlenecks in population size (in 1999, 2001, 2003 and 2006), probably resulting from years of low seed production in C. atlantica. Despite the occurrence of significant bottlenecks in the history of natural populations of M. schimitscheki, we however showed that individuals emerging from prolonged diapause may restore genetic diversity in further cohorts. To our knowledge, this is the first demonstration that prolonged diapause may constitute a genetic refuge in a stochastic environment.

Keywords: life-history trait, bottleneck, genetic refuge, Megastigmus schimitscheki, microsatellites.

Résumé

De nombreuses espèces soumises à des fluctuations spatiales ou interannuelles de leur environnement prolongent leur diapause, une forme de dormance, sur plus d'un an. Des études ont montrées que la diapause prolongée agit comme un refuge démographique dans les environnements stochastiques, mais à ce jour, nous ne connaissons pas son rôle potentiel dans la génétique des populations. Dans cette étude, nous avons étudié la façon dont la diapause prolongée pourrait diminuer ou favoriser les flux de gènes au sein d'une population naturelle d'un hyménoptère chalcidien séminiphage, Megastigmus schimitscheki, périodiquement soumis à des fluctuations drastiques de la ressource produite par son hôte Cedrus atlantica. Dans un premier temps, nous avons caractérisé le pattern de diapause prolongée de M. schimitscheki en suivant, au cours de cinq années consécutives, l'émergence d'adultes issus de 25 cohortes larvaires. Le pattern de diapause prolongée est bimodal, avec deux principaux pics d'émergences après deux ans (80%) et quatre ans (17%) de diapause. Les flux de gènes temporels ont été estimés au sein d'une population naturelle échantillonnée sur 10 années consécutives (1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008). Les résultats obtenus à l'aide de neuf marqueurs microsatellites polymorphes, ont montré que la forme bimodale du pattern n'est pas une barrière aux flux de gènes entre les cohortes produites une année paire et celles produites une année impaire (FST = 0,002). Cependant, nous avons observés des goulots d'étranglements pour quatre cohortes (1999, 2001, 2003 et 2006), résultant probablement de plusieurs années de faibles productions de graines par C. atlantica. Malgré des diminutions récurrentes de la taille efficace des populations naturelles de M. schimitscheki, nous avons montré que les individus émergeant de diapause prolongée pourraient restaurer la diversité génétique des cohortes futures. À notre connaissance, nous apportons la première démonstration que la diapause prolongée peut constituer un refuge génétique dans un environnement stochastique.

Mots clefs : traits d'histoire de vie, goulot d'étranglement, refuge génétique, Megastigmus schimitscheki, microsatellites.

Figure 1 :

 1 Figure 1 : Mean adult emergence percentage of a cohort of M. schimitscheki produced at year n

Figure 2 :

 2 Figure 2 : Assignment of individuals in more probable clusters of progeny wasps and 2year diapause wasps, and 4-year prolonged diapause wasps under admixture model. Color red and green represent cluster 1 and 2, each bar represents an individual.

  potential contribution of prolonged diapause to the restoration of genetic diversity after a population bottleneck, i.e. what we call here a genetic refuge, was assessed by estimating the level of genetic differentiation: (i) between 4-year and 2-year diapausing individuals emerging the same year (2008), with the 2-year ones having experienced a population bottleneck, and (ii) between these 2year and 4-year emerging individuals and the cohort produced in 2008. The existence of a significant genetic differentiation (FST = 0.09) in the first case but not in the second one may provide a strong support for a significant contribution of 4-year diapausing individuals to the genetic pool of the cohort produced in 2008, which may have counterbalanced the population bottleneck detected in the 2-year diapausing parental cohort. Conservation of genetic diversity is a

Table 1 : Number of individuals genotyped per cohort

 1

	COHORTS	SAMPLE SIZE
	1999	33
	2000	5
	2001	23
	2002	29
	2003	50
	2004	91
	2005	90
	2006	50
	2007	50
	2008	10

Table 2

 2 dNTP mix), and 0.02μL of each primer forward and reverse of 2μM. The PCR program was 95°C 15min, 94°C 30s, 57°C 90s, 72°C 90s for 30 cycles, final elongation at 72°C for 10min and 4°C for 1 min to stop Taq polymerase in an Eppendorf thermocycler.

) with the Qiagen® multiplex PCR kit as follow for each sample : 2μL diluted DNA, 1μL Q-solution(5x), 1.8μL RNAse-free water, 5μL QIAGEN Multiplex PCR Master mix 2x (6mM MgCl 2 , HotStarTaq ® DNA polymerase,

Table 2 : sequences of the primers used for the amplification reaction

 2

						DYE		SIZE
	MULTIPLEX	LOCUS	REFERENCE	PRIMER SEQUENCE (5'-3')	FORWARD	REPEAT	RANGE
					PRIMER		(BP)
			Boivin et al.,	F : GACTGCAAGCTCGACTCACAC		Fam	(CA)18	93 -
		MS1-alpha	2003	R : TTTCTCCTCGACGCTGAT
	MKA	MS2-162	2003 Boivin et al.,	R : TCTGCAGCACGATGTAAACG F : GCAGACCGGCGAATAAATAA		Fam	(AG)5TG(AG)35	122 -
	Multiplex	MW-34 MS1-110	Carcreff et al., 1998 2006 Boivin et al.,	F : CCCCGCCTCTACCAAATC R : TTGAAATTGCTCGGACCG R : AGTCGGCGTTATCGGTTATT F : TCAGCCCGACTTCGTCCTT		Tamra Hex	(AG)17(G)10 (AG)51	144 -208 -
		MS3-105	Fallour-Rubio,	F : ATGGTCGAGCCCGCTAC		Fam	(TC)17(ACTC)5	260 -
			2007	R:GAGGGAGAGACAGACGGCAAAT
	Multiplex MKB	MS3-99 MS1-43 MS3-98 MS3-91	Fallour-Rubio, 2007 Boivin et al., 2003 Fallour-Rubio, 2007 2007 Fallour-Rubio,	F : ATTGACTTTGCTCTCCGTCTC R : CCAAGTTAGCGCTTCAC F : GCAAGCCCTTCGCACAAC R:GCGCTTCACCGACCTCC F:GAGAGAGTCGCGTACGTGTAG R :TAACGGGTGCTCGAATCAAC R:CACGTTGCGGACAGAGAGCGA F:CGACACTTATACACCGGCATT		Tamra Hex Fam Tamra	(CT)52 (AC)15 (GA)28 (TC)41	99 -121 122 -203 -211 -
	Electrophoresis of PCR products was performed on ABI 3730XL sequencer as follows: 2μL of PCR
	product for each individual was diluted (3μL PCR product + 50μL H 2	O), combined with 8μL of
	GeneScan500 Size Standard (10μL GS500 (-250Liz) + 900μL Formamide), denatured at 95°C for 3
	minutes and then injected into the sequencer. The microsatellite profiles were analysed in
	GeneMapper® software version 4.1.

of adult emergence in a cohort of Megastigmus schimitscheki produced at year n Year of emergence Mean emergence percentage

	of Ventouret 7. The mean number of
	alleles per cohort ranged from 2.56 (in 2000) to 3.56 (in 2004 and 2007). Mean numbers of
	effective allele ranged from 1.81 (in 2000) to 2.49 (in 2003). Observed heterozygosities ranged
	from 0.55 (in 2006) to 0.69 (in 2008) whereas expected heterozygosities ranged from 0.45 (in 2000)

to 0.60

(in 2003, 2004 and 2007)

. The test for an excess of heterozygotes with inbreeding coefficient (FIS) was significant for cohorts produced in

2000, 2003, 2007 and 2008 (p = 0.05)

. The mean of allelic richness was low, with values ranging from 2.56 (in 2000) to 2.86 (in 2007) (Annexe 1).

Mean pattern

Table 3 : Pairwise genetic differentiation between cohorts

 3

. *p-value < 5%; **p-value < 1%.

Acknowledgements


.09

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  Photos internetPhotos et croquis: « L'Arbre et la Haie »


Otilia Dragomir

Rafael Gouriveau

Noureddine Zerhouni

Adaptive Neuro-Fuzzy Inference System for mid term prognostic error stabilization

Keywords: prognostic, neuro-fuzzy system, ANFIS, error of prediction

published or not. The documents may come

Adaptive Neuro-Fuzzy inference system for mid term prognostic error stabilization.

Introduction

Maintenance activity combines dierent methods, tools and techniques to reduce costs while increasing availability, reliability and security of equipments. That said, maintenance is far away to be only an industrial area of interest and researchers also show a growing attention to this thematic. The initial maintenance framework was delimited by the necessity of perceiving phenomena, next, of understanding them, and nally, of acting consequently. However, rather than understanding a phenomenon which has just appeared like a failure, it seems convenient to anticipate it's manifestation in order to act consequently and resort to protective actions. This is what could be dened as the prognostic process. Prognostic is nowadays recognized as a key feature in maintenance strategies. However, real prognostic systems are scarce in industry and that can be explained from dierent aspects. Firstly, prognostic still is not a stabilized concept: there is no consensual way of understanding it which makes harder the denition of tools to support it in real applications. Secondly, many approaches for prediction exist whose applicability is highly dependent of the available knowledge on the monitored system. Thirdly, the vagueness of prognostic process denition impedes to point out the inherent challenges for scientists. Thus, the purpose of this paper is to analyze and discuss the prognostic process from dierent points of view, and to propose a way of handling failure prognostics so that practitioners can act consequently. The paper is organized in two parts. First, the prognostic framework is delimited, starting with the prognostic denition, metrics, approaches and tools. Considering prognostic as the association of a prediction and an evaluation steps, a classication of prognostic metrics is given. The whole aims at giving a frame to perform (and develop) real prognostic systems. In the second section, the use of neuro-fuzzy predictor for prognostic purpose is briey justied and an illustration based on the adaptive neuro-fuzzy inference system is given. The proposed system performs good prediction and is built in order to reach the stabilization of mid term prognostic errors.

2 Performing prognostic: concept, measures and tools 2.1 Prognostic concept Although there are some divergences in literature (see [2]), prognostic can be dened as proposed by the International Organization for Standardization: prognostic is the estimation of time to failure and risk for one or more existing and future failure modes [START_REF]Condition monitoring and diagnostics of machines -prognostics -Part1: General guidelines[END_REF]. In this acceptation, prognostic is also called the prediction of a system's lifetime as it is a process whose objective is to predict the remaining useful life (RUL) before a failure occurs given the current machine condition and past operation prole [START_REF] Jardine | A review on machinery diagnostics and prognostics implementing condition-based maintenance[END_REF]. Two salient characteristics of prognostic can be pointed out [2]. 1) Prognostic is mostly assimilated to a prediction process (a future situation must be caught). 2) Prognostic is grounded on the failure notion, which implies that it is associated with a degree of acceptability (the predicted situation must be assessed with regard to a referential). Therby, at a prediction level, a prognostic system should be able to determine the future state of equipment as closely as possible to the future real state. Also, the control of the performance of prediction is the premise of a good global prognostic system. At an evaluation level, the predicted situation should be evaluated regarding the reference levels, RUL, condence, accuracy, etc., which implies the denition of prognostic measures.

Prognostic metrics

There is no general agreement as to an appropriate and acceptable set of metrics that can be employed eectively in prognostic applications, and researchers and CBM practitioners are still working on this [START_REF] Vachtsevanos | Intelligent Fault Diagnosis and Prognostic for Engineering System[END_REF].

1) The main objective of prognostic is to provide the ecient information that enable the underlying decision process, i.e., the choice of maintenance actions. Thus, a rst set of metrics are those that quantify the risks incured by the monitored system. This kind of metrics can be called the prognostic measures. As mentioned earlier, the main prognostic measure pursued is the predicted time to failure (TTF), also called the remaining useful life (RUL). In addition, a condence measure can be built to indicate the degree of certitude of the future predicted failure time. By extension, and considering that practitioners can be interested on assessing the system with regard to any performance limit, RUL and condence can be generalized: in Fig. 1(a), TTxx refers to the remaining time to overpass the performance limit Perf/xx, and Conf/xxT is the condence with which can be taken the asset TTxx > T.

2) Assuming that prognostic is in essence an uncertain process, it is useful to be able to judge from its quality in order to imagine more suitable actions. In this way, dierent indicators can be constructed: the prognostic system performance measures. The timeliness of the predicted time to failure (TTF) is the relative position of the probability density function (pdf) of the prediction model along the time axis with respect to the occurrence of the failure event. This measure evolves as more data are available and reveals the expected time to perform preventive actions [START_REF] Vachtsevanos | Intelligent Fault Diagnosis and Prognostic for Engineering System[END_REF] (see Fig. 1(b)). According to [START_REF] Goebel | Prognostic information fusion for constant load systems[END_REF], one needs to dene two dierent boundaries for the maximum acceptable late and early predictions. Accuracy measures the closeness of the predicted value to the actual value. It has an exponential form and is as higher as the error between the predicted value of TTF and the real one is smaller. Precision measure reveals how close predictions are grouped or clustered together and is a measure of the narrowness of the interval in which the remaining life falls. Precision follows from the variance of the predicted results for many experiments. The complementarity of accuracy and precision is illustrated in Fig. 1(c). Various prognostic approaches have been developed ranging in delity from simple historical failure rate models to high-delity physics-based models [START_REF] Vachtsevanos | Intelligent Fault Diagnosis and Prognostic for Engineering System[END_REF]. Similarly to diagnosis, prognostic methods can be associated with one of the following two approaches, namely model-based and data-driven. 1) Model-based methods assume that an accurate mathematical model for the analyzed system can be constructed. The main advantage of these approaches is their ability to incorporate physical understanding of monitored system. Moreover, if the understanding of the system degradation improves, the model can be adapted to increase its accuracy and to address subtle performance problems. But, this closed relation with a mathematical model may also be a strong weakness: it can be dicult, even impossible to catch the system's behavior.

2) Data-driven approaches use real data to track, approximate and forecast features revealing the degradation of components; in many applications, measured input/output data is the major source for a deeper understanding of the system degradation. Data-driven approaches can be divided into two categories: statistical techniques (multivariate statistical methods, linear and quadratic discriminators, etc.), and articial intelligence (AI) techniques (neural networks, fuzzy systems, etc.). The strength of data-driven techniques is their ability to transform highdimensional noisy data into lower dimensional information for decisions. In practice however, data-driven approaches are highly-dependent on the quantity and quality of operational data.

3 Building a neuro-fuzzy prognostic tool 3.1 ANFIS model: an adequate prediction tool Real systems are complex and their behavior is often non linear, non stationary. These considerations make harder a modeling step, even impossible. Yet, a prediction computational tool must deal with it. Moreover, monitoring systems have evolved and it is now quite easy to online gather data. According to all this, data-driven approaches have been increasingly applied to machine prognostic. More precisely, works have been led to develop systems that can perform nonlinear modeling without a priori knowledge, and that are able to learn complex relationships among inputs and outputs (universal approximators). Indeed, articial neural networks (ANNs) have been used to support the prediction process. Recent works focus on the interest of hybrid systems: many investigations aim at overcoming the major ANNs drawback (lack of knowledge explanation) while preserving their learning capability. In this way, neuro-fuzzy systems are well adapted. More precisely, rst order Tagaki-Sugeno (TS) fuzzy models have shown improved performances over ANNs and conventional approaches [START_REF] Wang | Prognosis of machine health condition using neurofuzzy systems[END_REF]. Thereby, they can perform the degradation modeling step of prognostic. A particular architecture of TS neuro-fuzzy systems is that of the adaptive neuro-fuzzy inference system (ANFIS) [START_REF] Jang | Neuro-fuzzy modeling and control[END_REF]. ANFIS is an inference system in which the parameters associated with specics memberships functions are computed using either a backpropagation gradient descent algorithm alone or in combination with a least squares method. Thanks to its structure and learning capability, ANFIS is tted to predict irregular or non-periodic time series. However, when used for mid term predictions purpose, ANFIS can make large residual errors. Next part of the paper emphasizes on this aspect.

Mid term prognostic error stabilization with ANFIS predictor

Since the occurrence of a failure is in essence uncertain, a prognostic tool should be enable to make predictions with quite the same accuracy at short, mid and long terms. This is the purpose of this part (assuming that ANFIS is an adequate short term prediction tool).

1) Here, prognostic is considered as a prediction process based on the aggregation of, naturally, past and present states of the system, but also, of the known future ones. Indeed, it appears to be useful to take into account future actions like the modication of the mission prole due to some extern interventions or like the inuence of a scheduled maintenance action. Consequently, the just-in-time-point (the time of failure when the life duration is [0%]) gets another dimension related to the starting point ([100%] of machine life duration) (see Fig. 2).

Figure 2: Inuence of scheduled maintenance actions on the prediction process 2) In most of the papers in which ANFIS is used as a prediction system, inputs are directly extracted from the data sets. Here, the Box-Jenkins furnace benchmark is used. There are originally 296 data samples {y(t), u(t)}, from t=1 to t=296. From the real process, CO2 concentration is considered as the output of the model y(t), and gas ow rate as the input u(t). In order to predict y(t) based on y(t-1), y(t-2), y(t-3), y(t-4), u(t-1), u(t-2), u(t-3), u(t-4), u(t-5), u(t-6), the number of eective data points becomes 290. A selection method must be used because all ten input variables generate to many rules and parameters to be updated on the learning phase: it would make the training data insucient and would obviously increase the computing time.

3) The choice of an error measure to compare prediction methods has been much discussed (see for example [START_REF] De Gooijer | 25 years of time series forecasting[END_REF]). In any case, error measures are only intended as summaries for the error distribution. This distribution is usually expected to be a normal white noise in a forecasting problem, but it probably is not so in a complex problem like load forecasting. The ANFIS architecture proposed by Jang with two and three selected inputs has satisfactory results for short term predictions. For mid and long terms ones, the obtained errors increase, which aects the prognostic performances (Fig. 3).

Figure 3: Error of prediction pdf -ANFIS model with two inputs 4) A rst way to stabilize the error of prediction is to construct serial architectures of ANFIS models. Indeed, linking two ANFIS enable to take over the growth tendency of error since the second one learns the error of the rst one. The eect of this modication is observed on the error values (see Fig. 4). The maximum measured error decreases signicantly and becomes satisfactory for mid term predictions as well as the prediction spread does.

Figure 4: Error of prediction pdf -serial architecture of two ANFIS models with two inputs 5) The known future solicitations of the system (mission prole) can then be injected as input information for the second ANFIS module in the serial architecture. The eect of taking into account the future for predictions has also inuence over error: the error spreading is signicantly reduced and the condence of the prediction process increases thereby consequently. Mid term predictions reect the improved quality of the approach (Fig. 5).

Conclusion and work in progress

In this article, prognostic is presented as the association of a prediction and an evaluation process.

Many approaches to support this activity exist, whose applicability and performance must be assessed to develop a prognostic tool. For that purpose, various prognostic metrics exist and are discussed in the paper. All of them are based on the evaluation of the prediction error spreading.

Following that, the prediction step of prognostic appears to be a critical one and controlling the performances of predictions is the premise for a good global prognostic system. According to all this, the interest of neuro-fuzzy system is pointed out and a new ANFIS architecture is proposed in order to ensure a certain stability of errors for mid term predictions. The system is based on a serial architecture of various ANFIS models, in order to overcome the local prediction errors (by learning them) and to inject the future scheduled maintenance actions. The obtained results are satisfactory from the industrials point of view since condence on predictions increases.

The work is still in progress and the developments are at present extended in four principal ways. First, the denition of new loss functions in the learning phase is studied. Secondly, the application of ANNs and NFs as tools for a global prognostic is been investigated. Thirdly, the interpretability of the obtained predictive system is been looked in a closely manner. Finally, the implementation of the studied framework is in progress at a French industrial partner for the monitoring of high speed trains motors.

Figure 1 :

 1 Figure 1: Metrics -(a) RUL, TTxx and condence, (b) timeliness, (c) accuracy and precision

Figure 5 :

 5 Figure 5: Error of prediction (%) for a serial architecture without (a) and with (b) future known solicitations injection


Hendrik Davi

Katalin Csillery

Sylvie Muratorio

Local adaptation to climatic gradients: application of a physio-demo-genetics simulation model to European beech populations

Forests are the most important terrestrial ecosystems, providing 75% of the terrestrial biomass and several ecosystem services. Climate change has already taken its toll on forests and the future of forests is unforeseen. Trees are key drivers of ecosystem processes and stability in forests, directly sustaining diverse plant and animal communities, and mediating nutrient and water ecological cycles Over the past two decades, extensive forest tree mortality triggered by droughts and extreme climatic conditions has been documented all around the world. Nonetheless, tree populations response to climate is still not sufficiently well understood to be able to make reliable predictions for the future. Besides migration, Forests response to climate will be determined by a combination of factors such as phenotypic plasticity and adaptive capacity that are largely unknown for many tree species.

Here we develop a new simulation approach to investigate the evolutionary potential of forest trees to adapt to ongoing climate change. We build an individual based model that accounts explicitly for the effect of individual genetic variability and climate on the variation of tree physiological traits. Selection is acting on functional traits involved in response to climate, such as the timing of budburst (TBB). We illustrate how this tool can be used to investigate the rate of evolutionary change in tree populations evolving for few generations along an environmental gradient.

Results

1) Five generations are enough for natural selection to result in genetic differentiation for the timing of budburst (TBB)

2) Phenotypic variation of TBB due to plasticity (20 days from 1000 to 1600 m) is higher than due to genetic adaptation (2 days from 1000 to 1200m).

3) In the presence of genetic adaptation population size is higher than without adaptation.

4) The patterns of phenotypic (TBB) and genotypic (Sum of degree days) variation across altitudes are not monotonic. 2) a population demographic module converting carbohydrate reserves into demographic rates for adult tree (growth, mortality and seed production) and simulating ecological processes across the life cycle (including seed and pollen dispersal, germination success, density-dependent mortality of seedlings).

Pollen Ovules

Genotype Phenotype

Climate

3) a quantitative genetic module (GENETICS) relating the genotype at quantitative trait loci (QTLs) to the phenotype of one or more functional traits, accounting for environmental effect. Individual fitness is an output of the model, calculated as the life time reproductive success resulting from a combination of functional traits.

Here we consider only the Timing of Budburst (TBB) = f (Sum of degree days , Temperature)

Case study

We parameterized the PDG model on a European beech (Fagus sylvatica) population colonising an altitudinal in South East of France on Mount Ventoux. Among the various traits contributing to fitness along the altitudinal gradient, we focussed on the timing of budburst (TBB) because: 1) TBB is a life history trait known to affect individual fitness in F. sylvatica: an earlier budburst extends the time during which photosynthesis occurs but it also increases the risk of late frosts. 2) TBB exhibits genetic differentiation across altitude in F. sylvatica: in common gardens trees from higher altitude budburst earlier

  projects http://capsis.cirad.fr/ http://www4.paca.inra.fr http://www.igv.fi.cnr.it/linktree/ of trees, the keystone elements of forests, to ongoing climate change is a critical issue.

  eco-physiological and physical module (CASTANEA) simulating carbohydrate and water fluxes at tree-level based on daily climate observations (real or simulated).