Keywords: paleoecology, population genetics, last interglaclial/glacial cycle, conifer, climate change, translocations

History of the European larch (Larix decidua Mill.)

Die Geschichte der Europäischen Lärche (Larix decidua Mill.)

Zusammenfassung

Diese Dissertation fokussiert die Auswirkungen vergangener Klimaschwankungen und anthropogener Einflüsse auf Populationen der Europäischen Lärche (Larix decidua Mill.) mittels Integration paläoökologischer und genetischer Daten. Solche retrospektiven Ansätze liefern eine wertvolle Grundlage für die Vorhersage möglicher Konsequenzen rezenter Entwicklungen. Eine wesentliche Beschränkung existierender Studien an Waldbäumen besteht darin, dass sie häufig ausschließlich die postglaziale Ausbreitungsgeschichte behandeln. Konsequenzen schnellerer Umweltveränderung, einschließlich rezenter Anpflanzungen oder abrupter Klimaereignisse des letzten Glazials auf Wälder, sind weithin vernachlässigt worden. Die vorliegende Studie basiert auf hochauflösenden genetischen Daten und präzisen vegetationsgeschichtlichen Archiven, korreliert mit hochauflösenden Klimarekords des letzten Interglazial/Glazial Zyklus (130 000 Jahre), um kurzzeitige und langzeitige Ereignisse, die die Geschichte der Europäischen Lärche beeinflussten, präzise zu dokumentieren. Für die genetischen Analysen wurden hochinformative nukleare Marker (Mikrosatelliten) entwickelt, die auf ein arealweites Sampling, bestehend aus 45 Populationen, angewendet wurden. Diese nuklearen Daten wurden zusammen mit mitochondrialen Daten analysiert, um eine Grundlage für Studien, die auf die Erforschung rezenter Translokationen zielen, zu schaffen. Die Ergebnisse zeigen, dass Larix decidua in erheblichem Maße angepflanzt worden ist, was zur Durchmischung autochthoner und allochthoner Populationen aus multiplen Quellen des Areals führte. Translokationsereignisse und Durchmischungsraten waren ungleichmäßig über das Areal verteilt und besonders häufig in Polen, der Slowakei und Tschechien, wo die Lärche eine verstreutere Verbreitung als in den Alpen aufweist. Einige der wertvollsten Populationen scheinen ernsthaft durch Translokationen bedroht. Die vegetationsgeschichtlichen Befunde zeigen, dass die Lärche den gesamten Interglazial/Glazialzyklus nahe ihres aktuellen Areal überdauerte, jedoch waren die Grenzen der Verbreitung hoch dynamisch und veränderten sich synchron mit kurzzeitigen als auch langzeitigen Klimaschwankungen, was im Einklang mit dem Pioniercharakter der Art steht. Die Verbreitung erreichte ihr maximales Ausmaß zur Zeit des ersten Frühweichsel-Interstadials (87 000 -109 000 Jahre), als Lärche boreale Wälder im nord-mitteleuropäischen Tiefland bildete. Reaktionen auf Kurzzeit-Klimaereignisse (Dansgaard-Oeschger-Zyklen, Heinrich-Events) waren extrem schnell. Für die Zeit der letzten maximalen Vereisung (LGM) wurden sieben Refugien, basierend auf Fossilien und genetischen Daten, identifiziert. Dies ermöglichte es, Rekolonisierungswege und begleitende Introgressions-und Homogenisierungprozesse zu ermitteln, was die Leistungsfähigkeit der gekoppelten populationsgenetischen und paläoökologischen Perspektive unterstreicht.

Schlüsselwörter

Résumé

Dans cette thèse, je m'intéresse aux conséquences sur les populations de mélèze d'Europe (Larix decidua Mill) des changements d'origine naturelle (climatique) et anthropique passés, en intégrant des données paléoécologiques et des données génétiques. Une telle étude rétrospective offre un exemple utile pour évaluer les conséquences possibles des changements actuels. Les études récentes disponibles sur les arbres forestiers sont généralement limitées à l'analyse des recolonisations postglaciaires. Les effets de changements plus rapides observés sur les forêts ont été largement négligés, par exemple les conséquences de plantations récentes ou d'événements climatiques brusques de la dernière période glaciaire. Dans cette étude, j'utilise des données génétiques riches ainsi que des inventaires précis de végétation liés à des relevés climatiques de haute résolution du derniers cycle interglaciaire/glaciaire (130 000 ans), afin d'analyser de façon détaillée les événements récents et plus anciens qui ont affecté l'histoire du mélèze d'Europe. Pour l'analyse génétique, j'ai mis au point des microsatellites, marqueurs génétiques informatifs, avec lesquels j'ai analysé un échantillon de 45 populations provenant de l'ensemble de l'aire de répartition actuelle du mélèze. J'ai analysé ces données de polymorphisme génétique nucléaire en même temps que des données de la diversité mitochondriale afin d'établir des cartes de référence de la diversité génétique naturelle me permettant de détecter des translocations récentes. Les résultats montrent que le mélèze a été fréquemment planté, ce qui a créé des mélanges entre des populations locales et d'autres introduites à partir de sources variées de l'ensemble de l'aire de répartition. Les événements de translocation et les taux de mélange sont répartis de façon hétérogène dans l'aire de répartition, avec une fréquence particulièrement élevée en Pologne, en Slovaquie et au Tchéquie, où le mélèze possède une répartition plus dispersée que dans les Alpes. Quelques-unes des populations de mélèzes présentant un intérêt écologique et économique majeur apparaissent sérieusement menacées par les translocations. Les résultats paléontologiques montrent que le mélèze est resté à proximité de son aire de répartition actuelle pendant le dernier cycle interglaciaire/glaciaire, mais que sa répartition s'est maintenue dans un équilibre dynamique avec les événements climatiques anciens mais aussi avec ceux plus récents, ce qui s'explique par les caractéristiques pionnières de l'espèce. L'amplitude de répartition de l'espèce a été maximale pendant le premier interstade Weichsélien (87 000 -109 000 ans) quand le mélèze a contribué à établir les forêts boréales des plaines européennes du nord et du centre. Les réponses aux événements climatiques brefs (événements Dansgaard-Oeschger et Heinrich) ont été extrêmement rapides. Sept refuges correspondant aux derniers maximums glaciaires ont été identifiés en utilisant les données fossiles et génétiques. Notre approche nous a permis d'identifier des voies de recolonisation et des événements d'introgression concomitants, illustrant la puissance de l'approche consistant à associer la génétique des populations et la paléoécologie.

Mots clés: paléoécologie, génétique des populations, conifère, dernier cycle interglaciaire/glaciaire, changement climatique, translocations tel-00880539, version 1 -6 Nov 2013

Résumé substantiel

L'étude des conséquences du changement climatique passé sur les forêts du monde fournit le contexte historique nécessaire pour évaluer les conséquences du changement climatique actuel. Les approches rétrospectives utilisant des données fossiles et génétiques sont particulièrement prometteuses parce qu'elles permettent des reconstructions très détaillées et robustes. Depuis quelques années la précision et la robustesse des reconstructions utilisant des fossiles a augmenté grâce à la disponibilité en plus grand nombre de séquences polliniques de haute résolution associées à des chronologies calendaires précises. Les tendances ont été similaires pour les macrofossiles, stomates et charbons. En parallèle, les reconstructions paléoclimatiques disponibles sont plus détaillées et plus précises, notamment celles du dernier cycle interglaciaire/glaciaire. Le Northern Greenland Ice Core Project (NGRIP) a contribué de façon importante à cette évolution en documentant les changements climatiques à long-terme et les événements plus rapides. Les corrélations constatées entre des changements de végétation observés dans les séquences polliniques et des changements climatiques enregistrés dans des archives paléoclimatiques comme le NGRIP (Northern Greenland Ice Core Project) ont montré que la forte variabilité du climat dans le passé a provoqué une forte variabilité de la végétation avec des réponses souvent instantanées. La période du dernier cycle interglaciaire/glaciaire est ainsi particulièrement informative pour l'évaluation des réponses de la végétation et des espèces qui la composent aux changements actuels. Les inférences historiques en génétique se basent principalement sur la structuration génétique des populations contemporaines. Les forêts d'Europe sont caractérisées par la fréquence des plantations importantes. Par conséquence, la structure génétique ancienne des arbres peut avoir été modifiée par les déplacements de matériel végétal liés à l'homme (= translocations). Dans l'aire de répartition d'une espèce, les translocations peuvent mettre en présence des individus d'origine locale et plus distante. L'identification des translocations requiert alors des outils moléculaires adaptés à une distinction des deux types d'individus. L'existence d'un tel outil fait souvent défaut, ce qui explique que les translocations au sein de l'aire de répartition d'une espèce sont peu étudiées, malgré leur importance sans cesse croissante pour les populations de plantes et d'animaux. Cependant les avancées techniques et méthodologiques dans le domaine de la génétique ces dernières années (développement de marqueurs, de nouvelles méthodes de génotypage, augmentation du nombre d'échantillons analysables, méthodes d'affectation) ouvrent de nouvelles perspectives pour l'étude des translocations et de la structuration génétique ancienne. Le mélèze, conifère européen (Larix decidua Mill.) est particulièrement adapté à une telle étude grâce à ses caractéristiques particulières : ses fossiles sont abondants, le pollen fossile est principalement d'origine locale du fait de sa faible aptitude à la dispersion, il a été massivement planté à une période de son histoire et c'est une espèce pionnière répondant rapidement aux changements climatiques.

Dans cette thèse j'ai reconstruit l'histoire de Larix decidua pendant le dernier cycle interglaciaire/glaciaire (130 000 années = 130 ka) en utilisant des données génétiques et fossiles et en procédant par étape : (i) synthèse des informations disponibles sur l'espèce et les outils génétiques existants (chapitre 2), (ii) développement de nouveaux multiplexes de microsatellites nucléaires (chapitre 3), (iii) identification des translocations dans des populations modernes à l'échelle de l'aire globale grâce à la génétique (chapitre 4), (iv) description de la dynamique de l'aire de répartition pendant les dernières 130 ka grâce aux données fossiles compilées pour ce travail (chapitre 5) et (v) reconstruction des refuges glaciaire et des voies de migration, analyse des effets du climat et de l'homme sur la répartition de l'espèce en utilisant les données fossiles et génétiques (chapitre 6).

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Chapitre 2 L'aire de distribution de Larix decidua est fragmentée et divisée en deux régions principales : la région des Alpes d'une part et la région des Carpates d'autre part, où l'espèce colonise principalement des altitudes élevées. Dans la partie est de l'aire (Sudètes, Carpates, Plaine de Pologne) la présence naturelle ou introduite de l'espèce est parfois difficile à trancher. Le mélèze L. decidua a en effet été largement planté au sein ou à proximité de son aire de répartition. Ces plantations ont commencé au XVI e siècle et ont culminé au XIX e siècle. L'intensification des plantations a été accompagnée par une expansion du chancre du mélèze, la maladie la plus grave touchant L. decidua. Le chancre a mené à l'échec d'une grande partie des plantations au XX e siècle, ce qui a été attribué à un mauvais choix de provenance. En effet, les provenances alpines sont généralement plus sensibles au chancre que les provenances de la partie est de l'aire. J'ai contribué, grâce à mes résultats de thèse, à un projet d'analyse des sensibilités au chancre du mélèze de différentes provenances (Appendice 1). En l'absence de marqueurs génétiques en nombre significatif chez le mélèze, des isozymes développées il y a 40 ans sont toujours utilisés actuellement. D'autres types de marqueurs nucléaires et cytoplasmiques ont été développés au niveau du genre Larix principalement pour étudier des questions sur les relations interspécifiques. Des polymorphismes nucléotidiques (Single Nucleotide Polymorphisms (SNP)) ont été identifiés chez L. decidua très récemment. Quelques études étaient basées sur des microsatellites (Simple Sequence Repeats (SSR)) nucléaires développés pour d'autres espèces du genre. Au début de cette thèse il manquait donc des marqueurs suffisamment polymorphes et de haute qualité pour des analyses génétiques fines. Ce type d'outil est également utile et important pour d'autres champs de recherche et pour des questions pratiques de foresterie (p. ex. adaptation, sélection, chancre du mélèze, traçabilité, conservation).

Chapitre 3

Nous avons développé deux kits multiplexe pour Larix decidua combinant des microsatellites très polymorphes. Ces multiplexes se composent de locus initialement développés pour L. kaempferi et des microsatellites développés spécialement pour L. decidua par nos soins en utilisant le pyroséquençage d'une librairie enrichie en motifs microsatellites suivi d'une sélection des meilleurs locus candidats. Pour cibler des marqueurs très polymorphes nous avons sélectionné uniquement les microsatellites possédant des motifs di-ou trinucléotidiques ayant un nombre de répétitions élevé (≥12). Une proportion importante des marqueurs possédait des bandes multiples, une mauvaise amplification ou un polymorphisme insuffisant. Ce genre de difficultés est classique lorsque l'on a affaire à des espèces ayant des génomes de grande taille, ce qui est le cas chez les conifères en général et chez le mélèze en particulier. Pour la validation des marqueurs nous avons appliqué les stratégies récentes recommandées pour le développement des microsatellites. Les locus les plus prometteurs ont été combinés en deux multiplexes, un 7-plex et un 6-plex. Ceux-ci ont été testés sur un échantillon de 413 individus appartenant à 18 populations distribuées à travers l'ensemble de l'aire de répartition. Les 13 locus ont révélé entre 9 et 36 allèles. Ces marqueurs ont été testés avec succès dans un autre laboratoire, ce qui confirme la robustesse de nos protocoles de biologie moléculaire. L'efficacité des marqueurs a également été testé sur six autres espèces venant de l'Asie et d'Amérique du Nord, 6 à 10 marqueurs se sont révélés utilisables pour chacune de ces espèces. Cette étude montre que même pour des espèces ayant de grande taille de génome et relativement peu de polymorphisme, des microsatellites peuvent être développés avec succès en utilisant du séquençage nouvelle génération, à condition que certaines précautions soient prises.

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Quand nous avons commencé la mise au point de nos marqueurs, la technique de pyroséquençage était encore peu fréquente. Il y avait peu d'expérience disponible et aucune stratégie particulière n'était préconisée. Dans notre cas, l'entreprise a été coûteuse en terme de temps (environ une année) et d'argent. En effet, un premier séquençage n'a pas permis de fournir des microsatellites aux motifs suffisamment longs. Par conséquent, pour obtenir des marqueurs polymorphes, il a fallu créer une autre librairie et séquencer une seconde fois.

Chapitre 4

Si les invasions biologiques sont actuellement très étudiées, ce n'est pas le cas des translocations de population à l'intérieur ou à proximité de l'aire de distribution d'une espèce. Il est en effet difficile de les identifier systématiquement. Larix decidua est caractérisé par une aire de distribution restreinte aux Alpes et à d'autres montagnes de l'Europe centrale. Il a été planté massivement depuis au moins 300 ans. Dans cette étude je me suis concentrée sur la caractérisation des translocations qui ont eu lieu à l'intérieur ou à la proximité de l'aire de répartition de cette espèce. Grâce à des données de séquence de deux fragments mitochondriaux et la variation de 13 locus microsatellites d'un échantillon représentatif de l'ensemble de l'aire, j'ai détecté une structuration spatiale forte avec les deux types de marqueurs. Deux méthodes complémentaires (affectation individuelle à des populations et détection de migrants de première génération) ont été utilisées pour identifier les événements de translocation. Les résultats montrent que des identifications sont possibles au niveau des génotypes individuels car elles sont confirmées par des données mitochondriales indépendantes. Les événements de translocation et de mélange (admixture) sont distribués de manière hétérogène à travers l'aire de répartition avec une fréquence particulièrement élevée en Europe centrale. En outre, les translocations impliquent de multiples sources géographiques, certaines provenances étant plus représentées que d'autres. Ces résultats soulignent l'importance d'une telle étude de translocation à l'échelle de l'ensemble de l'aire de répartition d'une espèce, indispensable pour analyser l'histoire ancienne, établir des système de traçabilité et assister d'éventuelles migration de populations à l'avenir.

Chapitre 5

Les études historiques utilisant des données fossiles permettent d'analyser les réponses d'espèces aux oscillations climatiques extrêmement variables du dernier cycle interglaciaire/glaciaire. Elles sont particulièrement informatives pour des évaluations des réponses des espèces au changement climatique actuel. Elles peuvent également servir à identifier des zones refuges, des voies de migrations et aussi les éventuels impacts anthropiques sur les populations. Dans cette étude nous avons retracé 130 ka de l'histoire de Larix decidua à l'échelle de l'aire globale en compilant des données fossiles (pollen, macrofossiles, stomates, charbons) de quatre sources (littérature, Alpine palynological database, Czech Quaternary palynological database et European pollen database). Parmi les 1026 sites fossiles analysés, 355 incluent des fossiles de Larix. Les occurrences fossiles sont visualisées sur une série de cartes chronologiques. Ces cartes montrent que pendant le premier interstadial du Weichsélien ancien (env. 87 -109 ka) la distribution du mélèze a été maximale dans la partie nord et centrale de l'Europe où il a contribué à établir des forêts boréales. Cette expansion exceptionnelle a été favorisée par un climat subcontinental concomitant à un événement froid (Montaigu event). Dans les Alpes le mélèze a formé des forêts d'altitude pendant cet interstadial. Pendant le deuxième interstadial du Weichsélien ancien la tel-00880539, version 1 -6 Nov 2013 distribution a été similaire mais avec une représentation plus faible dans la partie nord et centrale. À la fin de cet interstadial les forêts ont disparu dans cette partie tandis que dans les Alpes on trouve durant un troisième interstadial des forêts avec une contribution importante du mélèze. Pendant le MIS 4, caractérisé par des conditions climatiques extrêmement sévères, des toundras ont prédominé dans une grande partie de l'aire actuelle du mélèze. Les conditions climatiques plus favorables du MIS 3 ont mené à de nouvelles recolonisations par le mélèze. Dans deux sites localisés dans la région des Alpes on observe que l'expansion autour de ces sites a été favorisée par les événements Dansgaard-Oeschger 12 et 14. Pendant le maximum de la dernière glaciation (= LGM, env. 23-19 ka) le mélèze a persisté dans le piedmont des Alpes et des Carpates et dans la Plaine de la Pannonie. Les fossiles indiquent plusieurs refuges : la région de Turin, l'extrémité sud des Alpes, la région de la Vénétie, la région de la Carinthie, les Sudètes, les Carpates de l'ouest et les Carpates du sud. Après le LGM le mélèze s'établi sous forme de forêts pionnières en basse altitude. Cependant, il ne s'agit pas à ce moment là d'un développement majeur de ces forêts, freiné par les contraintes climatiques d'un événement froid caractérisant cette période (Heinrich 1). L'interstadial Bølling/Allerød (env. 14.5 -12.8 ka) a provoqué des recolonisations rapides des hautes altitudes des Alpes et des Carpates ainsi que de la plaine de Pologne. Toutefois, la limite altitudinale des arbres est restée inférieure au niveau actuel. C'est l'augmentation de température du début de l'Holocène qui a généré des recolonisations atteignant ou dépassant les niveaux actuels. Les forêts subalpines se sont établies pendant les deux premiers millénaires de l'Holocène. La distribution contemporaine est atteinte à environ 5 ka. Les impacts anthropiques négatifs (extinctions de populations) et positifs (espèce favorisée) sont devenus plus importants à partir de l'Age du Bronze et se prolongent jusqu'à aujourd'hui. Cette étude montre que le mélèze est resté proche de son aire actuelle mais que dans le même temps l'aire a été très dynamique. L'homme a créé des habitats artificiels et à conduit à une superposition des facteurs climatiques et anthropiques (p. ex. ouverture de paysage, feux, pâturage) influençant la distribution de l'espèce.

Chapitre 6

Les données génétiques et fossiles acquises dans les étapes précédentes fournissent une base solide pour évaluer l'impact climatique et anthropique sur la structuration génétique observée. L'étude révèle une structuration génétique exceptionnellement forte pour les deux génomes. Les données mitochondriales et nucléaires suggèrent une séparation ancienne des populations des deux régions principales (Alpes et Carpates/Sudètes/plaine de Pologne). Sept groupes nucléaires ont été identifiés: quatre dans les Alpes et trois dans la région est. Dans les Alpes les quatre groupes nucléaires et les deux haplotypes mitochondriaux majoritaires sont répartis longitudinalement. L'analyse de leur distribution suggère qu'il n'y a pas eu de flux de gènes importants entre les groupes de population correspondants. Cette structuration génétique et les fossiles trouvés pendant ou peu après le LGM (env. 23 -16 ka) indiquent que six des sept groupes nucléaires ont eu leur origine dans une des zones de refuge identifiées par des fossiles : groupe 1 dans la région de Turin, groupe 2 dans la région de la Vénétie, groupe 3 dans la région de Carinthie, groupe 5 dans les Sudètes, groupe 6 dans les Carpates de l'ouest, groupe 7 dans les Carpates du sud. Il n'y a pas de site fossile disponible correspondant au groupe 4 trouvé au bord des Alpes de l'est. Dans cette région ainsi que dans les Alpes maritimes les données génétiques suggèrent l'existence de refuges non encore identifiés par des fossiles (refuges cryptiques). La recolonisation des Alpes a commencé dans les trois ou quatre zones refuges identifiées et s'est poursuivie le long de plusieurs voies de migration traversant les Alpes. Dans la partie est de l'aire de distribution, les populations contemporaines proviendraient de populations locales par simple déplacement altitudinal.

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Les méthodologies paléoécologiques et génétiques combinées dans cette thèse permettent d'évaluer les effets climatiques et anthropiques sur les populations à des échelles temporelles et spatiales multiples, donnant ainsi des perspectives différentes. L'approche génétique n'utilise que les populations actuellement en place et montre que la distribution des lignées est expliquée d'une part par le climat (structure ancienne) et d'autre part par des translocations récentes (superposées à la structure ancienne). Elle montre que les translocations ont un impact important sur les populations contemporaines. Dans certains cas les données fossiles peuvent confirmer la preuve génétique des translocations. Mais les détails des processus évolutifs accompagnant les changements climatiques et anthropiques (mélange, introgression, etc.) ne peuvent être étudiés que par l'approche génétique. Dans les populations de mélèzes les mélanges artificiels sont abondants et même alarmants dans quelques cas, notamment en Slovaquie, Tchéquie et Pologne. Le fait que les provenances ont des sensibilités différentes vis-à-vis du chancre du mélèze implique que des translocations de provenances mal adaptées peuvent mener à des effondrements de populations, un point conforté par l'étude sur le chancre du mélèze menée parallèlement à cette thèse. Cette analyse a confirmé que les outils génétiques développés dans cette thèse et associés à un échantillonnage au niveau de l'ensemble de l'aire de répartition permettent de répondre à des questions de foresterie (p.ex. sensibilité différente des provenances de mélèze face au chancre, traçabilité). L'approche paléoécologique permet de révéler et de dater d'autres effets anthropiques, par exemple le pâturage favorisant ou défavorisant le mélèze à partir de l'Age de Bronze. Cette approche va donc plus loin dans le passé et montre que entre 130 ka et env. ............................................................................

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Scientific background

Studies of the consequences of past climate change on forests worldwide provide a necessary historical context for evaluating the possible consequences of ongoing climate change, an emerging field of research (e.g. [START_REF] Colombaroli | Species responses to fire, climate and human impact at the tree line in the Alps as evidenced by palaeoenvironmental records and a dynamic simulation model[END_REF][START_REF] Petit | Forests of the Past: A Window to Future Changes[END_REF]Tinner & Ammann 2005). For instance, palaeoclimatic studies have identified periods of rapid changes, with temperature shifts up to 16°C on a centennial or even decadal timescale [START_REF] Wolff | Millennial-scale variability during the last glacial: The ice core record[END_REF].

Reconstructions of past forest distributions and dynamics can advance our understanding of relevant processes such as tree colonization, adaptation, and extinction in response to climatic change. A detailed understanding of these processes is necessary to accurately model future vegetation dynamics under climatic conditions for which no modern analogues exist. The advantages of such retrospective approaches are obvious in view of the difficulties to experiment with forests and climate.

In Europe, the availability of large amounts of fossil data coupled with modern reconstruction and dating techniques have enabled detailed and robust reconstructions of the last glacial and postglacial vegetation and climate based on pollen and macrofossils. Similarly, phylogeographic studies and population genetic surveys of several dominant forest trees (e.g. Quercus, Fagus sylvatica, Pinus sylvestris, Abies alba, Picea abies) have been performed. These studies have helped identifying glacial refugia and postglacial colonisation routes (reviewed in [START_REF] Hu | Paleoecology meets genetics: deciphering past vegetational dynamics[END_REF]). Side-by-side comparisons of genetic and fossil data have been used to reconstruct the history of these tree species since the last glacial maximum [START_REF] Cheddadi | Imprints of glacial refugia in the modern genetic diversity of Pinus sylvestris[END_REF][START_REF] Liepelt | Postglacial range expansion and its genetic imprints in Abies alba (Mill.) -a synthesis from paleobotanic and genetic data[END_REF][START_REF] Magri | A new scenario for the Quaternary history of European beech populations: palaeobotanical evidence and genetic consequences[END_REF][START_REF] Petit | Identification of refugia and post-glacial colonisation routes of European white oaks based on chloroplast DNA and fossil pollen evidence[END_REF][START_REF] Tollefsrud | Genetic consequences of glacial survival and postglacial colonization in Norway spruce: combined analysis of mitochondrial DNA and fossil pollen[END_REF]. In comparison, the effects of more rapid changes on forests, including those caused by recent plantations or by abrupt climatic events, have been largely neglected. New, more powerful genetic markers (e.g. Guichoux et al. 2011a) and precise vegetation records that can be correlated to high-resolution climate records of the last glacial cycle [START_REF] Fletcher | Millennial-scale variability during the last glacial in vegetation records from Europe[END_REF] must now be used and combined to assess long-term and short-term history using different perspectives.

For such studies, suitable biological models are needed. Conifers combine several important advantages. They are typically abundant and represent keystone species for vegetation reconstruction. Thanks to their large biomass, they are susceptible to leave many fossils in sediments. In particular, these wind pollinated species produce considerable amounts of pollen that can be identified in sediments from peat bogs, ponds or lakes to reconstruct in detail their past distribution through time. Another advantage is that they have three different tel-00880539, version 1 -6 Nov 2013 genomes, each with a specific mode of inheritance (biparental for the nuclear genome, maternal for the mitochondrial genome and paternal for the chloroplast genome), which provide contrasting and complementary information on their history if the data is appropriately analysed. They are therefore very promising candidates for the application of such an approach. They should provide precise and accurate insights into past vegetation dynamics and help identify the underlying processes and lead to important improvements of existing vegetation and climate models for simulating future scenarios.

We propose as case study the European larch (Larix decidua). This deciduous conifer species has not been subject to recent historical studies, but it has some unique features that make it very suitable to study recent and ancient history (see chapters 2, 4 and 5).

Structure of the thesis

This thesis is an interdisciplinary study joining population genetics and palaeoecology. Prior to this work, knowledge on the range-wide genetic structure of the species was very limited.

To prepare the population genetic part, I undertook a detailed literature review focused on existing markers for the species. This review was published in a report for the European project "Trees for Future" (http://www.trees4future.eu) whose aim is to provide genetic tools for future forest management. A modified version of this review is presented in chapter 2.

Based on this and on a technical expertise on genetic marker development I had acquired before (Guichoux et al. 2011a;Guichoux et al. 2011b;[START_REF] Lefèvre | Multiplex kits of microsatellite markers for genetic studies of beech[END_REF], I started the design of nuclear microsatellites, as explained in chapter 3. This turned out to be, as in other conifers characterized by a large genome, a difficult and time-consuming technical challenge, for which I could take advantage of the development of next-generation sequencing techniques. These markers were eventually applied on a range-wide sample of 45 modern populations (24 individuals/population) and analysed in parallel with mitochondrial DNA data that had been produced by cooperation partners (S. Liepelt and co-workers, Marburg University). The aim was to identify and characterize recent translocations without any reference to fossil data (chapter 4).

The palaeoecological work also included an extensive literature survey to compile and correlate at the European scale larch fossil over the last 130,000 years. Based on this and on fossil data from different databases I reconstructed the history of larch including its responses to climate oscillations (short-term, long-term) and to anthropogenic influences without tel-00880539, version 1 -6 Nov 2013 making reference to the genetic data (chapter 5). In the final chapter 6, I synthesize fossil and genetic data to identify last glacial maximum refugia and to compare anthropogenic impacts identified by genetic tools versus by fossils. I also mention some ideas emerging from a related project dealing with larch canker, the most serious larch disease. Tolerance to this disease strongly differs between populations from different parts of the range, suggesting that recent translocations could have serious consequences if maladapted provenances are transplanted. A preliminary report of this project is provided in Appendix 1.

All chapters are conceptualized to allow an independent understanding.

Scientific contributions and acknowledgements

This study was funded by the German Research Foundation (DFG LI 582/18-1) and conceived as a bi-nationally supervised thesis. The paleoecological part was directed by The chapters of this thesis represent the state of the art of my research and are at different stages of advancement: chapters 2 and 3 are published (http://www.trees4future.eu, Wagner et al. 2012). They appear in slightly modified versions in this thesis. Chapter 4 will be submitted after some final analyses are performed, whereas chapters 5 and 6 are preliminary reports.

Species description

Larch (Larix) belongs to the Pinaceae family and is one of the most abundant coniferous genera of the northern hemisphere. The genus comprises 10 species occurring in Eurasia and North America [START_REF] Farjon | A Handbook of the World's Conifers[END_REF]. European larch (Larix decidua, Miller 1768) is endemic to Europe and is characterized by a strongly disjunctive distribution (Fig. 1, Fig. 2) (e.g. [START_REF] Bauer | Die Sudetenlärche[END_REF][START_REF] Geburek | Larix decidua Miller[END_REF][START_REF] Mccomb | The European Larch: Its races, site requirements and characteristics[END_REF][START_REF] Rubner | Die pflanzengeographischen Grundlagen des Waldbaus Neumann[END_REF]). The first and major continuous distribution area is located in the European Alps and is centred in the subalpine belt of the continental Central Alps but larch also occurs in other parts of the Alps over a wide altitudinal range. In the Western Alps, the upper limit is 2,300 m or even 2,900 m if one includes krummholz trees (i.e. small trees that do not grow into large canopy trees). The lower limit is 1,300 m in the south-western part of the Alps and 450 m in the north-western part. In the easternmost Alps the distribution is centred at lower altitudes with more humid climates. The upper limit in this part of the range is 1,600 m and the lower limit 300 m. Morphologically and ecophysiologically differentiated ecotypes are found in each of the areas described above. These ecotypes were previously treated as subspecies of L. decidua (ssp. decidua, ssp. sudetica, ssp. carpathica and ssp. polonica). [START_REF] Farjon | A Handbook of the World's Conifers[END_REF] identifies only three varieties (var. decidua, var. carpathica, and var. polonica). A crucial result of a multisite L. decidua provenance trial was the finding that Central European provenances, in particular the Sudety ones as well as alpine provenances from lower altitudes, are less canker sensitive than the other alpine provenances, especially the western Alpine provenances [START_REF] Schober | Vom II. internationalen Lärchenprovenienzversuch: Ein Beitrag zur Lärchenherkunftsfrage[END_REF]Schober 1985). Larch canker is transmitted by the ascomycete Lachnellulla willkomii (Hartig) Dennis [START_REF] Schober | Die Lärche -Eine ertragskundlich-biologische Untersuchung[END_REF][START_REF] Willkomm | Die mikroskopischen Feinde des Waldes[END_REF]) and is the most serious of European larch diseases.

Delimitation of the natural range of European larch is very challenging. This is caused by a long history of planting, starting in the 16 th century and culminating in the 19 th century (e.g. [START_REF] Rubner | Die pflanzengeographischen Grundlagen des Waldbaus Neumann[END_REF][START_REF] Schober | Die Lärche -Eine ertragskundlich-biologische Untersuchung[END_REF][START_REF] Tschermak | Die natürliche Verbreitung der Lärche in den Ostalpen[END_REF]. During this time, larch was extensively planted within and beyond its natural range, leading to uncertainties about the autochthony of populations, in particular in the Central European region. An unexpected attendant phenomenon of these extensive plantations was the expansion of larch canker, which contributed in a major way to the decrease of plantations in the 20 th century. Later on these problems were attributed to the use of canker-sensitive material in afforestation (e.g. [START_REF] Schober | Vom II. internationalen Lärchenprovenienzversuch: Ein Beitrag zur Lärchenherkunftsfrage[END_REF]. Japanese larch (Larix kaempferi (Lamb.) Carr.) and hybrids of European and Japanese larch (Larix × eurolepis Henry) have also been planted in Europe, but only since the middle of the 20th century. The hybrid combines favourable properties of the parental species such as stem straightness, drought-tolerance and low canker sensitivity [START_REF] Acheré | Chloroplast and mitochondrial molecular tests identify EuropeanxJapanese larch hybrids[END_REF][START_REF] Bauer | Die Sudetenlärche[END_REF][START_REF] Geburek | Larix decidua Miller[END_REF]. European larch is a monoecious, diploid (2n=24), mainly outcrossing, deciduous pioneer tree, which becomes mature after 20-30 years [START_REF] Dieckert | Selbststerilität und Selbstfertilität bei Picea abies Karst und Larix decidua Mill[END_REF][START_REF] Geburek | Larix decidua Miller[END_REF]). Flowering period is from (February) March to April (May). Its wind-dispersed pollen does not have air sacs like the pollen of other conifers, resulting in less extensive dispersal [START_REF] Sjögren | The development of composite dispersal functions for estimating absolute pollen productivity in the Swiss Alps[END_REF]Sjögren et al. 2008). Seeds are ripe in autumn of the flowering year.

They are enclosed in cones within winged nutlets. Cones stay on the tree over 2-3 (5) years and shed seeds under dry weather conditions. Seeds are mostly wind-dispersed and mast seeding years occur approximately every 10 years in this species [START_REF] Rameau | Flore forestière française -Guide écologique illustré 2: montagnes[END_REF]. Clonal propagation resulting in small clusters of clonal trees can occur when sapling branches touch the ground and get rooted [START_REF] Pluess | Pursuing glacier retreat: genetic structure of a rapidly expanding Larix decidua population[END_REF]. Larch is a light-demanding pioneer tree with a low competitive ability. It is outcompeted by other species in climax stages whereas its persistence is favoured in different kinds of disturbance-driven ecosystems. Under optimal conditions and tel-00880539, version 1 -6 Nov 2013 free standing it can reach maximum ages of 600-850 years. Under forest conditions, it reaches only about 150 years [START_REF] Bauer | Die Sudetenlärche[END_REF][START_REF] Geburek | Larix decidua Miller[END_REF]. Even though European larch is currently less planted than some other conifers such as Norway spruce or Douglas-fir, it is still very much appreciated because of its fast juvenile growth, low sensitivity to most diseases as well as to wind, snow, fire, air pollution and chemicals. Its wood is characterized by a high density, long durability, good elasto-mechanical properties, and good aesthetic value. For these and for other reasons it is used for afforestation, reforestation and as construction-timber for outdoor and indoor equipments or barrels for conservation of chemicals. Examples illustrating its excellent construction properties are the world's highest wooden tower (118 m, Gliwice, Poland) and several Venise bridges [START_REF] Miranda | Bridges in Venise -architectural and structural engeneering aspects[END_REF][START_REF] Grosser | Das Holz der Lärche -Eigenschaften und Verwendung[END_REF].

Concerning the material to be traced it would be of great interest to have markers allowing to elucidate populations' status (native/non-native) and to evaluate the potential effects of introgression (inter-or intraspecific), in particular in regions that have been strongly impacted by recent introductions. This would allow a better delimitation of its natural range. Second, it would be important to trace material used in breeding populations and in forest reproductive material (FRM) to ensure the use of only well performing provenances and varieties for plantation. Third, plantations showing unexpectedly high canker sensitivity should be studied to clarify their origin. Another type of material that could be profitably analysed is wood logs of unknown origin to fight illegal logging.

Review of existing genetic markers

For some reason marker development in European larch has been lagging behind that observed in other European tree species, although its three differently inherited genomes (nuclear: biparentally inherited, mitochondria: maternally inherited, chloroplast: paternally inherited) offer ideal preconditions for powerful marker combinations. Genetic studies started about 40 years ago with the development of isozyme markers [START_REF] Mejnartowicz | Genetic studies on European larch (Larix decidua Mill.) employing isozyme polymorphisms[END_REF]. Isozyme markers are still used today [START_REF] Konnert | Proof of identity of forest reproductive material based on reference samples[END_REF][START_REF] Müller-Starck | Genetische Variation in altbeständen der Lärche und ihrer natürlichen Verjüngung im alpenraum (Genetic variation in adult stands of european larch and its natural regeneration in the alpine habitat)[END_REF]. Other nuclear and organelle markers have primarily been designed for the genus to address issues at the interspecific level (e.g. [START_REF] Acheré | Chloroplast and mitochondrial molecular tests identify EuropeanxJapanese larch hybrids[END_REF][START_REF] Mc | Species-diagnostic markers in Larix spp. based on RAM and nuclear, cpDNA, and mtDNA gene sequences, and their phylogenetic implications[END_REF]Semerikov & Lascoux 2003). Only very recently nuclear species-specific SNP markers have been developed (Mosca et al. 2012b). Other markers (oleoresins) have been tested with some tel-00880539, version 1 -6 Nov 2013 success. They allowed the distinction between Alpine and other provenances and between European larch from Japanese larch as well as from hybrids of European and Japanese larch [START_REF] Lang | Unterschiede in der Monoterpenzusammensetzung des Harzes einjähriger Lärchenzweige[END_REF][START_REF] Weissmann | Identifizierung von Hybridlärchen mit Hilfe chemischer Merkmale[END_REF].

Material analysed, DNA extraction and fragment analysis

DNA has been successfully isolated from various tissues such as megagametophytes (e.g. Mosca et al. 2012b), seedlings (e.g. Semerikov & Lascoux 2003), leaves (e.g. Pluess 2011), buds [START_REF] Scheepers | Identification of larch species (Larix decidua, Larix kaempferi and Larix X eurolepis) and estimation of hybrid fraction in seed lots by RABD fingerprints[END_REF] and embryogenic mass (L. Pâques, pers. comm.). For isozyme analyses megagametophytes have been commonly used (e.g. Lewandowski & Mejnartowicz 1991a). DNA isolation mostly followed CTAB [START_REF] Devey | A genetic linkage map for Pinus radiata based on RFLP, RAPD, and microsatellite markers[END_REF][START_REF] Doyle | Isolation of small amount of plant tissues[END_REF] or manufactured isolation kits such as DNeasy Plant Mini Kit (Qiagen, Gros-Louis et al. 2005) or DNAeasy 96 Plant kit [START_REF] Pluess | Pursuing glacier retreat: genetic structure of a rapidly expanding Larix decidua population[END_REF].

Organelle markers (mitochondrial and chloroplast DNA)

Organelle markers applicable on Larix decidua have been developed for questions such as phylogeny and species diagnostics. Chloroplast DNA variation has been studied based on sequence variation [START_REF] Mc | Species-diagnostic markers in Larix spp. based on RAM and nuclear, cpDNA, and mtDNA gene sequences, and their phylogenetic implications[END_REF][START_REF] Qian | Genetic relationships among larch species based on analysis of restriction fragment variation for chloroplast DNA[END_REF][START_REF] Wei | Phylogentic split of Larix: evidence from paternally inherited cpDNA trnT-trnF region[END_REF], PCR-RFLPs and microsatellites (Semerikov and Lascoux 2003;[START_REF] Acheré | Chloroplast and mitochondrial molecular tests identify EuropeanxJapanese larch hybrids[END_REF]). Mitochondrial DNA variation has been studied using direct PCR, PCR-RFLPs [START_REF] Acheré | Chloroplast and mitochondrial molecular tests identify EuropeanxJapanese larch hybrids[END_REF]Semerikov & Lascoux 2003;[START_REF] Semerikov | Mitochondrial DNA variation pattern in larches of eastern Siberia and the far east[END_REF]) and sequencing [START_REF] Mc | Species-diagnostic markers in Larix spp. based on RAM and nuclear, cpDNA, and mtDNA gene sequences, and their phylogenetic implications[END_REF]. These studies have identified useful markers and marker combinations for species discrimination. By combining a mitochondrial and a chloroplast marker, L. decidua and L. kaempferi could be discriminated [START_REF] Acheré | Chloroplast and mitochondrial molecular tests identify EuropeanxJapanese larch hybrids[END_REF][START_REF] Jagielska | Zastosowanie markerów genetycznych w identyfikacji gatunkowej modrzewia europejskiego (Larix decidua Mill.) i japońskiego (Larix kaempferi Sarg.) oraz ich mieszańców [Application of genetic markers in species identification of European and Japanese larch and their hybrids[END_REF]. In another study, L. decidua and L. kaempferi could be discriminated from L. sibirica and L. laricina by sequencing four chloroplast regions (matK, trnL-intron, trnT-trnL trnL-trnF) and all four species could be identified by sequencing five mitochondrial introns (cox1-1, matR-1, nad1b/c, nad3-1 and nad5-1; [START_REF] Mc | Species-diagnostic markers in Larix spp. based on RAM and nuclear, cpDNA, and mtDNA gene sequences, and their phylogenetic implications[END_REF]. Note that in the same study [START_REF] Mc | Species-diagnostic markers in Larix spp. based on RAM and nuclear, cpDNA, and mtDNA gene sequences, and their phylogenetic implications[END_REF] also obtained nuclear markers discriminating the four species (see nuclear markers section below).

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Nuclear markers

AFLPs (amplified fragment length polymorphisms) [START_REF] Arcade | Application of AFLP, RAPD and ISSR markers to genetic mapping of European and Japanese larch[END_REF] constructed single-tree genetic linkage maps of European and Japanese larch using 114 AFLPs resulting from 5 AFLP primer combinations. Note that in the same study they also used 149 RAPD and 3 ISSR loci. AFLPs were also used in phylogenetic reconstruction of the genus Larix (Semerikov et al. 2003). In the latter case the authors applied six primer combinations and scored 442 polymorphic fragments in 11 species.

Isozymes

Until 2011 isozymes have been the only nuclear markers used to study intraspecific variation in European larch. A common set of 13 isozyme markers is used in the German Züf certification system to trace forest reproductive material [START_REF] Konnert | Proof of identity of forest reproductive material based on reference samples[END_REF]. First attempts for studying intraspecific genetic variation across the range have been made by Lewandowski and Mejnartowicz (1991a) and [START_REF] Maier | Genetic variation in European larch[END_REF]. These studies led to some valuable information but as marker resolution is limited and sampling was not representative (both studies relied on seven populations), further investigations are necessary. One suggestion coming from Maier's study is that populations from the Eastern Alps are genetically closer to the Central European ones than to the Western Alpine ones. On the regional scale, a study in the Western Alps region of Piedmont found that larch populations from the Western Alps (Torino) and the Italian Central Alps (Verbania) form a common genetic group whereas a population from the southernmost Maritime Alps (southern Cuneo) was clearly different [START_REF] Belletti | Genetic variability among European larch (Larix decidua Mill.) populations in Piedmont, north-western Italy[END_REF]). However, sampling was uneven and should be refined to confirm the results. Another study included 26 populations from across the Alps and showed that there is no important loss of genetic diversity between old stands and their natural regeneration. It further revealed that genetic variation in L. decidua is lower than in Picea abies and Pinus mugo but higher than in Abies alba [START_REF] Müller-Starck | Genetische Variation in altbeständen der Lärche und ihrer natürlichen Verjüngung im alpenraum (Genetic variation in adult stands of european larch and its natural regeneration in the alpine habitat)[END_REF][START_REF] Müller-Starck | Empfehlungen zur genetisch nachhaltigen Waldbewirtschaftung -Beispiele aus dem Gebirgswald[END_REF]. Finally, a study carried out in Romania highlighted genetic peculiarities and relationships of the five fragmented Romanian centres of natural larch occurrence [START_REF] Meusel | Genetic diversity and breeding of larch (Larix decidua Mill.) in Romania[END_REF].

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Microsatellites Microsatellites in larch have been developed for different larch species [START_REF] Chen | Development and characterization of microsatellite loci in western larch (Larix occidentalis Nutt.)[END_REF][START_REF] Isoda | Isolation and characterization of microsatellite loci from Larix kaempferi[END_REF][START_REF] Khasa | Contrasting microsatellite variation between subalpine and western larch, two closely related species with different distribution patterns[END_REF][START_REF] Khasa | Isolation, characterization, and inheritance of microsatellite loci in alpine larch and western larch[END_REF]. Microsatellite development for European larch has been performed in this study (see Chapter 3).

SNP

SNPs have first been used in larch in a study aiming at distinguishing species (i.e. L. decidua, L. sibirica, L. kaempferi and L. laricina; [START_REF] Mc | Species-diagnostic markers in Larix spp. based on RAM and nuclear, cpDNA, and mtDNA gene sequences, and their phylogenetic implications[END_REF]. They resulted in the identification of three gene loci with fixed interspecific polymorphisms implicating 17 SNPs and two indels. Recently Mosca et al. (2012b) used a candidate gene approach to study nucleotide diversity and genes departing from neutral expectation in the four alpine species L. decidua, Pinus cembra, Pinus mugo and Abies alba. 800 genes originally sequenced in Pinus taeda were resequenced in each the four species, resulting in 307 SNPs for L. decidua.

This study was rather exploratory for larch as sequencing success was relatively low and sampling was not representative. A subsequent study dealt with geographical and environmental determinants of genetic diversity of the four alpine conifer species mentioned above (Mosca et al. 2012a). For L. decidua this was based on 267 SNPs discovered in the first study and revealed three genetic clusters across the Alps.

ITS

ITS sequencing of ITS1, the 5.8S ribosomal RNA gene and ITS2 was used in phylogenetic reconstruction of the genus Larix [START_REF] Gernandt | Internal transcribed spacer region evolution in Larix and Pseudotsuga (Pinaceae)[END_REF]Semerikov et al. 2003). ITS and AFLP phylogenies were very similar and reveal a basal position of L. decidua in the clade of Eurasian larch species.

ISSR

Three ISSR (inter-SSR) regions were used in combination with RADPs and AFLPs for genetic mapping of European and Japanese larch [START_REF] Arcade | Application of AFLP, RAPD and ISSR markers to genetic mapping of European and Japanese larch[END_REF].

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RAPD RAPD fingerprints were used to discriminate L. decidua, L. kaempferi and their hybrid L. x eurolepis to investigate hybrid performance [START_REF] Arcade | Heterozygosity and hybrid performance in larch[END_REF][START_REF] Scheepers | Identification of larch species (Larix decidua, Larix kaempferi and Larix X eurolepis) and estimation of hybrid fraction in seed lots by RABD fingerprints[END_REF].

Furthermore they were used in combination with AFLP and ISSR for genetic mapping of European and Japanese larch [START_REF] Arcade | Application of AFLP, RAPD and ISSR markers to genetic mapping of European and Japanese larch[END_REF].

Levels of differentiation

Phylogeny, evolutionary history, distinction of species, subspecies and hybrids

The phylogeny of the genus Larix has been reconstructed using various molecular markers [START_REF] Gernandt | Internal transcribed spacer region evolution in Larix and Pseudotsuga (Pinaceae)[END_REF][START_REF] Qian | Genetic relationships among larch species based on analysis of restriction fragment variation for chloroplast DNA[END_REF]Semerikov et al. 2003). All studies showed three monophyletic clades: a North American clade, a South Asian clade and a North Eurasian clade, with L. decidua having a basal position in the Eurasian clade. Special attention has been paid to the var. polonica that has been hypothesized to be a hybrid of L. decidua and L. sibirica larch [START_REF] Bobrov | History and systematics of larches[END_REF]. Hybrids of European and Japanese larch (L. x eurolepis) have been artificially introduced in the 20 th century.

To date conclusions about levels of genetic differentiation in Larix decidua have been drawn from studies focusing on the local and regional scales. On a very local scale (<5km) [START_REF] Pluess | Pursuing glacier retreat: genetic structure of a rapidly expanding Larix decidua population[END_REF] has reported an F ST value of 0.014 based on nuclear SSR data. On a broader scale (distance between the populations up to 200km), Mosca et al. (2012a) have obtained an F ST value of 0.011 based on nuclear SNP data. Finally, at the scale of the Italian Alpine region (populations located up to 600 km from each other), Mosca et al. (2012a) have reported a mean F ST of 0.04, indicating a higher degree of differentiation that can be explained by the subdivision of the studied populations in three major genetic groups, as revealed by Bayesian cluster analysis.

Glacial refugia, biogeographic history

So far, biogeographic history of European larch has not been studied in detail as it is the case for the other European tree species e.g. Quercus [START_REF] Petit | Identification of refugia and post-glacial colonisation routes of European white oaks based on chloroplast DNA and fossil pollen evidence[END_REF], Fagus sylvatica [START_REF] Magri | A new scenario for the Quaternary history of European beech populations: palaeobotanical evidence and genetic consequences[END_REF] and Pinus sylvestris [START_REF] Cheddadi | Imprints of glacial refugia in the modern genetic diversity of Pinus sylvestris[END_REF]) A detailed reconstruction of its history tel-00880539, version 1 -6 Nov 2013 of the last 130,000 years is done in this study using newly compiled fossils and genetic data (see Chapter 4 and Chapter 5).

Stand, seed and pollen dispersal, small scale genetic structure [START_REF] Pluess | Pursuing glacier retreat: genetic structure of a rapidly expanding Larix decidua population[END_REF] has performed a landscape-scale analysis of L. decidua along the lateral moraine and the adjacent valley slope of a glacier in the Swiss Alps at 1700-2240 m a.s.l.. Nine SSR markers were used for this purpose. All sampled individuals (N = 730) formed a single genetic cluster indicating homogenizing gene flow despite spatial genetic structure (SGS) up to 80 m. No evidence for selfing or for inbreeding was found in adults or in juveniles (heterozygote deficit was not significantly different from zero). SGS among juveniles was found at up to 30 m in the older sub-population whereas no SGS was found in the younger, recently established sub-population. A maximum likelihood paternity assignment revealed local gene dispersal in the ancient part (2-48 m) and intermediate-tolong distance dispersal into the recently colonized part (115-3132 m), pointing to intensive mixing of the genes in this expanding population and suggesting that genetic diversity can be maintained during rapid population expansion driven by climate warming.

Individual tree, parent trees

Genetic distances among European and Japanese larch clones were estimated based on RAPD markers to investigate the link between heterozygosity and heterosis in hybrid larch [START_REF] Arcade | Heterozygosity and hybrid performance in larch[END_REF].

Use of a traceability system

Currently 13 isozyme loci are used for European larch in the German ZüF (Zertifizierungsring für überprüfbare Forstliche Herkunft Süddeutschland e.V.) certification system [START_REF] Konnert | Proof of identity of forest reproductive material based on reference samples[END_REF].

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Traceability systems in use and future needs

Species identification

In European larch several markers have been tested for species identification. First, there is a combination of one chloroplast and one mitochondrial marker that can discriminate L. decidua and L. kaempferi and determine the parental species of their hybrid L. x eurolepis.

This approach is more powerful than isozymes and generally results are satisfying. However, there is the disadvantage that chloroplast DNA analysis is based on the presence (L. kaempferi) or absence (L. decidua) of DNA fragments, which in some cases leads to ambiguous results [START_REF] Acheré | Chloroplast and mitochondrial molecular tests identify EuropeanxJapanese larch hybrids[END_REF][START_REF] Jagielska | Zastosowanie markerów genetycznych w identyfikacji gatunkowej modrzewia europejskiego (Larix decidua Mill.) i japońskiego (Larix kaempferi Sarg.) oraz ich mieszańców [Application of genetic markers in species identification of European and Japanese larch and their hybrids[END_REF]. The use of pure species parents controls partly solves this problem. Second, sequencing organelle and nuclear markers can discriminate the four species L. decidua, L. kaempferi, L. sibirica and L. laricina [START_REF] Mc | Species-diagnostic markers in Larix spp. based on RAM and nuclear, cpDNA, and mtDNA gene sequences, and their phylogenetic implications[END_REF]; cf. Organelle marker section). Moreover infrared spectroscopy (NIRS) of needles and wood is currently tested as an alternative (L. Pâques pers. comm.). A future task will be to evaluate discrimination power of the microsatellites developed in this study for identification of different species as well as advanced hybrid generations. This is particularly needed as future seed orchards will frequently work with material from up to three hybrid generations including backcrosses.

Identification of the origin of FRM

The only method to identify the origin of seedlings declared in certification documents is to compare the seedlings to a reference sample. In Germany such a comparison is performed with the Züf System. An ad hoc assignment of FRM to its provenance region would be crucial for future silviculture preventing the choice of maladapted provenances. The markers and the range-wide reference sample of this study could be used for attempts in this direction (see Chapter 4).

Inference of the number of mother trees from a seed lot by molecular methods

Genetically improved seed lots from clonal seed orchards could be controlled with highly polymorphic markers using parentage analyses. This would allow tracing this material to the seed orchards where it was produced (or possibly ruling out such an origin) and enumerating the number of parental clones involved in the composition of each seed lot. The only tel-00880539, version 1 -6 Nov 2013 reference needed would be one representative of each clone. In principle, such an approach could also be used on unknown seed lots, although more developments would be necessary.

Clone identification, including clonal mixtures

Other than for hybrids of European and Japanese larch there are to our knowledge no commercial plantations with clonal material of European larch. However, clones are used in seed orchards to produce inter-and intraspecific hybrid seeds. Clonal identification with the help of sufficiently variable nuclear markers could help assessing the exact composition of seed orchards to control propagation operations.

Identification of introgression

Identification of introgression would be an important topic to address in regions where exotic provenances of European larch or Japanese larch have been detected.

Wood identification in relation to illegal logging

Illegal logging of old growth larch forests in Siberia is a topic of concern. Developing methods to trace this material with DNA isolated from logs (cambium or wood) would be of high relevance. However L. sibirica is also planted in some European countries makes it more complicated. Nevertheless, checking the conformity of the logs to the species could be a first useful step, as in Siberian forests several larch species occur.

Conclusions

Marker development and studies of European larch are not very numerous and as a consequence knowledge about this species has stayed limited. Powerful markers (SNPs, SSR) are urgently needed to open new perspectives for a number of practical applications in forest research and in forest management. They should enable precise monitoring of material produced in seed orchards as well as the identification of native and introduced material from across the range. This should guide plantation forestry and help focus conservation efforts in a species with a particularly intense history of provenance translocations.

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Supporting Information

Introduction

Larix decidua is an endemic European conifer with a highly fragmented montane to subalpine distribution in the Alps, the Sudety, the Tatra and the Carpathians as well as some exceptional lowland occurrences in Poland [START_REF] Mccomb | The European Larch: Its races, site requirements and characteristics[END_REF][START_REF] Rubner | Die pflanzengeographischen Grundlagen des Waldbaus Neumann[END_REF]. As these regions, especially the mountainous ones (e.g. [START_REF] Colombaroli | Species responses to fire, climate and human impact at the tree line in the Alps as evidenced by palaeoenvironmental records and a dynamic simulation model[END_REF]Tinner & Kaltenrieder 2005), are strongly exposed to climate change, there is an urgent need for detailed range-wide genetic studies that can provide information for preservation of valuable genetic resources and sustainable forest management. So far, range-wide genetic studies based on nuclear markers in L. decidua have exclusively relied on allozyme markers. Such studies have yielded useful information about genetic relationships among populations (Lewandowski et al. 1991;Lewandowski & Mejnartowicz 1991a, b;[START_REF] Maier | Genetic variation in European larch[END_REF]). However, to better understand past population dynamics of the species an increased resolution is needed. We have therefore tel-00880539, version 1 -6 Nov 2013 endeavoured to develop highly polymorphic nuclear microsatellites. The development was based on the transfer of existing markers from sister species and the selection and design of new markers based on 454 pyrosequencing. First, we tested existing markers from the Asian sister species L. kaempferi [START_REF] Isoda | Isolation and characterization of microsatellite loci from Larix kaempferi[END_REF] and the North American species L. occidentalis [START_REF] Chen | Development and characterization of microsatellite loci in western larch (Larix occidentalis Nutt.)[END_REF][START_REF] Khasa | Isolation, characterization, and inheritance of microsatellite loci in alpine larch and western larch[END_REF]. As this did not lead to a satisfactory number of markers matching recommended quality criteria, e.g. sufficient polymorphism and clear binning of alleles (Guichoux et al. 2011a), we developed new markers based on pyrosequencing of an enriched microsatellite library. This approach can be substantially more cost-effective as the conventional method based on the screening of cloned libraries by Sanger sequencing (e.g. [START_REF] Santana | Microsatellite discovery by deep sequencing of enriched genomic libraries[END_REF]. However, in our study, we had to face two difficulties. First, initial tests had shown that markers with <12 repeats (i.e. for the individual which was initially sequenced) were characterized by low variation. This low variation might in part be attributed to the fact that trees and shrubs, which have relatively long generation times, generally show lower rates of molecular evolution than related herbaceous plants [START_REF] Smith | Rates of Molecular Evolution Are Linked to Life History in Flowering Plants[END_REF]. To augment the chances to find variable markers we focused on microsatellites with a high number of repeats (≥12) as microsatellite polymorphism is known to increase exponentially with the number of microsatellite repeats [START_REF] Ellgren | Microsatellite mutations in the germline: implications for evolutionary inference[END_REF][START_REF] Kelkar | The genome-wide determinants of human and chimpanzee microsatellite evolution[END_REF]. Second, in coniferous species with large genomes such as L. decidua (11,198 Mb, Greibhuber 1986), a large proportion of microsatellites can be expected to be located within repetitive DNA sequences, leading to marker candidates that are difficult to amplify (e.g. [START_REF] Pfeiffer | Identification and characterization of microsatellites in Norway spruce (Picea abies K.)[END_REF]. We therefore targeted a large number of marker candidates to select those showing both clear amplification profiles and sufficient polymorphism. Finally, we decided to combine the markers in multiplex reactions, i.e. PCR reactions amplifying several markers simultaneously, because once such multiplexes are established they enormously reduce laboratory costs and labour time and thus enable high throughput analyses and promote accuracy and precision of the genetic result (Guichoux et al. 2011a;Guichoux et al. 2011b;[START_REF] Lefèvre | Multiplex kits of microsatellite markers for genetic studies of beech[END_REF]. By following all these steps, we obtained two highly variable multiplex for Larix decidua (one 7-plex and one 6-plex). tel-00880539, version 1 -6 Nov 2013

Materials and methods

Plant material and DNA isolation

In 2010, we collected phloem and needle samples from 18 populations forming a gradient over the natural distribution range (Table 2). Eight had been collected in situ and 10 ex situ in four German provenance trials. In the latter case, each population sample (consisting in 24 individuals) originated from one single trial. Further samples for marker validation as well as for transferability tests were provided by colleagues. These included six progenies (each comprising one female parent and seven offspring) that were collected in a progeny trial (Planches, France). Note that progeny tests in this study only give a rough insight into Mendelian segregation but should help detect null alleles. Originally we planned to start with 12 progenies (12 female parents and 7 offspring) based on seeds. Due to problems with material, we were had to work with the six progenies described above. Furthermore, we obtained DNA samples of another six Larix species. These were L. sibirica (21 individuals from one population), L. kaempferi (12 individuals from 12 populations) and L. gmelinii var. japonica (12 individuals from 12 populations), all from Eurasia. From North America, we obtained samples from L. laricina (10 individuals from 10 populations), L. lyallii (4 individuals from 4 populations), and L. occidentalis (4 individuals from 4 populations). For the samples that we collected ourselves, we mostly relied on phloem as tree height (up to 40m in the trials) made it difficult to collect needles. Phloem was sampled by using a hammer and small leather punch (Ø=1cm, length=10cm). The sampling technique we developed was rapid and easy. The leather punch was positioned between the bark scales (which can be very thick) and with one to three slight hammer strokes a small but sufficient sample (Ø=1cm, depth 1.5cm) was recovered. Damage to the tree was minimal and fast regeneration was ensured by sampling during the growing season. Samples were then put into tea bags that were stored in sealed plastic bags with 10g of silica gel. We isolated DNA from all individuals using 96-well plates. Starting material was mostly phloem (1cm disc 0.5mm thick), but in some cases needles were used (1-3 needles, cut into 2mm pieces). For material disruption, we added two 4mm-tungsten beads to the wells with the starting material. The plates were frozen during 1min in liquid nitrogen before a 1:30 min disruption by a Mixer Mill (Retsch, Germany). This step was repeated once. An Invisorb DNA 96 plant HTS kit (Invitek, Germany) was then used for DNA isolation following the manufacturer protocol. After isolation, DNA quality was evaluated on a 1% (w/v) agarose gel stained with GelRed (Biotium, USA). DNA concentration was determined by an eight channel Nanodrop spectrometer and adjusted to 10ng/µl on a STARTlet 8 channel robot (Hamilton, USA).

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Multiplex optimization

Multiplex optimization followed the recommendations given in Guichoux et al. (2011a) to guarantee high marker quality, sufficient polymorphism and unambiguous allele binning.

Screening for marker candidates started by a literature search of available markers from the closest sister species Larix kaempferi [START_REF] Isoda | Isolation and characterization of microsatellite loci from Larix kaempferi[END_REF]) and the North American species L. occidentalis [START_REF] Chen | Development and characterization of microsatellite loci in western larch (Larix occidentalis Nutt.)[END_REF][START_REF] Khasa | Isolation, characterization, and inheritance of microsatellite loci in alpine larch and western larch[END_REF]. These were tested in simplex reactions. As this did not lead to a sufficient number of suitable candidates, we designed new markers based on 454 sequencing of an enriched microsatellite library. Sequencing was done by the Swiss company ecogenics GmbH. In this sequencing approach, fragments were selected according to their size from genomic DNA enriched with simple sequence repeat (SSR) motifs by using magnetic streptavidin beads and biotin-labelled CT and GT repeat oligonucleotides. The SSR enriched library was analyzed on a Roche 454 platform using the GS FLX titanium reagents. We worked on 1/16th of a 454 run. For the selection of candidate tel-00880539, version 1 -6 Nov 2013 markers, we used as main criterion the number of dinucleotide repeats (≥12). Note that this decision was taken after a failed attempt based on 454 sequencing of another enriched library produced by another company (data not shown). The problem had been that most of the marker candidates had had less than 12 repeats and were monomorphic or displayed limited variation (≤4 alleles). Those having 12 or more repeats (15 out of 100) had showed bad amplification or multiple bands. Hence, this experiment was abandoned. The main reason for the failure of the first experiment seemed to be poor sequencing quality and insufficient read length, making it difficult to design primers in the flanking regions of microsatellites with the targeted repeat unit number. In the second attempt, more candidates were obtained.

Polymorphism and profile quality were first checked in simplex reactions using the M13technique [START_REF] Schuelke | An economic method for the fluorescent labeling of PCR fragments[END_REF]. The PCR products were separated on a capillary sequencer (ABI-3730, Applied Biosystems, USA). Each marker was tested on seven individuals from across the range and on an additional DNA pool composed of 12 individuals from different geographic origins. Markers with low polymorphism (<5 alleles/7 individuals, unless additional alleles could be found in the pooled DNA sample) and profiles of low quality (multiple bands, bad amplification) were discarded. Second, the remaining candidates were tested for the presence of null alleles and for large allele dropout (e.g. non amplification of the longer allele due to differential amplification success) by using segregation analyses. For this purpose, six families composed of the female parent and seven offspring were screened.

Third, based on observations of optimal annealing temperature for the markers and by taking into account the size range of each locus, we composed the two multiplexes. For the multiplex reaction, we used the Qiagen Multiplex PCR kit (Qiagen, Germany). Final volume and final concentration of Mastermix were optimized to reduce final costs (Guichoux et al. 2011a).

PCR mix for both multiplexes was composed of 4.75µl of sterile water, 4µl of Qiagen Multiplex Buffer (2×), 1.25µl of primer premix and 3µl of DNA (10ng/µl). Concentrations of the primer pairs in the primer premix are given in

Results and Discussion

Multiplex optimization

We tested the nine most promising candidate loci originating from the literature in simplex reactions. Most promising means that these loci had already been proven to amplify well and to be polymorphic [START_REF] Pluess | Pursuing glacier retreat: genetic structure of a rapidly expanding Larix decidua population[END_REF]. The 454 sequencing approach resulted in 27,041 reads with an average length of 184 base pairs. Of these, 3,311 contained a microsatellite insert with a tetra-or a trinucleotide of at least six repeat units or a dinucleotide of at least 10 repeat units.

Primer design was possible in 312 reads, of which 100 were tested. Simplex PCR conditions had to be adjusted for each marker. This was because some candidates with long motifs needed more PCR cycles than others to be successfully amplified whereas such an increase resulted in non-specific products for other candidates. Testing marker profile quality and polymorphism (with a target of ≥5 alleles) over seven individuals and an additional DNA pool resulted in the selection of seven out of nine loci originating from the literature and 26 out of the 100 newly designed candidates. For identifying the latter, we started by excluding 34 markers with stutter bands or bad amplification, as identified on agarose gels. After genotyping the 66 remaining candidates with ABI capillary sequencer, we removed loci with multiple bands (19 markers), bad amplification (9 markers) or insufficient polymorphism (12 markers), leaving only 26 suitable markers. Low success rates have also been reported in other plant species with large genomes (e.g. [START_REF] Pfeiffer | Identification and characterization of microsatellites in Norway spruce (Picea abies K.)[END_REF][START_REF] Röder | Abundance, variability and chromosomal location of microsatellites in wheat[END_REF]. In particular, a detailed investigation of the causes of amplification failures of microsatellite markers in Picea abies using Southern blot experiments showed that, in six out of seven cases tested, loci showing high quality profiles corresponded to single or low-copy sequences [START_REF] Pfeiffer | Identification and characterization of microsatellites in Norway spruce (Picea abies K.)[END_REF]. This suggests that to identify sufficient candidate loci in species with large genomes, careful evaluation of the sequencing depth is necessary. Indeed, many markers will fail to produce clear PCR if the flanking regions (used to design the primers) are repeated elsewhere in the genome.

The other evaluation steps included allele binning as well as tests of Mendelian segregation to search for large allele dropout or null alleles. This led to the selection of a subset of 22 candidates for multiplexing for which binning was clear. There was no indication for nonamplification corresponding to large allele dropout. Null alleles were apparent at some markers (Ld50, bcLK189, bcLk263, Ld42, Ld45, Ld30, Table 3 andTable S1, Supporting Information). They were identified by comparing the female parent genotype with tel-00880539, version 1 -6 Nov 2013 the genotypes of the offspring. As there were too few loci combining sufficient polymorphism and zero mismatches in the progeny test, we retained those loci with no more than two Mendelian discrepancies and sufficient polymorphism. The number of possible multiplex combinations was limited as allele size ranges of the markers were large and as we had to take into account heterogeneities in PCR conditions. We were finally able to design one 6-plex and one 7-plex that we validated over the 413 individuals coming from across the range.

Laboratory protocols were successfully tested in another laboratory (INRA Orléans, UR AGPF, Vanina Guérin, personal communication), confirming their robustness.

Genotype scoring and analyses

Clear binning of alleles was confirmed over the whole sample. The marker allelograms, i.e. diagrams showing all detected allele raw sizes ranked in increasing order, are shown in Figures 3 and4. They indicate that most markers are characterized by a clear succession of dinucleotide repeats over large size ranges (between 28 and 74bp, mean 42bp) and a remarkably high number of alleles (9-36, mean of 20). Off-ladder microvariants, i.e. allele sizes that are in between the sizes expected from the repetition of dinucleotide units, were only observed for one of the 13 loci (Ld45, see Figure 4) and as these intermediate size variants were clearly separated from the neighbouring size classes, they did not cause problems for binning. In fact, such clearly defined off-ladder microvariants can improve the precision of the analyses when they are correctly identified (Guichoux et al. 2011b). There was no incoherency across repetitions (positive and random controls). Type A and Type B error rates ranged between 0 and 0.19% (mean of 0.05%) for multiplex 1, and between 0 and 1.3% (mean 0.32%) for multiplex 2. The frequencies of cases that could not be scored were slightly higher. They ranged from 0 to 2.1% (mean of 0.32%) for multiplex 1 (mainly caused by an unspecific product being amplified at locus bcLK211) and from 0 to 0.94% (mean of 0.16%) for multiplex 2 (mainly caused by excessive stuttering of some long alleles at locus bcLK263

Conclusions and perspectives

This study shows that even for species with very large genome size and low overall polymorphism, new highly informative microsatellites can be developed based on 454 sequencing, provided that some precautions are taken. These precautions involve (i) verifying if the initial sequencing of the enriched microsatellite library can guarantee a sufficient read length and sequencing depth to identify enough candidates and (ii) focusing from the beginning on microsatellites with high number of repeats. The two Larix decidua multiplexes developed in this study are currently applied on a range-wide sample of 43 populations to resolve in detail past population dynamics, which should help identify and protect valuable genetic resources.

Introduction

Translocations are defined as anthropogenic movements of living organisms from one place to another (IUCN 1987). They increasingly affect the integrity of native plant and animal populations through hybridization and genetic swamping resulting in growing conservation concerns [START_REF] Allendorf | The problems with hybrids: setting conservation guidelines[END_REF][START_REF] Hufford | Plant ecotypes: genetic differentiation in the age of ecological restoration[END_REF]. Despite their importance, they are understudied. In particular, research on translocations is clearly lagging behind that on biological invasions, which has received considerable attention during the last 15 years (for review see [START_REF] Ellstrand | Hybridization as a stimulus for the evolution of invasiveness in plants[END_REF][START_REF] Lee | Evolutionary genetics of invasive species[END_REF][START_REF] Mooney | The evolutionary impact of invasive species[END_REF][START_REF] Sakai | The population biology of an invasive species[END_REF][START_REF] Suarez | The evolutionary consequences of biological invasions[END_REF]. Some cases of translocations have been classified as "cryptic invasions" (e.g. [START_REF] Kolbe | Genetic variation increases during biological invasion by a Cuban lizard[END_REF][START_REF] Kolbe | Admixture determines genetic diversity and population differentiation in the biological invasion of a lizard species[END_REF][START_REF] Rosenthal | Evidence for multiple sources of invasion and intraspecific hybridization in Brachypodium sylvaticum (Hudson) Beauv. in North America[END_REF][START_REF] Saltonstall | Cryptic invasion by a non-native genotype of the common reed, Phragmites australis, into North America[END_REF][START_REF] Saltonstall | Remnant native Phragmites australis maintains genetic diversity despite multiple threats[END_REF][START_REF] Williams | Chloroplast and microsatellite DNA diversities reveal the introduction history of Brazilian peppertree (Schinus terebinthifolius) in Florida[END_REF]. However, they represent a rather restricted subset of cases of long-distance, often intercontinental translocations where the introduced material turns out to be selectively favoured. Translocations have broader significance but explicit studies of translocations remain surprisingly limited, except in the particular case of restocking in fishes or of plantations in forest trees (e.g. [START_REF] Deguilloux | Checking the geographical origin of oak wood: molecular and statistical tools[END_REF][START_REF] Gum | Discriminating the impact of recent human mediated stock transfer from historical gene flow on genetic structure of European grayling Thymallus thymallus L[END_REF][START_REF] König | Chloroplast DNA variation of oaks in western Central Europe and genetic consequences of human influences[END_REF][START_REF] Lowe | The utility and limitations of chloroplast DNA analysis for identifying native British oak stands and for guiding replanting strategy[END_REF][START_REF] Amandine | Environmental factors correlate with hybridization in stocked brook charr (Salvelinus fontinalis)[END_REF]Neville & Dunham 2011;[START_REF] Scribner | Hybridization in freshwater fishes: a review of case studies and cytonuclear methods of biological inference[END_REF][START_REF] Winkler | Hybridization and restricted gene flow between native and introduced stocks of Alpine whitefish (Coregonus sp.) across multiple environments[END_REF]. To date, none of these relatively few studies has explored in detail translocations across a complete distribution range. Moreover, few have investigated details of the introduction history per se.

To establish a baseline for future research on this topic, it would be useful to identify a biological model with suitable characteristics. First, at least some non-introgressed genotypes should have persisted in most populations, thereby facilitating the identification of introduced or introgressed material. Second, the species should have a strong ancestral genetic structure to facilitate discrimination of native, introduced and admixed genotypes. Finally, a focus on the range-wide scale should help develop a systematic strategy for the detection and sourcing of introduced material, thereby allowing a better understanding of the translocation process.

Trees should be particularly good models for translocations studies as some species have been planted for centuries [START_REF] Lefèvre | Le peuplier noir : une ressource génétique à l'interface entre habitats naturels d'intérêt communautaire et sylviculture intensive[END_REF]. Moreover, many of them are characterized by large populations and long generation time [START_REF] Petit | Some Evolutionary Consequences of Being a Tree[END_REF], which should favour persistence of native genotypes. Translocation studies in trees have mostly relied on maternally inherited organelle markers, taking advantage of the strong genetic structure often detected in these species with such markers [START_REF] Petit | Comparative organization of chloroplast, mitochondrial and nuclear diversity in plant populations[END_REF]. However, combining nuclear and organelle data in trees would allow investigating cytonuclear disequilibrium, which can be of even greater tel-00880539, version 1 -6 Nov 2013 support to detect recent introductions [START_REF] Asmussen | Definition and properties of disequilibrium statistics for associations between nuclear and cytoplasmic genotypes[END_REF]. Cytonuclear disequilibrium should be maximal immediately after introduction and then decrease quickly following random mating, as it gets halved at each generation.

A good candidate tree for such a study is European larch (Larix decidua Mill.). It mostly grows at high altitudes and is characterized by a restricted native distribution range subdivided into two main areas, the Alpine and the Central European region. This fragmented distribution, whose origin has been investigated using fossils [START_REF] Huntley | An atlas of past and present pollen maps for Europe: 0-13000 years ago Cambridge[END_REF][START_REF] Lang | Quartäre Vegetationsgeschichte Europas: Methoden und Ergebnisse Fischer[END_REF], should have promoted the development of a strong geographic genetic structure by limiting gene flow across the different parts of the range. Most importantly, the species has been widely planted within and outside its range in Europe since the 16 th century, with the intensity of plantations culminating in the 19 th century. In Canada and the United States the species has become naturalized at different places [START_REF] Li | Breeding strategies for Larix decidua, L. leptolepis and their hybrids in the United States[END_REF][START_REF] Little | Checklist of United States trees (native and naturalized)[END_REF][START_REF] Seymour | The Flora of New England[END_REF][START_REF] Voss | Gymnosperms and monocots[END_REF]. In Europe, the popularity of European larch as plantation species has reached such a level that it received a name in forest literature: "Lärchenmanie" (German) or "manie du mélèze" (French; Engl. = larch mania) [START_REF] Fourchy | Vegetation und Paläoklima der Weichsel-Kaltzeit im nördlichen Mitteleuropa -Ergebnisse paläobotanischer, faunistischer und geologischer Untersuchungen[END_REF]Münch 1936;[START_REF] Rubner | Die pflanzengeographischen Grundlagen des Waldbaus Neumann[END_REF][START_REF] Schober | Die Lärche -Eine ertragskundlich-biologische Untersuchung[END_REF][START_REF] Tschermak | Die natürliche Verbreitung der Lärche in den Ostalpen[END_REF]).

This study focuses on intraspecific translocations using a range-wide Larix decidua sample.

The issues we address are (i) the systematic detection of translocations using combinations of well-validated methods, (ii) the identification of admixture events in populations where translocations had occured, (iii) the characterization of the frequency and intensity of plantations, (iv) the sourcing of the material used for plantation and finally (v) the reconstruction of the original genetic structure and its comparison with the current (postplantation) genetic structure.

Material and methods

Plant material and DNA isolation

In view of the long history of plantations involving Larix decidua and hence of the dubious status of larch populations in several regions, we carefully selected 40 presumably native populations from across the range (Fig. 5, Table S2, Supporting information), taking advantage of the existence of a multi-site L. decidua provenance trial established 1957/1958 [START_REF] Schober | Vom II. internationalen Lärchenprovenienzversuch: Ein Beitrag zur Lärchenherkunftsfrage[END_REF]Schober 1985). This trial includes populations from most parts of the historical range and aims to study important phenotypic traits for forestry (e.g. growth tel-00880539, version 1 -6 Nov 2013 rate, canker sensitivity). During its establishment special care had been taken to select native populations, taking into account available historical information on plantations (e.g. Münch 1936; [START_REF] Rubner | Die pflanzengeographischen Grundlagen des Waldbaus Neumann[END_REF]. Moreover, by sampling material gathered 50 years ago, one should reduce the risk of working with non-native populations. Three populations correspond to the variety or subspecies polonica, located in the Swietokrzyskie Mountains and listed as vulnerable on the IUCN Red List [START_REF] Lewandowski | Genetic relationships between European and Siberian larch, Larix spp. (Pinaceae), studied by allozymes. Is the Polish larch a hybrid between these two species?[END_REF]. We also included five populations located beyond the acknowledged species distribution range (72, 73, 78, 79, and 80). In 2010 we collected phloem and needle samples from these 45 populations (24 individuals per population). Most of these populations (32, i.e. 71%) originated from eight German provenance trial sites and the remaining ones (13, i.e. 29%) were sampled in situ. For comparison, we also included one L. sibirica population from a Swedish provenance trial.

Details about sampling technique and DNA isolation had been described previously by [START_REF] Wagner | Two highly informative dinucleotide SSR multiplexes for the conifer Larix decidua (European larch)[END_REF].

Mitochondrial DNA

To obtain a representative sample for mitochondrial variation, we sequenced eight individuals per population at two mtDNA regions: UBC460 and atpA. These mtDNA regions had been successfully used for similar purpose in other Larix species [START_REF] Polezhaeva | Cytoplasmic DNA variation and biogeography of Larix Mill. in Northeast Asia[END_REF][START_REF] Semerikov | RAPD-derived, PCR-based mitochondrial markers from Larix species and their usefulness in phylogeny[END_REF]. Primer sequences for UBC460 were published in [START_REF] Semerikov | RAPD-derived, PCR-based mitochondrial markers from Larix species and their usefulness in phylogeny[END_REF] while primer sequences for atpA (5'-GCGGCTGCCTATAGATACGA-3' and 5'-GCTACCGAGGCAGATATGGA-3') were designed by V. Semerikov (pers. comm.). PCR reactions were performed in a volume of 25 µl with 1× PCR buffer (DreamTaq Green, Fermentas), 0.24 mM of each dNTP, 0.16 mg/ml of BSA, 0.2 µM of each primer, 1 U of Taq polymerase (DreamTaq Green, Fermentas) and 20 ng of genomic DNA. PCR programs started with an initial denaturation at 94 °C for 4 min followed by 30 (UBC460) or 28 (atpA) cycles of denaturation at 94°C for 1 min, annealing at 65 °C (UBC460) or 57 °C (atpA) for 45 s and elongation at 72°C for 2 min 50 s (UBC460) or 1 min 50 s (atpA). The cycles were followed by a final elongation at 72°C for 10 min. PCR products were sent to LGC Genomics for cleanup and sequencing. Sequence editing and alignment was performed using CodonCode Aligner Version 4.0.3. For each mtDNA region, forward and reverse sequences of each sample were aligned and edited, if necessary, according to the strand with the better sequence quality at the respective base position. All edited good quality sequences from each region were aligned using the ClustalW algorithm.

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Variable sites were checked for artefacts and sequence errors. A table of confirmed differences was exported and haplotypes were assigned using GENALEX 6.41 (Peakall & Smouse 2006). Using differences from both genes, combined haplotypes were determined.

For reconstructing phylogenies and minimum spanning networks, artificial sequences were generated based on the variable sites. The atpA region contained a minisatellite with two different repeat motifs and a variable number of repeats. This variation was coded into single nucleotide positions to compute a minimum spanning network using TCS 1.21 [START_REF] Clement | TCS: a computer program to estimate gene genealogies[END_REF] with a fixed 12-step connection limit.

Microsatellite genotyping and cluster analysis

24 individuals per population were genotyped at 13 highly variable microsatellite loci combined in two multiplex following [START_REF] Wagner | Two highly informative dinucleotide SSR multiplexes for the conifer Larix decidua (European larch)[END_REF] [START_REF] Falush | Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies[END_REF]Pritchard et al. 2000) with K values ranging from one to 10, and 10 runs for each K. We worked on the implemented admixture model, a burn-in of 200 000 and subsequent 1 000 000 iterations. To determine the most likely value of K we computed the posterior probabilities and the second order rate of change L''(K) with STRUCTURE HARVESTER [START_REF] Earl | STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method[END_REF][START_REF] Evanno | Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study[END_REF]. We explored the inter-cluster relationships by deriving a neighbour joining tree from the pairwise distance matrix of the STRUCTURE output using the R package APE (Paradis et al. 2004).

Evaluation of assignment accuracy

To evaluate assignment accuracy we simulated 5000 individuals under different scenarios (cf. Results section) using HYBRIDLAB version 1.0 (Nielsen et al. 2006).

Subsequently 1000 individuals were chosen at random from these simulations and analyzed with STUCTURE with the same parameters as in the original analysis. We included the original genotypes with assignment values (q-values) ≥0.75 as a reference (USEPOPINFO model), so that the assignment scores of the simulated individuals could be compared with those from the observed genotypes in the original analysis. We also included as controls the 263 individuals that had q-values <0.75, to check if assignment scores remain the same across analyses.

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Cytonuclear disequilibrium and nuclear admixture analysis

In the combined mito-nuclear analysis we tested for the association between mitochondrial and nuclear lineages. We used a z-test to compare admixture proportions among mitochondrial groups and a one-sided student test to compare q-values among populations with and without indication of recent translocations (cf. Results section).

Translocation frequency, intensity and source areas

We compared observed translocation frequencies with theoretical expectations from the Poisson law and tested if there was a difference with a χ 2 -test. Existence of regional differences in translocation frequency was tested by comparing proportions of purebred and admixed individuals found in the Alpine and the Central European region using a z-test. To test if translocations often involved multiple sources, we counted the number of different nuclear cluster per population and compared results among populations with and without indication for translocation using a Wilcoxon rank-sum test.

Systematic detection of first-generation migrants

To detect cases of recent translocations separately in each population, we applied a Bayesian approach implemented in GENECLASS [START_REF] Piry | GENECLASS2: A Software for Genetic Assignment and First-Generation Migrant Detection[END_REF]. It allows identifying first-generation migrants in a population and provides likelihoods of belonging to a given reference population. With this approach, recent translocations, especially those across very divergent populations, are assimilated to first generation migrants. If admixture has taken place, the model assumptions are not longer fulfilled and introduced genotypes should no longer be identified. We relied on 10,000 simulated individuals (Paetkau et al. 2004) and a type I error of 0.01. We ran GENECLASS three times with the same parameters. After each run, we excluded the detected migrant individuals.

Composition of introduced material and reconstruction of ancient genetic structure

To investigate the composition of introduced material, we calculated mean q-values of each cluster for different groups of introduced individuals corresponding to different combinations of source and release areas. These were compared to mean q-values of presumed native Alpine and Central European populations that were obtained by removing individuals tel-00880539, version 1 -6 Nov 2013 considered to result from translocation or associated admixture (identified using either STRUCTURE or GENECLASS, cf. Results section). The remaining individuals were mapped and summary statistics including classical measures such as allelic richness (N A ), gene diversity (H S ), inbreeding coefficient (F IS ) and fixation index (F ST ) were calculated and compared with the original using FSTAT version 2.9.3.2 [START_REF] Goudet | FSTAT, a program to estimate and test gene diversities and fixation indices (version 2.9.3)[END_REF]) with 15,000 permutations for nuclear data and using PERMUT (http://www.pierroton.inra.fr/genetics/labo/Software/) for mtDNA data [START_REF] Pons | Measuring and testing genetic differentiation with ordered versus unordered alleles[END_REF].

Results

Mitochondrial DNA

A total of 22 mtDNA haplotypes were detected in 363 Larix decidua samples from 43 populations, 10 of which resulted from simple sequence variation and 12 from minisatellite variation (Fig. 5A). The eight L. sibirica samples had a single private haplotype (haplotype 23) that was however not very divergent from those found in L. decidua. The L. decidua haplotypes formed two main groups, each of which was composed of two frequent haplotypes and of some less frequent ones. In group 1 (haplotypes 16, 17, 18 and 22), haplotypes 16 and 18 were found in 58% of the individuals. In group 2 (haplotypes 8, 9, 10, 19, 20, 21 and minisatellite variants), haplotypes 9 and 10 occurred in 28% of the individuals.

Haplotypes of group 1 were mostly found in the Alpine region and those of group 2 in Central Europe (Fig. 5B). However, there were 37 individuals (10%) with group 1 haplotypes found in Central Europe and 14 individuals (4%) with group 2 haplotypes found in the Alpine S3, Supporting information). However, some statistical support and clear individual assignments were also found for K = 7 (Fig. 6A, Fig. S2, Supporting information). The seven clusters were mainly distributed mainly in an east-west direction (Fig. 6B). Yet, in some populations, there were individuals that had high assignment probability to clusters more frequent in other regions. For instance, in population 18, there were three individuals assigned to clusters 5 and 7, whereas all other individuals where assigned to cluster 2. Furthermore, three Central European populations (73, 78 and 79) were entirely composed of individuals assigned to clusters 2 and 3, which were more frequent in the Alpine region. For subsequent analyses, we combined the seven nuclear clusters into two larger groups of related clusters (group 1: cluster 1-4, group 2: cluster 5-7;

Fig. S2, Supporting information).

Evaluation of assignment accuracy

To evaluate the robustness of the assignments, we simulated genotypes using HYBRIDLAB, considering two scenarios. One was based on panmixia within each of the seven identified cluster. In the other scenario, we simulated panmixia within the two cluster groups. To derive allelic frequencies for each cluster or cluster group, genotypes from the original sample were selected according to their q-values in the STRUCTURE analysis. We selected individuals with q-values of ≥ 0.75 for the respective cluster and ≥ 0.875 for the respective cluster group (Alps versus Central Europe), the latter being the sum of the q-values of all individual clusters making up the respective cluster group. A threshold value of 0.875 corresponds to the optimal theoretical assignment threshold to distinguish backcross from purebred individuals, whereas the relaxed threshold of ≥ 0.75 used for each of the seven individual clusters corresponds to the optimal threshold to distinguish F1 from purebreds. In the second scenario, individuals were selected only based on q-values ≥ 0.875 for cluster groups, i.e. it included all individuals admixed among clusters belonging to the same cluster group. Simulations were used to quantify the frequency with which genotypes produced under each scenario were falsely assigned to the alternative purebred category (q ≥ 0.875) or to the admixed category (0.125 ≤ q < 0.875) (Table S4, Supporting information).

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We found that, under both scenarios, not a single individual was assigned to the alternative purebred category, pointing to a very low risk to misassign purebred category. In contrast, some purebred individuals were falsely assigned to the admixed category. Their proportion depended on the cluster and on the scenario but it remained limited. Genotypes from group 1 were falsely assigned to the admixed category in 1.3-4.6% of the cases in the first scenario and in 13.3% in the second scenario. For group 2, values ranged between 5.4% and 11.1% in the first scenario and reached 12.3% in the second. Comparison of the 263 original admixed genotypes included in the STRUCTURE analysis and in the subsequent run with the simulated genotypes revealed that there was no important change in the q-values between the two analyses (mean difference of 0.034), indicating that the addition of simulated genotypes did not modify clustering criteria, as expected given our settings in the STRUCTURE analysis (USEPOPINFO model).

Cytonuclear analysis

We checked for association between the two nuclear cluster groups and the two mtDNA lineages using 363 individuals for which both type of information was available. All nuclear genotypes assigned to group 1 purebreds carried group 1 mtDNA haplotypes, and all but one group 2 purebred carried group 2 mtDNA haplotypes, indicating nearly total cytonuclear association (Table 5, bold numbers first two lines). We also analysed the same association within each region. In both cases, regionally uncommon nuclear purebreds were associated with regionally uncommon mtDNA lineages (Table 5, bold numbers lines 4 and 5), which we interpret as evidence for recent establishment of genotypes originating from the other region.

We then focused on admixed individuals in both regions. We reasoned that if introgression was caused by translocations, there should be a higher proportion of introgressed individuals characterized by the regionally uncommon mtDNA lineage (i.e. presumably introduced) than by the regionally dominant lineage. In the Alpine region we found 14 individuals with group 2 haplotypes. Seven of them (50%; Table 5, underlined) were introgressed (0.125 ≤ q < 0.875). This contrasts with the corresponding proportion in individuals with group 1 haplotypes (13%, z = -3.68, p < 10 -3 ). In Central Europe, results were similar. A total of 37 individuals had group 1 haplotypes and 16 of them (43%, Table 5, underlined) were introgressed, whereas the proportion of introgressed individuals with group 2 haplotypes was significantly lower (24%, z = -2.26, p = 0.03). Altogether, mtDNA and nuclear data crosssupported each other, suggesting that in both regions, uncommon nuclear and mitochondrial tel-00880539, version 1 -6 Nov 2013 lineages resulted from translocations. We decided to use only the nuclear criteria for the detection of translocations and of admixture events, as nuclear data was available indistinctly for all genotypes, in contrast to mtDNA. Using the nuclear criterion for purebreds, we detected 89 (8.6%) exotic genotypes (i.e. purebreds belonging to the cluster group that is regionally uncommon), of which 23 were found interspersed in populations with common genotypes and 66 in the three populations predominantly composed of exotic genotypes (Fig. 7, black coloured proportions).

Nuclear admixture analysis

We reasoned that in populations where translocation events had been detected, more admixed individuals should be found than in populations where no translocation event was detected.

To test this hypothesis, populations were assigned into two categories: one with at least one exotic genotype, the other one with populations lacking such evidence. The analysis was also performed separately in each region (Alps, Central Europe). In each population studied we excluded purebreds originating from the other region to direct the analysis on admixture. The prediction was that in populations with indication of translocation, average q-values should be higher. In both regions differences in levels of admixture between the two categories (with versus without evidence for translocation) were highly significant (Alps: q-value = 0.08 versus 0.05, p<10 -4 , Central Europe: q-values = 0.19 versus 0.09, p<10 -4 , Fig. 8). This suggests a relationship between translocation and admixture. Overall we found 155 (15%) admixed individuals using the selected thresholds (Fig. 7, grey coloured proportions).

Translocation frequency, intensity and number of source areas

We then tried to better describe the process of translocation, i.e. its frequency, intensity and distribution. First, we tested if the probability of occurrence of translocation events can be considered to have the same probability across populations or if it is higher in some populations than in others. For this purpose, we counted the number of translocation events per population, excluding the five non-native populations. The number of introduced individuals per population ranged from zero to five (mean = 0.5). As the overall frequency of exotic individuals was low (2%), we considered translocations as rare events and compared their distribution with the theoretical expectations derived by the Poisson law. The distribution did not match with Poisson law predictions (χ² = 259, p ≈ 0; Fig. S 3, Supporting information). Observed frequency of populations with individual introduced was lower than expected whereas the observed frequency of populations with 0, 2 and 5 introduced individuals was higher than expected. This points to a deficit of cases with no or few introductions and an excess of cases with multiple introductions, compared to a scenario where introductions would have taken place with the same probability across all populations.

Second, we checked if translocations were equally abundant in the two regions (Alps, Central Europe). In each region, we compared the proportion of exotic purebreds and of regionally admixed individuals. For both values the proportion was significantly lower in the Alps than in Central Europe (2% versus 5%, z = -3.04, p°=°0.002 and 10% versus 37%, z = -8.43, p°<10 -5 , respectively). Third, we checked if translocation events involved material from a single source area or from several ones. For this purpose, we focused on the Alpine region where geographic genetic structure is stronger than in Central Europe. As previously, we compared populations with and without indication of translocation events. In each population, the clusters were counted on the basis of a critical q-value of ≥ 0.5. In the group where no exotic individual was detected, we considered all clusters minus one (the most frequent one).

In the group where exotic individuals were detected, we considered all clusters minus two (the most frequent in the population and the most frequent among introduced individuals, i.e.

assuming introduction from a single geographic origin). We then compared the number of extra clusters found in the two groups. We found a mean number of 0.8 and 1.8 extra clusters in populations without and with translocation events, respectively. To test if cases of interregional translocations were associated with cases of intraregional translocations, we performed the same comparison but focusing only on extra Alpine clusters. We found in this case on average 0.7 and 1.2 extra clusters in populations without and with translocation events. Differences were significant in both cases (p<0.001), suggesting that introductions often involve material from several geographic sources or from already admixed sources.

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Systematic detection of first-generation migrants

The approach based on STRUCTURE could only be applied at the interregional scale due to a lack of suitable mtDNA variation at finer scales to validate it. However, there is no reason to believe that translocations have not taken place also within each region, as suggested by the study of the number of extra clusters in populations with indication of translocation. To recover other types of translocation events, we used the GENECLASS approach [START_REF] Piry | GENECLASS2: A Software for Genetic Assignment and First-Generation Migrant Detection[END_REF]. Three successive runs led to the detection of 92 (9%) first-generation migrants among all samples (Fig. 9). A total of 40 of these 92 individuals corresponded to interregional translocation events, only 13 of which had been previously detected. The remaining 52 individuals corresponded to intraregional translocation events. The classification of translocation events was based on the likelihoods of each multilocus genotype to belong to each cluster group or each of the seven clusters. The intraregional translocations could be further subdivided in 23 inter-and 28 intra-cluster translocation events. The latter occurred mostly (8 out of 12 cases) together with translocation events at larger geographic scales. We checked if the detection power of the GENECLASS approach was lower in populations in which admixture had been detected with the previous approach. We found that this was the case: in strongly admixed populations, translocations typically remained undetected (Fig. S 4) which can also explain why we detected only one third of the translocations detected previously, as mentioned above. tel-00880539, version 1 -6 Nov 2013

Composition of introduced material and reconstruction of ancient genetic structure

To investigate the genetic composition of introduced material, we assigned all exotic genotypes into four categories representing the four possible combinations of source and release areas (Alps to Central Europe, Central Europe to Alps, intra-Alps, intra-Central Europe). For consistency, we considered only individuals with q-values ≥0.875 for one of the two cluster groups. The comparison between presumably natural populations of the Alpine region and material inferred to have been introduced from the Alps revealed that genotypes from cluster 2 were more frequently involved than genotypes from other Alpine clusters (intra-Alps: 48%, Alps to Central: 60%; Fig. S 5, Supporting information). For material introduced from Central Europe, genotypes from cluster 5 were frequently involved, especially considering the limited distribution of native populations where this cluster predominates (intra-Central Europe: 24%, Central Europe to Alps: 39%).

Altogether, using both approaches (STRUCTURE and GENECLASS), 322 (31%) individuals distributed in 43 populations were removed in an attempt to recover the original genetic structure (Fig. 5 C, Fig. 6 C). This included exotic purebreds and admixed individuals detected by the STRUCTURE approach as well as the individuals considered to be first generation migrants using the GENECLASS approach. Four of the five populations sampled beyond the acknowledged species range (populations 73, 78, 79 and 80) did not match with the overall pattern. The fifth populations (72) had high heterozygote deficit (F IS =0.09), and no distinct genetic composition despite its location away from the main species range. Thus, we excluded these five populations. Global summary statistics for nuclear data with the original sample (N A = 6.6, H S = 0.76, F IS = 0.05, F ST = 0.08) and with the final sample (N A = 6.2, H S = 0.74, F IS = 0.02, F ST = 0.11) differed significantly (p <0.001 for all measures), confirming the need to systematically remove anthropogenic artefacts to correctly estimate genetic parameters. After correction, a few populations still have exceptionally high heterozygote deficit (F IS > 0.08: populations 3, 53, 66 and 84, see Table S 6, Supporting information). For at least some of them, the existence of so far cryptic intraregional translocations appears likely on the basis of mtDNA or nuclear variation, pointing to the need for further refinements of the approach. Similarly, for mtDNA, genetic diversity was lower and genetic structure higher with the final sample, obtained after removing introduced material (h S = 0.25, G ST = 0.67) than with the original one (h S = 0.37, G ST = 0.52).

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Discussion

Systematic detection of translocations

Selecting appropriate methods and tools to detect translocations and investigate their consequences remains challenging as this implies differentiating conspecific individuals originating from different parts of the range. Clearly, in most cases individuals from nearby populations should be more difficult to differentiate than those originating from distant locations. In our study, we took advantage of suitable features of European larch, including strong ancestral genetic structure at both mitochondrial and nuclear genomes and the persistance of native genotypes in most populations. We detected two mitochondrial lineages differentiating most individuals from the Alpine and the Central European parts of the range.

Scattered occurences of mtDNA haplotypes that did not fit with this pattern suggested that translocations might had been involved. In accordance with this, individuals with mtDNA haplotypes not belonging to the regionally frequent lineage also happened to be characterized by genotypes assigned to nuclear clusters more frequent in the other region. This is a very strong evidence in favour of recent contact and hence of the artificial nature of the mixed populations as a few generations should erase such cytonuclear disequilibrium. The methodology for the detection of cases of long-distance translocation, based on 13 highly polymorphic microsatellites and a comprehensive screening across the range, provides clearcut results, with virtually no risk to misassign purebred genotypes from one region to the other region. However, such a strategy only allowed differentiating individuals originating from the Alps versus from Central Europe, i.e. at the interregional scale. Yet we showed that, in the Alpine range, translocations from Central Europe were associated with the introduction of material from other parts of the Alpine region. This suggests that translocations are often complex, with material introduced ultimately coming from several sources, both distant and less distant ones.

To systematically detect recent translocations across all scales, we used an approach based on assignment criteria to exclude reference populations as the origin of diploid individuals on the basis of multilocus genotype data [START_REF] Piry | GENECLASS2: A Software for Genetic Assignment and First-Generation Migrant Detection[END_REF]. With this method, we detected 92 cases of recent translocations across the range, from the most distant to the most local. The advantage of this approach is that it can be used for species lacking a strong geographic genetic structure as it works on the basis of data from the population of interest only.

Although in principle first-generation migrants can also occur naturally, the broad geographic sampling scale of the study makes this hypothesis unlikely in our survey. However, in tel-00880539, version 1 -6 Nov 2013

situations where native and introduced genotypes have resulted in admixture, the approach failed to detect introduced material, indicating that both methods used have limitations and that future developments to detect non-native genotypes should focus both on comparisons with other populations and on analyses of the internal structure of each target population.

Admixture events

Numerous admixture events were detected, representing 15% of the individuals. Although some false positives are certainly included, admixture events were overall more frequent in populations where translocations were detected. In some populations, introduced and admixed individuals made up more than 50% of the individuals, indicating massive plantation or high success of introduced genotypes in subsequent generations. This finding points to relatively ancient translocation events, in line with the known history of plantations of the species in Europe (e.g. [START_REF] Fourchy | Vegetation und Paläoklima der Weichsel-Kaltzeit im nördlichen Mitteleuropa -Ergebnisse paläobotanischer, faunistischer und geologischer Untersuchungen[END_REF]Münch 1936;[START_REF] Rubner | Die pflanzengeographischen Grundlagen des Waldbaus Neumann[END_REF][START_REF] Schober | Die Lärche -Eine ertragskundlich-biologische Untersuchung[END_REF][START_REF] Tschermak | Die natürliche Verbreitung der Lärche in den Ostalpen[END_REF]).

The translocation process

The admixture corresponds to the later steps of a translocation. Although it is conditioned by the initial input of exotic material, it is a largely autonomous process. In this respect, it can be compared with the expansion phase of a biological invasion. In contrast, the translocation per se depends on human agency and can be compared with the first establishment step of an invasion. We found that plantation pressure had been very heterogeneous across the range, with some areas where human input has been reduced and others where plantations have been frequent. In fact, exotic material was introduced at particularly high rates in Central Europe.

Compared to the Alpine region, the native distribution of European larch in Central Europe is particularly fragmented with populations of rather small size, making them more vulnerable to genetic swamping/admixture.

Translocations and associated processes uncovered in this study can be assumed to be related to the intensive periods of larch plantation during the 18 th and 19 th century described in forest literature [START_REF] Fourchy | Vegetation und Paläoklima der Weichsel-Kaltzeit im nördlichen Mitteleuropa -Ergebnisse paläobotanischer, faunistischer und geologischer Untersuchungen[END_REF]Münch 1936;[START_REF] Rubner | Die pflanzengeographischen Grundlagen des Waldbaus Neumann[END_REF][START_REF] Schober | Die Lärche -Eine ertragskundlich-biologische Untersuchung[END_REF][START_REF] Tschermak | Die natürliche Verbreitung der Lärche in den Ostalpen[END_REF]). The

Reconstruction of ancient genetic structure

Our sampling included five populations sampled away from the acknowledged species range, but whose exotic status is sometimes questioned. No argument was found to modify the currently acknowledged species distribution range in Europe. The genetic structure at nuclear and especially mtDNA that was revealed after removing the individuals considered to have been introduced or to be the result of admixture was much stronger than that found prior to removing these individuals. Lower within population diversity and greater F ST and overall geographic structure were noted. The pre-plantation genetic structure is likely even stronger as our method did not allow us to detect all cases of intraregional translocations or admixture events and as there is still a relatively high heterozygote deficit in some populations, pointing to Wahlund effect due to subpopulation structure possibly caused by plantation

Conclusions and Perspectives

Our study revealed several aspects of the translocation process in the European larch.

Although it is to our knowledge one of the most comprehensive studies of translocation and of its consequences, it did not cover all possible aspects. Among the issues that still remain to be addressed, one can cite the development of specific demo-genetic methods to identify simultaneously recent introductions and admixture and their timing. For instance, determination of the earliest translocations and of the peak period of plantations would help determine if they match with historical records. Another important objective would be to test if introduced genotypes or genes have differential fitness compared to local ones. Our results underpin the importance of investigating translocations from a range-wide perspective as this is the only way to efficiently and accurately trace introduced material of different geographic origins.

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Supporting Information [START_REF] Fletcher | Millennial-scale variability during the last glacial in vegetation records from Europe[END_REF][START_REF] Goñi | Millennial-scale climate variability and vegetation changes during the Last Glacial: Concepts and terminology[END_REF]. Climatic oscillations and coincident forest development have been analyzed for the last and current interglacial (e.g. [START_REF] Berglund | Palaeoecological events during the last 15000 years[END_REF][START_REF] Drescher-Schneider | Die Vegetations-und Klimaentwicklung im Riß/Würm-Interglazial und im Früh-und Mittelwürm in der Umgebung von Mondsee. Ergebnisse der pollenanalytischen Untersuchungen[END_REF][START_REF] Zagwijn | An analysis of Eemian climate in western and central Europe[END_REF]. In contrast, short-term climate events, i.e. changes within <200 years [START_REF] Goñi | Millennial-scale climate variability and vegetation changes during the Last Glacial: Concepts and terminology[END_REF], and their influence on vegetation cover are still understudied, due to the limited resolution of the pollen records. The situation has started to change with the availability of the high resolution Greenland ice-core record NGRIP (e.g. [START_REF] Andersen | High-resolution record of Northern Hemisphere climate extending into the last interglacial period[END_REF][START_REF] Johnsen | Irregular glacial interstadials recorded in a new Greenland ice core[END_REF][START_REF] Wolff | Millennial-scale variability during the last glacial: The ice core record[END_REF]) and marine cores (e.g. [START_REF] Fletcher | Millennial-scale variability during the last glacial in vegetation records from Europe[END_REF][START_REF] Roucoux | Combined Marine Proxy and Pollen Analyses Reveal Rapid Iberian Vegetation Response to North Atlantic Millennial-Scale Climate Oscillations[END_REF][START_REF] Goñi | Contrasting impacts of Dansgaard-Oeschger events over a western European latitudinal transect modulated by orbital parameters[END_REF][START_REF] Goñi | Synchroneity between marine and terrestrial responses to millennial scale climatic variability during the last glacial period in the Mediterranean region[END_REF] registering numerous short-term climate events during the last glacial period. Vegetational responses to these events have been quasi-instantaneous and very heterogeneous. While the general history of European forests has now started to be investigated at this resolution for the period of the last glacial [START_REF] Fletcher | Millennial-scale variability during the last glacial in vegetation records from Europe[END_REF][START_REF] Pini | The vegetation and climate history of the last glacial cycle in a new pollen record from Lake Fimon (southern Alpine foreland, N-Italy)[END_REF][START_REF] Sümegi | Responses of terrestrial ecosystems to Dansgaard-Oeshger cycles and Heinrich-events: A 28,000-year record of environmental changes from SE Hungary[END_REF], it seems timely to study the flexibility of individual tree taxa towards short-term climate oscillations as this can assist in preparing forests for future climate change.

The most significant climate variation of the Quaternary is the alternation of glacial and interglacial climates that occurred regularly with an approximately 100 ka periodicity since ~800 ka. This variation is explained by external orbital parameters leading to changes in latitudinal and seasonal patterns of insolation modulated by internal factors such as ice-sheet extent or greenhouse gas concentration. Short-term changes are superimposed on long-term changes and occur at the millennial scale and with irregular periodicity (Sánchez [START_REF] Goñi | Millennial-scale climate variability and vegetation changes during the Last Glacial: Concepts and terminology[END_REF][START_REF] Wolff | Millennial-scale variability during the last glacial: The ice core record[END_REF]. They are particularly striking during the last glacial between 73.5 and 14.7 ka. There are two types of such rapid climate events. First, Dansgaard-Oeschger (D-O) events [START_REF] Dansgaard | North Atlantic climatic oscillations revealed by deep Greenland ice cores[END_REF] correspond to abrupt warming registered in the Greenland ice-core record (e.g. [START_REF] Andersen | High-resolution record of Northern Hemisphere climate extending into the last interglacial period[END_REF][START_REF] Johnsen | Irregular glacial interstadials recorded in a new Greenland ice core[END_REF][START_REF] Wolff | Millennial-scale variability during the last glacial: The ice core record[END_REF]. Second, Heinrich events (HE) [START_REF] Heinrich | Origin and consequences of cyclic ice rafting in the northeast Atlantic ocean during the past 130,000 years[END_REF] (data provided by M. Sánchez Goñi, published in Sánchez [START_REF] Goñi | Contrasting impacts of Dansgaard-Oeschger events over a western European latitudinal transect modulated by orbital parameters[END_REF]; above: regional biostratigraphic units [START_REF] Woillard | Grande Pile peat bog: A continuous pollen record for the last 140,000 years[END_REF][START_REF] Behre | Eine Folge von Eem und 4 Weichsel-Interstadialen in Oerel/Niedersachsen und ihr Vegetationsablauf[END_REF][START_REF] Beaulieu | The transition from temperate phases to stadials in the long upper Pleistocene sequence from Les Echets (France)[END_REF] [START_REF] Landais | A continuous record of temperature evolution over a sequence of Dansgaard-Oeschger events during Marine Isotopic Stage 4 (76 to 62 kyr BP)[END_REF]. Apart from this event other events during MIS 4 and MIS 2 were relatively weak, whereas they were more pronounced during MIS 3. The final D-O event is generally considered to be synchronous with the Bølling-Allerød interstadial at the beginning of MIS 1 (~14.7 -12.8 ka). Ice-core derived temperature estimates for different D-O events range between 8°C and 16°C. Heinrich events occurred during MIS 2, 3 and 4. They seem to have been particularly severe during MIS 2 and MIS 4 when global ice volume was maximal and insolation was minimal. For the Holocene (since ~11.7 ka) there are no D-O or Heinrich events registered. However, there were some rapid climate changes in the early Holocene, the most prominent being the 8.2 ka cold event likely caused by the final collapse of the Laurentide ice sheet of northeastern North America [START_REF] Ellison | Surface and Deep Ocean Interactions During the Cold Climate Event 8200 Years Ago[END_REF]. Climate changes documented in the ice cores and marine cores (including marine pollen records) from the North Atlantic can be correlated with continental pollen records to reassess vegetational development documented based on regional biostratigraphic units (Fig. 10).

In Europe, the availability of large amounts of fossil data has enabled detailed reconstructions of late glacial and Holocene tree histories. In most of these investigations, paleontological data have been combined with modern genetic data, e.g. Quercus, Fagus sylvatica, Pinus sylvestris, Abies alba, Picea abies [START_REF] Cheddadi | Imprints of glacial refugia in the modern genetic diversity of Pinus sylvestris[END_REF][START_REF] Falush | Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies[END_REF][START_REF] Liepelt | Postglacial range expansion and its genetic imprints in Abies alba (Mill.) -a synthesis from paleobotanic and genetic data[END_REF][START_REF] Magri | A new scenario for the Quaternary history of European beech populations: palaeobotanical evidence and genetic consequences[END_REF][START_REF] Petit | Identification of refugia and post-glacial colonisation routes of European white oaks based on chloroplast DNA and fossil pollen evidence[END_REF][START_REF] Tollefsrud | Genetic consequences of glacial survival and postglacial colonization in Norway spruce: combined analysis of mitochondrial DNA and fossil pollen[END_REF]. To date, however, the impact of past abrupt climate events on tree histories has not been investigated. Yet, such knowledge could be an important foundation to understand trees flexibility towards future climate change.

Pioneer tree species could represent useful models for such studies as their response to climate should be particularly rapid due to their high rate of spread and modest soil and temperature requirements. However, pollen in these taxa is typically dispersed at long distances and its detection, at least at low concentration, cannot therefore be used to testify for the presence of a species in a given locality. Interestingly, European larch (Larix decidua Mill.), a pioneer tree species, does not disperse very well its pollen (e.g. [START_REF] Jankovská | Larix and its natural occurence in the Central Europe from the point of view of palaeoecology[END_REF][START_REF] Pelánková | Surface pollen-vegetation relationships in the forest-steppe, taiga and tundra landscapes of the Russian Altai Mountains[END_REF][START_REF] Sjögren | The development of composite dispersal functions for estimating absolute pollen productivity in the Swiss Alps[END_REF]Sjögren et al. 2008a;Sjögren et al. 2008b).

Furthermore, its needles are well preserved as macrofossils. To date, Larix has not been the subject of recent historical studies. Its modern distribution range is disjunct and comprises four distinct areas, the Alps, the Sudetes Mts, the Tatra Mts, and central Poland. In addition, it tel-00880539, version 1 -6 Nov 2013 has a more sporadic distribution in the eastern and southern Carpathians and in the Bihar mountains of Romania [START_REF] Rubner | Die pflanzengeographischen Grundlagen des Waldbaus Neumann[END_REF]. Altitudinal distribution varies widely. The Central Alps with most prominent larch occurrences are definitely continental whereas occurrences in the northern Alps or in the Sudetes Mountains differ from the continental climate type [START_REF] Mccomb | The European Larch: Its races, site requirements and characteristics[END_REF][START_REF] Rubner | Die pflanzengeographischen Grundlagen des Waldbaus Neumann[END_REF]. However, all occurrences are situated in montane to subalpine regions except a few populations located in the Polish lowlands.

This study focuses on a range-wide reconstruction of the history of the European larch during the last 130,000 years based on a new comprehensive fossil compilation. The issues we address are (i) the chronological documentation of range changes since the last interglacial, (ii) the identification of the consequences of D-O events and Heinrich events on larch populations, (iii) the identification of LGM refugia and postglacial migration pathways, and

(iv) the characterization of the origin (natural versus anthropogenic) of changes in larch geographic distribution and abundance.

Materials and methods

Data compilation

Pollen, macrofossil, stomata and charcoal data for Larix at the European scale are compiled over the last 130 ka. For 130 -19 ka data are newly assembled from literature. During literature compilation we first evaluated if the investigator has been able to recognize Larix pollen. This is necessary because Larix pollen is hard to identify and has remained unnoticed in some earlier investigations due to its similarity with spores.

For the last 19 ka years macrofossil, stomata, charcoal and pollen data from literature are combined with pollen data from the Alpine Palynological Database (ALPADABA), the Czech Quaternary Palynological database (PALYCZ, http://botany.natur.cuni.cz/palycz/, [START_REF] Kuneš | Czech Quaternary Palynological Database -PALYCZ: review and basic statistics of the data[END_REF]) and the European pollen database (EPD), which has recently benefitted from new chronologies [START_REF] Giesecke | Towards mapping the late Quaternary vegetation change of Europe[END_REF]. Data are compiled in this sequence from the corresponding databases. In addition stomata records stored in the EPD (http://www.europeanpollendatabase.net/) are included.

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Pollen productivity, dispersion, identification

As pointed out before, pollen productivity and dispersal of Larix are limited, suggesting that even single pollen counts can indicate the local presence of the species. However, we cannot completely exclude that scarce single counts result from long-distance wind-transport, especially at the high altitudinal belts of high mountain systems that may be characterized by strong winds. After carefully checking all sequences one by one, we decided to include single pollen data as an indication for the local presence of the species, unless the findings stayed exceptional. Correspondingly, we documented for each record if the findings were scattered or more or less continuous and transferred the information on the maps.

Chronological mapping

After compilation all data are assigned to successive time intervals representing biostratigraphic units (130 -11.7 ka). For the Holocene (since 11.5 ka) 1000 year intervals are used. For each time interval positive and negative evidence given by pollen, macrofossils, stomata and charcoals are mapped using ArcGIS 9.3 (ESRI 2009).

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Results and discussion

Data compilation

1026 fossil sites of which 355 include Larix evidence have been compiled (Fig. 11, Table S 6 For MIS 5, Alpine and north-central European profiles are studied based on the regional biostratigraphic units that are correlated to the NGRIP chronology to identify climate events from the northern hemisphere that should be isochronous across Europe (Fig. 10).

Last interglacial sensu stricto (~130 -112 ka, Sánchez [START_REF] Goñi | European climate optimum and enhanced Greenland melt during the Last Interglacial[END_REF] For the last interglacial (Eemian) 41 fossil sites are found. 14 of them include scattered Larix pollen and macrofossils in the Alpine and the north-central European regions (Fig. 12, Table S 6 Supporting information). Larch fossils are only found at the transitions of this interglacial. Larch likely had a wider distribution range than today as fossils not only occurred in the Alpine region but also in the north-central European lowlands. In addition, larch can be expected to have persisted in the Carpathian region. However this cannot be proven due to the lack of fossil sites.

In the Alpine region the first evidence is found at the transition from the penultimate glacial (Rissian) to the last interglacial (Eemian). Fossils occur for instance in Mondsee (540 m a.s.l.)

in the northern Alpine foreland and in Lake Fimon (23 m a.s.l.) in the southern Alpine foreland (Drescher-Schneider 2000; [START_REF] Pini | The vegetation and climate history of the last glacial cycle in a new pollen record from Lake Fimon (southern Alpine foreland, N-Italy)[END_REF]. At this time Larix was growing in light forests dominated by Pinus and Betula. These pioneer formations were replaced by rapidly expanding thermophilous trees favoured by climatic warming. All Larix fossil sites of the Alpine region are located below 700 m; the species disappeared from these sites during the temperate part of the Eemian. The absence of Larix fossils during the temperate phases could be due to the upward shift of the species and the lack of data at higher altitudes, by analogy with the temperate phase of the current interglacial (Holocene), during which such an upward shift could be traced with fossils (see below).

New evidence from Mondsee and Lake Fimon as well as from other sites of the northern Alpine foreland, for instance Jammertal in Southern Germany [START_REF] Müller | A Late-Pleistocene Polle sequence from the Jammertal, south-western Germany with particular reference to location and altitude as factors determinig Eemian forest composition[END_REF] or Gondiswil in Switzerland [START_REF] Wegmüller | Vegetationsgeschichtliche und stratigraphische Untersuchungen an Schieferkohlen des nördlichen Alpenvorlandes Birkhäuser[END_REF], is found at the transition to the first last glacial stadial At the transition to Mélisey I Larix, together with Pinus and Picea, replaced less cold-tolerant deciduous trees. For the Alpine region, it has been proposed that these coniferous forests prevailed during about 5 ka and were then replaced by a tundra-like vegetation with prevailing non-arboreal components [START_REF] Müller | Vegetation dynamics in southern Germany during marine isotope stage 5 (~ 130 to 70 kyr ago)[END_REF]. The Mondsee record demonstrates that small groups of trees including Larix and other cold-tolerant arboreal species (Picea, Pinus, Pinus cembra, Salix, Juniperus, Alnus viridis) persisted during the Mélisey 1 period at mid altitudes [START_REF] Drescher-Schneider | Die Vegetations-und Klimaentwicklung im Riß/Würm-Interglazial und im Früh-und Mittelwürm in der Umgebung von Mondsee. Ergebnisse der pollenanalytischen Untersuchungen[END_REF].

In the north-central European lowlands Larix pollen was found at the transition from the penultimate glacial (Saalian) in one site located in central Poland (Horoszki Duze, 180 m a.s.l., [START_REF] Granoszewski | Late Pleistocene vegetation history and climate changes at Horosziki Duzé, eastern Poland: a palaeobotanical study[END_REF]. As in the Alpine region, evidence during the temperate phases is lacking. The species is found again at the transition to the first last glacial stadial (Herning) in several Polish and northern German sites (e.g. [START_REF] Erd | Pollenanalytischer Gliederung des Pleistozäns der DDR[END_REF][START_REF] Granoszewski | Late Pleistocene vegetation history and climate changes at Horosziki Duzé, eastern Poland: a palaeobotanical study[END_REF][START_REF] Menke | Das Eeminterglazial und das Weichselfrühglazial von Rederstall/Dithmarschen und ihre Bedeutung für die mitteleuropäische Jungpleistozän-Gliederung[END_REF] in communities dominated by Pinus and Betula.

The first last glacial stadial in northwestern and central Europe, which is unfortunately poorly documented, was characterized by tundra environments with predominating steppic taxa (for review see Caspers & Freund 1997). Scarce Larix pollen evidence from different locations together with other arboreal components like Pinus, Betula, Betula nana, Juniperus [START_REF] Behre | Botanical macro-remains and insects from the Eemian and Weichselian site of Oerel (northwest Germany) and their evidence for the history of climate[END_REF][START_REF] Behre | Eine Folge von Eem und 4 Weichsel-Interstadialen in Oerel/Niedersachsen und ihr Vegetationsablauf[END_REF][START_REF] Erd | Pollenanalytischer Gliederung des Pleistozäns der DDR[END_REF][START_REF] Granoszewski | Late Pleistocene vegetation history and climate changes at Horosziki Duzé, eastern Poland: a palaeobotanical study[END_REF][START_REF] Menke | Das Eeminterglazial und das Weichselfrühglazial von Rederstall/Dithmarschen und ihre Bedeutung für die mitteleuropäische Jungpleistozän-Gliederung[END_REF] suggest scattered occurrence of larch but species persistence cannot be proven conclusively due to insufficient resolution of the profiles.

First early Weichselian interstadial (~109 -87 ka, Müller & Sanchez Goñi 2007) The forests corresponding to the first last glacial interstadial can be studied in 42 fossil sites including 28 with Larix evidence (Fig. 12 , Table S 7 In the Alpine region larch pollen and macrofossils are found in the northern and southern Alpine foreland, e.g. in Füramoos (Germany), Mondsee (Austria) and Lake Fimon (Italy) [START_REF] Drescher-Schneider | Die Vegetations-und Klimaentwicklung im Riß/Würm-Interglazial und im Früh-und Mittelwürm in der Umgebung von Mondsee. Ergebnisse der pollenanalytischen Untersuchungen[END_REF][START_REF] Müller | Die Vegetations-und Klimaentwicklung im jüngeren Quartär anhand ausgewählter Profile aus dem südwestdeutschen Alpenvorland[END_REF][START_REF] Müller | Vegetation response to rapid climate change in Central Europe during the last 140,000 yr based on evidence from Füramoos pollen record[END_REF][START_REF] Pini | The vegetation and climate history of the last glacial cycle in a new pollen record from Lake Fimon (southern Alpine foreland, N-Italy)[END_REF]. Larch was a component of coniferous formations largely dominated by Picea and Pinus. Generally evidence was strongest during early successional stages and during climatic cooling at the tel-00880539, version 1 -6 Nov 2013 transition to the subsequent stadial (Mélisey 2) suggesting an upward shift during the climatically most favourable times (e.g. Mondsee, Samerberg and Gondiswill, Drescher-Schneider 2000;[START_REF] Grüger | Spätriss, Riss/Würm und Frühwürm am Samerberg in Oberbayern -ein vegetationsgeschichtlicher Beitrag zur Gliederung des Jungpleistozäns[END_REF][START_REF] Wegmüller | Vegetationsgeschichtliche und stratigraphische Untersuchungen an Schieferkohlen des nördlichen Alpenvorlandes Birkhäuser[END_REF]. In some sites (e.g. Füramoos and Lake Fimon, [START_REF] Müller | Die Vegetations-und Klimaentwicklung im jüngeren Quartär anhand ausgewählter Profile aus dem südwestdeutschen Alpenvorland[END_REF][START_REF] Müller | Vegetation response to rapid climate change in Central Europe during the last 140,000 yr based on evidence from Füramoos pollen record[END_REF][START_REF] Pini | The vegetation and climate history of the last glacial cycle in a new pollen record from Lake Fimon (southern Alpine foreland, N-Italy)[END_REF], Larix was clearly favoured by the Montaigu event, an abrupt cooling episode in the second third of St. Germain 1, St. Germain 1b, between 105 ka and 103 ka [START_REF] Müller | Vegetation dynamics in southern Germany during marine isotope stage 5 (~ 130 to 70 kyr ago)[END_REF][START_REF] Woillard | Grande Pile peat bog: A continuous pollen record for the last 140,000 years[END_REF]. At La Grande Pile in the Vosges Mountains, a massif that lies outside the current species range, the same trend is observed (De Beaulieu & Reille 1992a).

In the north-central European lowlands the presence of Larix is evidenced in multiple sites in Germany and Poland during this first last glacial interstadial (Brørup). Macrofossils are found at several places and pollen percentages were conspicuously high for larch with maximum values up to 18% (e.g. Osterwanna, Oerel and Keller, [START_REF] Behre | Die Vegetation im Spätpleistozän von Osterwanna/Niedersachsen[END_REF][START_REF] Behre | Botanical macro-remains and insects from the Eemian and Weichselian site of Oerel (northwest Germany) and their evidence for the history of climate[END_REF][START_REF] Behre | Eine Folge von Eem und 4 Weichsel-Interstadialen in Oerel/Niedersachsen und ihr Vegetationsablauf[END_REF][START_REF] Menke | Ergebnisse der Pollenanalyse zur Pleistozän-Stratigraphie und zur Pliozän-Pleistozän Grenze in Schleswig-Holstein[END_REF] indicating that it was a major component of boreal forests in which it occurred together with Picea, Pinus and Betula. Colonization of the Central European lowlands by larch started from the southeast and proceeded to the northwest. After its first establishment, the species was favoured by a cold event located within the Brørup interstadial, corresponding to the Montaigu event, which affected competing species (e.g. [START_REF] Caspers | Die Vegetation im Weichsel-Früh-und Hochglazial von Groß Todtshorn (Kr. Harburg; Niedersachsen) -Geologische und palynologische Untersuchungen zu Vegetation und Klimaverlauf der letzten Kaltzeit[END_REF]Caspers & Freund 1997;[START_REF] Menke | Das Eeminterglazial und das Weichselfrühglazial von Rederstall/Dithmarschen und ihre Bedeutung für die mitteleuropäische Jungpleistozän-Gliederung[END_REF]. After this cold event, Larix reached its maximal distribution in the second half of the Brørup.

At the transition to the second stadial (Rederstall) Larix declined, probably as a consequence of increased continentality (e.g. Caspers & Freund 1997;[START_REF] Menke | Das Eeminterglazial und das Weichselfrühglazial von Rederstall/Dithmarschen und ihre Bedeutung für die mitteleuropäische Jungpleistozän-Gliederung[END_REF][START_REF] Ricken | Vegetationsentwicklung, Paläoböden, Seespiegelschwankungen: Untersuchungen an eem-und weichselzeitlichen Sedimenten von Südrand des Harzes[END_REF]). Considering the whole last interglacial/glacial interval the period between 105 ka (beginning of Montaigu event) and 100 ka (end of Brørup) seems to be the period of maximal larch distribution in the north-central European lowlands.

In the second early Weichselian stadial (Mélisey 2/Rederstall) tundra-like formations prevailed in the Alpine and the north-central European region. However, there is still some pollen and macrofossil evidence of Larix found in several sites, in particular in the Alpine region, indicating that scattered trees still occurred together with other cold-tolerant trees such as Picea, Pinus, Pinus cembra, Betula, Juniperus (e.g. [START_REF] Drescher-Schneider | Die Vegetations-und Klimaentwicklung im Riß/Würm-Interglazial und im Früh-und Mittelwürm in der Umgebung von Mondsee. Ergebnisse der pollenanalytischen Untersuchungen[END_REF][START_REF] Erd | Pollenanalytischer Gliederung des Pleistozäns der DDR[END_REF]Hahne et al. 1994b;[START_REF] Müller | A Late-Pleistocene Polle sequence from the Jammertal, south-western Germany with particular reference to location and altitude as factors determinig Eemian forest composition[END_REF][START_REF] Müller | Die Vegetations-und Klimaentwicklung im jüngeren Quartär anhand ausgewählter Profile aus dem südwestdeutschen Alpenvorland[END_REF]. It seems likely that in the Alpine region boreal trees did not only occur in the analysed intermediate altitudinal sites but that they also colonized the lowlands and built mosaic landscapes with steppic taxa.

tel-00880539, version 1 -6 Nov 2013

Second Weichselian interstadial (~83 -78.2 ka, Sanchez Goñi pers. comm.)

The second early Weichselian interstadial (St. Germain 2/Odderade) can be studied in 34 sites, 20 of which contained Larix evidence (Fig. 12, Table S 8, Supporting information). The fossil distribution and the role of larch in the coniferous forests of the Alpine and northcentral European region are similar to those from the first interstadial. Some differences can be detected in the vegetational succession and in the reduced pollen and macrofossil representation in the central European lowlands, pointing to a weaker abundance than during the Brørup (e.g. [START_REF] Behre | Botanical macro-remains and insects from the Eemian and Weichselian site of Oerel (northwest Germany) and their evidence for the history of climate[END_REF][START_REF] Behre | Eine Folge von Eem und 4 Weichsel-Interstadialen in Oerel/Niedersachsen und ihr Vegetationsablauf[END_REF]Caspers & Freund 1997;[START_REF] Menke | Das Eeminterglazial und das Weichselfrühglazial von Rederstall/Dithmarschen und ihre Bedeutung für die mitteleuropäische Jungpleistozän-Gliederung[END_REF].

In north-central Europe the second interstadial was followed by the final decline of early Weichselian woodland and the spread of tundra and steppe vegetation. In contrast, in the Alpine region there was another forested interstadial (Ognon 1 ~ DO 20 ~ 76.4 -75.5 ka, [START_REF] Wolff | Millennial-scale variability during the last glacial: The ice core record[END_REF]) that was characterized by conspicuous Larix advances observed at several places [START_REF] Drescher-Schneider | Die Vegetations-und Klimaentwicklung im Riß/Würm-Interglazial und im Früh-und Mittelwürm in der Umgebung von Mondsee. Ergebnisse der pollenanalytischen Untersuchungen[END_REF][START_REF] Grüger | Spätriss, Riss/Würm und Frühwürm am Samerberg in Oberbayern -ein vegetationsgeschichtlicher Beitrag zur Gliederung des Jungpleistozäns[END_REF][START_REF] Grüger | Riss/Würm und würmzeitliche Ablagerungen im Wurzacher Becken (Rheingletschergebiet)[END_REF][START_REF] Müller | Die Vegetations-und Klimaentwicklung im jüngeren Quartär anhand ausgewählter Profile aus dem südwestdeutschen Alpenvorland[END_REF][START_REF] Müller | Vegetation response to rapid climate change in Central Europe during the last 140,000 yr based on evidence from Füramoos pollen record[END_REF][START_REF] Wegmüller | Vegetationsgeschichtliche und stratigraphische Untersuchungen an Schieferkohlen des nördlichen Alpenvorlandes Birkhäuser[END_REF]Welten 1982a). In Gondiswil for instance pollen values reached 20% [START_REF] Wegmüller | Vegetationsgeschichtliche und stratigraphische Untersuchungen an Schieferkohlen des nördlichen Alpenvorlandes Birkhäuser[END_REF]). After the Ognon 1 interstadial forest vegetation was finally replaced by steppic communities, including in the Alpine region.

MIS 4 (~73.5 -59.4 ka)

For MIS 4 there are only few fossil sites (Fig. 13, Table S 9, Supporting information).

Scattered Larix pollen is found north (Beerenmösli, Wegmüller 1992) and south of the Alps [START_REF] Pini | The vegetation and climate history of the last glacial cycle in a new pollen record from Lake Fimon (southern Alpine foreland, N-Italy)[END_REF] as well as in central Poland [START_REF] Komar | Spatial vegetation patterns based on palynological records in the loess area between the Dnieper and Odra Rivers during the last interglacial-glacial cycle[END_REF] indicating that the species should have persisted in the Alpine region as well as in Central Europe.

At the sites where Larix was found it occurred together with prevailing tundra components pointing to small tree populations growing within open vegetation. In Füramoos north of the Alps [START_REF] Müller | Die Vegetations-und Klimaentwicklung im jüngeren Quartär anhand ausgewählter Profile aus dem südwestdeutschen Alpenvorland[END_REF][START_REF] Müller | Vegetation response to rapid climate change in Central Europe during the last 140,000 yr based on evidence from Füramoos pollen record[END_REF], Larix and other tree taxa disappeared during MIS 4, indicating harsh climate conditions. Considering the unfavourable climatic character of MIS 4, due to low insolation and the Heinrich 6 event, this stage can be assumed to have had dramatic consequences for larch populations in all parts of its range by causing major extinctions and strongly restricting population size. In the Alpine region different aspects of larch history are documented in the northern Alpine site Füramoos and in the southern Alpine sites of Lake Fimon and Lago della Costa [START_REF] Kaltenrieder | Environmental and climatic conditions at a potential Glacial refugial site of tree species near the Southern Alpine glaciers. New insights from multiproxy sedimentary studies at Lago della Costa (Euganean Hills, Northeastern Italy)[END_REF][START_REF] Müller | Vegetation response to rapid climate change in Central Europe during the last 140,000 yr based on evidence from Füramoos pollen record[END_REF][START_REF] Pini | The vegetation and climate history of the last glacial cycle in a new pollen record from Lake Fimon (southern Alpine foreland, N-Italy)[END_REF][START_REF] Wick | Full-to late-glacial vegetation and climate changes and evidence of glacial refugia in the south-eastern Alps (Italy)[END_REF]). In the southern sites macrofossils corroborate pollen evidence, pointing to local persistence of larch during MIS 3 within a mosaic of boreal forests and steppes. In addition, individually dated charcoals were found at the southern Alpine fringe in the provinces of Verona and Como [START_REF] Maspero | Dati sulla vegetazione del periodo glciale: Antracologgia dei siti paleolitici del nord Italia[END_REF] between ~ 46 ka and 36 ka. This suggests a relatively wide species distribution range in the southern Alpine foreland and would fit well with studies reporting alternating episodes of spread of boreal and temperate trees [START_REF] Canali | Vegetational and environmental changes in the eastern Venetian coastal plain (Northern Italy) over the past 80,000 years[END_REF][START_REF] Drescher-Schneider | Palaeobotanical investigations at the mammoth site of Niederweningen (Kanton Zürich)[END_REF][START_REF] Pini | Pollen stratigraphy, vegetation and climate history of the last 215 ka in the Azzano Decimo core (plain of Friuli, north-eastern Italy)[END_REF][START_REF] Preusser | Luminescence dating of the Niederweningen mammoth site, Switzerland[END_REF]. Inferences on Larix response to D-O events can be made on the basis of the Füramoos pollen record [START_REF] Müller | Die Vegetations-und Klimaentwicklung im jüngeren Quartär anhand ausgewählter Profile aus dem südwestdeutschen Alpenvorland[END_REF][START_REF] Müller | Vegetation response to rapid climate change in Central Europe during the last 140,000 yr based on evidence from Füramoos pollen record[END_REF]. This record reveals that Larix together with Betula was favoured by D-O events 14 and 12, which date back to 54.2 ka and 46.8 ka, respectively [START_REF] Wolff | Millennial-scale variability during the last glacial: The ice core record[END_REF]. In contrast, earlier D-O events 17 (~59.4 ka) and 16 (~58.2 ka) did not elicit such a response. This is consistent with a recent study demonstrating that at latitudes above 40°N D-O 12 and 14 triggered the most prominent forest advances (Sánchez [START_REF] Goñi | Contrasting impacts of Dansgaard-Oeschger events over a western European latitudinal transect modulated by orbital parameters[END_REF]. In Lake Fimon individual D-O events cannot be discerned as general forest development was strong due to orographic rainfall.

However, pollen curves do suggest that a cluster of D-O events including D-O 12 and D-O 14

favoured Larix as well as other taxa (Picea, Betula and Alnus).

For the Carpathian region persistence of Larix during MIS 3 is proven by the pollen sequences of Šafárka (600 m a.s.l.) and (in part) of Jablůnka (350 m a.s.l.) stemming from two well protected intermountain basins of the Western Carpathians [START_REF] Jankovská | Vegetační poměry Slovenska a Českých zemí v posledním glaciálu jako přírodní prostředí člověka a fauny [Vegetation of Slovakia and Czechia during the last glacial as and environment of human and fauna[END_REF][START_REF] Jankovská | Šafárka -first palaeobotanical data on vegetation and landscape character of Upper Pleistocene in West Carpathian (North East Slovakia)[END_REF][START_REF] Jankovská | Forest vegetation of the last full-glacial period in the Western Carpathians (Slovakia and Czech Republic)[END_REF]. Pollen records supported by macrofossil show values up to 20% in the bottom part of the Šafárka sequence and indicate predominance of larch together with Betula and Pinus cembra followed by predominance of Picea since ~30 ka [START_REF] Kuneš | Interpretation of the last-glacial vegetation of eastern-central Europe using modern analogues from southern Siberia[END_REF]. The pollen assemblages from both sites have been compared to modern analogues from southern Siberia. Results have revealed that the Western Carpathians had been covered by varying vegetation complexes encompassing taiga forests, hemiboreal forests, forest tundra, forest steppes, tundra or steppes [START_REF] Kuneš | Interpretation of the last-glacial vegetation of eastern-central Europe using modern analogues from southern Siberia[END_REF]. Responses to D-O tel-00880539, version 1 -6 Nov 2013 events cannot be studied as resolution and dating are insufficient. The complementary information given by single dated pollen probes, macrofossils and charcoals from between ~55 ka and 28 ka (e.g. [START_REF] Damblon | Radiocarbon chronology of reprensentatove Upper Palaeolithic sites in the central European Plain: a contribution to the SC-004 project[END_REF][START_REF] Damblon | New datings and considerations on the chronology of Upper Palaeolithic sites in the Great Eurasiatic Plain[END_REF][START_REF] Erd | Pollenanalytischer Gliederung des Pleistozäns der DDR[END_REF][START_REF] Geyh | Neue chronologischenAngaben der Würm-Vereisung in Ungarn[END_REF][START_REF] Komar | Spatial vegetation patterns based on palynological records in the loess area between the Dnieper and Odra Rivers during the last interglacial-glacial cycle[END_REF][START_REF] Krolopp | Absolute chronological data of the Quaternary sediments in Hungary[END_REF][START_REF] Mamakowa | New data about the profile of young Quaternary deposits at Brzeznica on the Wisloka River, the Carpathian foreland (SE Poland)[END_REF]Musil 2003;Opravil 1994;[START_REF] Rybníčková | The environment of the Pavlovian: palaeoecological results from Bulhary,South Moravia[END_REF][START_REF] Willis | Trees or no trees? The environments of central and eastern Europe during the Last Glaciation[END_REF] suggest great abundance of larch in the surroundings of the Carpathian Mountains and in the Pannonian Plain, within a mosaic of more or less open forest tundra, forest steppe or small forests.

MIS 2 (~27. 8 -14.7 ka) Pre-LGM and LGM period

For the part of MIS 2 preceding the LGM (~27.8 -23.5 ka, Fig. 13) and the LGM (~23.5 -19 ka, Fig. 14) [START_REF] Mix | Environmental processes of the ice age: land, oceans, glaciers (EPILOG)[END_REF][START_REF] Goñi | Millennial-scale climate variability and vegetation changes during the Last Glacial: Concepts and terminology[END_REF], there are only few fossil sites. However, they clearly demonstrate the persistence of Larix in the Alpine and the Carpathian region as well as in the Pannonian basin.

Two Larix sites were studied in the Alpine region: Lake Fimon and Lago della Costa [START_REF] Kaltenrieder | Environmental and climatic conditions at a potential Glacial refugial site of tree species near the Southern Alpine glaciers. New insights from multiproxy sedimentary studies at Lago della Costa (Euganean Hills, Northeastern Italy)[END_REF][START_REF] Pini | The vegetation and climate history of the last glacial cycle in a new pollen record from Lake Fimon (southern Alpine foreland, N-Italy)[END_REF]. In Lago della Costa Larix pollen and macrofossils [as well as pollen of other light demanding woody and herbaceous pioneers (Juniperus, Betula, Artemisia, Poaceae)] slightly increased in the period before the LGM. The authors suggest that steppic vegetation dominated the Po Plain whereas Larix colonized slopes around the lake. A similar landscape opening with occurrence of Larix and domination by xerophytes was documented in Lake Fimon. These results demonstrate that Larix was favoured during the Heinrich event 2 between 26.5 ka and 24.3 ka (Sánchez [START_REF] Goñi | Millennial-scale climate variability and vegetation changes during the Last Glacial: Concepts and terminology[END_REF]. Lake

Fimon does not cover the LGM whereas in Lago della Costa pollen and macrofossils of Larix are also found during the period of the LGM between 23.5 ka and 19.5 ka [START_REF] Mix | Environmental processes of the ice age: land, oceans, glaciers (EPILOG)[END_REF].

Hence, Lago della costa (Euganean Hills, Venetia) is identified as LGM refuge.

In the Pannonian Basin detailed inferences on the pre-LGM period are possible thanks to a recent investigation of Lake Fehér (86 m a.s.l.) in the Hungarian Plain [START_REF] Sümegi | Responses of terrestrial ecosystems to Dansgaard-Oeshger cycles and Heinrich-events: A 28,000-year record of environmental changes from SE Hungary[END_REF].

As in the two Alpine sites Larix was favoured by Heinrich event 2 during which forest declined and steppe expanded. Similar deforestation and desertification coinciding with tel-00880539, version 1 -6 Nov 2013 Heinrich event 2 has also been observed in other European regions (e.g. Northwestern Iberia, Naughton et al. 2007), suggesting that Heinrich 2 event may also have affected Larix across its range. Moreover, studies in Lake Fehér also document two warm intervals leading to an increase of Picea and deciduous trees and a concomitant decrease of Larix. The authors assigned these warm intervals to D-O events 2 and 3 and corroborated the palynological evidence with malacological results [START_REF] Sümegi | Responses of terrestrial ecosystems to Dansgaard-Oeshger cycles and Heinrich-events: A 28,000-year record of environmental changes from SE Hungary[END_REF]. This study shows particularly well how millennial scale climate oscillations repeatedly impacted larch population in the early MIS 2.

During the LGM Larix can still be found in Lake Fehér and in addition in Nagymohos (394 m a.s.l.) located in the north-eastern Hungarian Mountains [START_REF] Magyari | Climate versus human modification of the Late Quaternary vegetation in Eastern Hungary PhD thesis[END_REF][START_REF] Magyari | Palynological and plant macrofossil data on Late Pleistocene short term climatic oscillations in North-Eastern Hungary[END_REF]. There are also charcoals from the Hungarian Plain dated to the LGM [START_REF] Gaborí-Csánk | A ságvári telep abszolút kormeghatározása[END_REF][START_REF] Geyh | Neue chronologischenAngaben der Würm-Vereisung in Ungarn[END_REF][START_REF] Vogel | Groningen radiocarbon dates V[END_REF][START_REF] Willis | Trees or no trees? The environments of central and eastern Europe during the Last Glaciation[END_REF]) identified as Picea-Larix. As both species were present in Lake Fehér this may indicate other Larix occurrences. In addition LGM persistence of larch is proven in the Western Carpathians by a continuous pollen curve and additional macrofossils from Šafárka as well as in the southeast Polish Bieszczady Mountains by a single probe with pollen and macrofossils from Smerek (600 m a.s.l.) dated back to ~20 ka (16925 ± 325 uncal. BP, Ralska-Jasiewiczowa 1980; [START_REF] Wacnik | Larix decidua (Mill.) -European larch. In: Late Glacial and Holocene History of vegetation in Poland based on isopollen maps[END_REF]. There is also early pollen evidence dated ~20 ka from one site Labský důl (1039 m a.s.l.) in the Sudetes (PALYCZ; [START_REF] Engel | Sedimentary evidence of landscape and climate history since the end of MIS 3 in the Krkonoše Mountains, Czech Republic[END_REF][START_REF] Jankovská | Krkonoše v době poledové -vegetace a krajina [Giant Mountains in Postglacial -vegetation and landscape[END_REF]).

However, the early part of the profile was poorly dated, and hence LGM persistence in the Sudetes cannot be proven solely on the basis of this record.

Post-LGM -onset of MIS 1 (19 -14.7 ka, [START_REF] Mix | Environmental processes of the ice age: land, oceans, glaciers (EPILOG)[END_REF][START_REF] Svensson | A 60 000 year Greenland stratigraphic ice core chronology[END_REF] The period between the LGM and the onset of MIS 1 (~19 -14.7 ka) can be studied based on a much larger number of sites compared to previous periods, including 88 with Larix evidence (Fig. 14, Table S 12 includes low altitudinal sites south to the Alps but scarce evidence was also found at some higher elevation sites and along the Adige-Inn valley, indicating first upward movements and Alpine transmigration starting from several places (Lake Garda region, Lake Como region, Turin region).

Fossil wood dated back to 18.3 ka is found in Lago Piccolo di Avigliana (353 m a.s.l), a finding embedded in a sediment layer above an even older but undated needle evidence.

Together with conspicuous pollen and stomata evidence assigned to different phases of the Oldest Dryas (e.g. Lago di Viverone, 220 m a.s.l., Torfsee, 270 m a.s.l., Schneider 1978) this suggests that larch was already present in the lowlands when deglaciation started (Schneider 1978;[START_REF] Vescovi | Interactions between climate and vegetation during the Lateglacial period as recorded by lake and mire sediment archives in Northern Italy and Southern Switzerland[END_REF]. Data suggest that the Turin region was the initial area of subsequent recolonization. Evidence from higher altitudes is very scarce and may indicate the presence of a few (krummholz-like) individuals near the site or the presence of trees at lower elevations.

There is a time lag between early deglaciation and pioneer forest establishment that has been dated back to ~ 15.8 -16 ka [START_REF] Vescovi | Interactions between climate and vegetation during the Lateglacial period as recorded by lake and mire sediment archives in Northern Italy and Southern Switzerland[END_REF]). This may be explained by climatic constraints caused by Heinrich event 1 (~18 -15.6 ka, Sánchez Goñi & Harrison 2010), which has been shown to have caused desertification in Western Iberia (Naughton et al. 2007). For this time treeline was estimated at ~400 -500 m (Tinner 2007).

A first single but noteworthy pollen finding in the Maritime Alps was made in Selle di Carnino at ~16.2 ka (1905 m a.s.l.;ALPADABA;[START_REF] Beaulieu | Contribution pollenanalytique à l'histoire tardiglaciaire et Holocene de la végétation des Alpes méridionales françaises[END_REF]. It remains unclear whether it was wind-transported or of local origin. However, this raises the question of the origin of current larch populations from the Maritime Alps in France. Two hypotheses have been formulated [START_REF] Beaulieu | Contribution pollenanalytique à l'histoire tardiglaciaire et Holocene de la végétation des Alpes méridionales françaises[END_REF]Ortu 2002). First, they could originate from the Italian populations that survived in the Turin region. Second, the source populations could have persisted the LGM in the Maritime Alps, a region that was less glaciated than other parts of the Alps. De [START_REF] Beaulieu | Contribution pollenanalytique à l'histoire tardiglaciaire et Holocene de la végétation des Alpes méridionales françaises[END_REF] suggested for instance the French Mercantour as a possible refuge. A combination of both hypotheses can also be imagined.

In the regions of Lombardy and Ticino larch fossils are less abundant after the LGM which may be explained by the less continental conditions favouring other tree taxa (e.g. Lago di Biandronno, Suossa, Schneider 1978;Zoller & Kleiber 1971). Further east, in the Trentino-Adige and Venetian region, macrofossils and pollen findings are more abundant and indicate Larix growing in pioneer forests. Buried Larix trunks were found for instance in the Venetian tel-00880539, version 1 -6 Nov 2013

Prealps and dated back to 18.1 -17 ka [START_REF] Casadoro | Un deposito Tardowurmiano con tronchi subfossili alle Fornaci di Revine (Treviso)[END_REF][START_REF] Friedrich | Paleo-environment and radiocarbon calibration as derived from Lateglacial/Early Holocene tree-ring chronologies[END_REF][START_REF] Kromer | Segments of atmospheric 14 C change as derived from Lateglacial and Early Holocene tree-ring series[END_REF]. In addition, macrofossil, stomata and pollen records are documented in the Ragogna Lake located at the southern Alpine margin in northwestern Italy since ~ 17.1 ka [START_REF] Monegato | Evidence of a two-fold glacial advance during the last glacial maximum in the Tagliamento end moraine system (eastern Alps)[END_REF][START_REF] Wick | Lateglacial and Early-Holocene palaeoenvironments in Brianza, N Italy[END_REF]. Evidence from the area around Lake Garda and scarce findings along the Adige-Inn valley suggest a migrational pathway across the Alps leading west to the Dolomites along a line from Lake Garda to Innsbruck through the Brenner pass, i.e. the lowest pass in this part of the Alps.

In the eastern Alpine margin (>14° E), fossil sites are scarce during this period. However, pollen evidence from ~18.7 ka exists in the Austrian region of Carinthia in the site Längsee [START_REF] Schmidt | Late and post-glacial history of meromictic Längsee (Austria), in respect to climate change and anthropogenic impact[END_REF]. Given that maximum glacier extent during the LGM did not entirely cover the eastern Alpine margin and in view of the fact that many herbaceous endemics associated to upper montane coniferous forests grow there [START_REF] Tribsch | In search for Pleistocene refugia for mountain plants: patterns of endemism and comparative phylogeography confirm palaeo-environmental evidence in the Eastern European Alps[END_REF], it can be assumed that the Längsee record also points to a refuge of larch.

In the southern Carpathian region pollen and stomata are found in two high elevation sites in the Romanian Retezat mountains that are part of the Southern Carpathians (Lake Brazi, 1740 m a.s.l.; Lake Galeş, 1990 m a.s.l., [START_REF] Magyari | Rapid vegetation response to Lateglacial and early Holocene climatic fluctuation in the South Carpathian Mountains (Romania)[END_REF]. There is evidence for the presence of larch in both profiles since the onset of the record at ~15 ka. In the Retezat mountains maximum glacier advance was centred ~16-18 ka and the early high altitudinal larch occurrences seem to be best explained by refuges at lower elevations [START_REF] Magyari | Rapid vegetation response to Lateglacial and early Holocene climatic fluctuation in the South Carpathian Mountains (Romania)[END_REF]). In the western Carpathians and their forelands as well as in the Sudetes larch fossils became abundant at this time suggesting that larch was more widespread in this region than today.

MIS 1 (since ~14.7 ka)

During MIS 1, which includes the late-glacial interstadial (~14.5 -12.8 ka, [START_REF] Ammann | Late Glacial Multidisciplinary Studies[END_REF][START_REF] Litt | Correlation and synchronisation of Lateglacial continental sequences in northern central Europe based on annually laminated lacustrine sediments[END_REF][START_REF] Rasmussen | A new Greenland ice core chronology for the last glacial termination[END_REF], the Younger Dryas (~12. 8 -11.7 ka, Ammann et al. 2006;[START_REF] Litt | Correlation and synchronisation of Lateglacial continental sequences in northern central Europe based on annually laminated lacustrine sediments[END_REF][START_REF] Rasmussen | A new Greenland ice core chronology for the last glacial termination[END_REF]) and the Holocene (since ~ 11.7 ka) Larix is observed in 283 fossil sites. The data show a great diversity of responses to climatic and anthropogenic changes depending on the geographical location, the topography of the mountain massifs and additional local factors. In the following the most important features of this history are described without going into the details from each individual site.

tel-00880539, version 1 -6 Nov 2013

Late-glacial interstadial (Bølling/Allerød, 14.5 -12.8 ka) During the Bølling/Allerød (D-O 1) fossil abundance increases considerably indicating a conspicuously wide distribution range covering the Alps, the Sudetes, the Carpathians, the Pannonian Plain and the Polish lowland where the species is found far to the north (Fig. 14).

In the Alpine region the abrupt warming at the onset of the Bølling/Allerød triggered an altitudinal shift to higher elevations, documented by larch macrofossil, stomata and pollen records. Macrofossil occurrences above 1000 m a.s.l. were for instance found in the Pilaz peat bog (1900 m a.s.l.) in the Aosta Valley [START_REF] Brugiapaglia | Paleobiogeografia della Valle d'Aosta a partire dall'ultima glaciazione[END_REF][START_REF] Brugiapaglia | Evoluzione altitudinale spazio-temporale degli alberi durante GLI ultimi 12000 anni in valle d'Aosta[END_REF], the Totenmoos (1718 m a.s.l.) in the Adige Valley [START_REF] Heiss | The Ulten Valley in South Tyrol, Italy: Vegetation and settlement history of the area, and macrofossil record from the Iron Age cult site of St. Walburg[END_REF] and the Palughetto mire (1040 m a.s.l.) in the Venetian Prealps [START_REF] Avigliano | A new late glacial to early Holocene palaeobotanical and archaeological record in the Eastern Pre-Alps: the Palughetto basin (Cansiglio Plateau, Italy)[END_REF]. However, it has been shown that Alpine treelines with Larix and other treeline species remained about 600 m below the current level [START_REF] Gobet | Early-Holocene afforestation processes in the lower subalpine belt of the Central Swiss Alps as inferred from macrofossil and pollen records[END_REF]Tinner 2007;[START_REF] Tobolski | Macrofossils as records of plant responses to rapid Late Glacial climatic changes at three sites in the Swiss Alps[END_REF] and that subalpine larch forests became established only after the Bølling/Allerød. At lower elevations Larix is still found, although other tree species became predominant (e.g. Finsinger et al. 2006;[START_REF] Monegato | Evidence of a two-fold glacial advance during the last glacial maximum in the Tagliamento end moraine system (eastern Alps)[END_REF]Schneider 1978).

In the Carpathian region a detailed focus on altitudinal distribution is not possible due to the scarcity of sites >1000 m a.s.l. However, the two high altitudinal southern Carpathian sites and sites at 600 -800 m a.s.l. in the northeastern and western Carpathians (Feurdean & Bennike 2004;[START_REF] Jankovská | Late Glacial finds of Pinus cembra L. in the Lubovnianska kotlina Basin[END_REF][START_REF] Jankovská | The development of the vegetation in Inner Carpathian Basin of the Tatra foreland cover at the end of Glacial period till the present time[END_REF][START_REF] Magyari | Rapid vegetation response to Lateglacial and early Holocene climatic fluctuation in the South Carpathian Mountains (Romania)[END_REF][START_REF] Ralska-Jasiewiczowa | Late-glacial and Holocene vegetation of the Bieszczady Mts[END_REF][START_REF] Wohlfarth | Reconstruction of climatic and environmental changes in NW Romania during the early part of the last deglaciation (~15,000-13,600 cal yr BP)[END_REF] demonstrate the presence of larch in the mountains and an increased abundance during this warming phase.

Younger Dryas (~12. 8 -11.7 ka) The cold episode of the Younger Dryas (~12. Fig. 15) led to heterogeneous responses of larch. This can be illustrated by the considerable variation of the upper forest line in the Alps [START_REF] Gobet | Early-Holocene afforestation processes in the lower subalpine belt of the Central Swiss Alps as inferred from macrofossil and pollen records[END_REF] and in the Carpathians. Larch responses ranged from a complete decline in some high altitudinal sites (e.g. [START_REF] Heiss | The Ulten Valley in South Tyrol, Italy: Vegetation and settlement history of the area, and macrofossil record from the Iron Age cult site of St. Walburg[END_REF][START_REF] Jankovská | Late Glacial finds of Pinus cembra L. in the Lubovnianska kotlina Basin[END_REF][START_REF] Jankovská | The development of the vegetation in Inner Carpathian Basin of the Tatra foreland cover at the end of Glacial period till the present time[END_REF] to stability or even expansion at lower elevations [START_REF] Andrič | A multi-proxy Late-glacial palaeoenvironmental record from Lake Bled, Slovenia[END_REF][START_REF] Bortenschlager | Beiträge zur Vegetationsgeschichte Tirols. Inneres Ötztal und unteres Inntal[END_REF][START_REF] Koperowa | The history of the Late-Glacial and Holocene vegetation in Nowy Targ Basin[END_REF]. The first two millennia of the Holocene can hence be viewed as the key period to account for modern larch distribution. Moreover, during these two millennia larch dominated subalpine forests in the central Alps as it was favoured by high summer insolation and low winter insolation that limited the spread of Pinus cembra (Tinner & Kaltenrieder 2005). Treeline studies indicate that between 10 ka and 6 ka larch should have reached its uppermost position (Tinner 2007) and it has been shown that during this time larch forests were in dynamic equilibrium with climate including rapid changes such as the 8.2 cold event that led to the decline of larch populations in a site near the treeline (Tinner 2007;Tinner & Kaltenrieder 2005).

Since ~ 6 ka the timberline started to drop as a response to anthropogenically and climatically induced changes. While some human interventions led to the extinction of larch, others favoured the expansion of the species, which was observed in sites from across the range (e.g. [START_REF] Feurdean | The usefulness of a long-term perspective in assessing current forest conservation management in the Apuseni Natural Park, Romania[END_REF][START_REF] Kaltenrieder | Zur Langzeitökologie des Lärchen-Arvengürtels in den südlichen Walliser Alpen[END_REF]Ortu et al. 2005;Schneider & Tobolski 1985). Prehistorical and historical epochs that had a major impact on larch were the Bronze Age, the Roman Age, the Middle Age and the Modern Age. During the Roman Age larch was favoured together with plants that are known to have long cultural histories. The species appears for instance in one site of the Maritime Alps during the Roman Age contemporaneously with chestnut (Castanea), walnut (Juglans), vine (Vitis) and cereals (Ortu et al. 2003), a coincidence that was also described in other regions (e.g. Schneider & Tobolski 1985). Land use changes, the role of fire and other anthropogenic disturbances favouring or limiting larch expansion are described in numerous publications. They will not be reviewed in this chapter, but it should be stressed that recent translocation history described in chapter 4 encompassing the last centuries has to be seen as one aspect of a 6 ka lasting anthropogenically shaped history during which multiple introductions and re-introductions of larch can be expected, even if they cannot be traced with paleontological tools. During the last 50 years larch history continued to be influenced by plantations and land abandonment in the Alpine region, suggesting that the species is currently expanding. tel-00880539, version 1 -6 Nov 2013

Ancient genetic structure

The study uncovered an exceptionally strong genetic structure using both nuclear and mitochondrial DNA markers in the European larch (see CHAPTER 4). Seven nuclear genetic clusters have been identified (Fig. 21): four in the Alpine region and three in the Central European region. The genetic distance between nuclear clusters from these two regions is particularly large. The analysis of the mitochondrial genome, which evolves very slowly in plants including Conifers [START_REF] Hipkins | Organelle genomes in conifers: structure, evolution, and diversity[END_REF], also indicates a clear separation between Alpine and Central European lineages. Altogether, this suggests that the split between Alpine and Central European populations is very ancient. In the Alpine region, the four nuclear clusters as well as the two most common mitochondrial haplotypes (haplotypes 16 and 18) are distributed longitudinally. Moreover, the mitochondrial haplotypes alternate along the same direction (with the following sequence of haplotypes from west to east:

16/18/16/18). In most cases, nuclear clusters are characterized by one major mitochondrial haplotype (nuclear cluster 1 with haplotype 16, cluster 2 with haplotype 18, cluster 3 with haplotype 16, cluster 4 with haplotype 18), indicating that there has been no important gene flow, whether by seed or by pollen, between the corresponding groups of populations.

However, we found two populations in the Swiss central Alps in the region of Valais (populations 27 and 83) that depart from this pattern. These populations were assigned to nuclear cluster 1 and carry haplotype 18. This suggests that the bulk of the ancestors of these populations originate from the southwestern Alps but have become introgressed by a mitochondrial haplotype originating from a refuge located further east, in the Venetian region.

Another important result was the identification of a rare haplotype (haplotype 22) only found in the Maritime Alps and slightly further to the north (fixed in population 81 and mixed with haplotype 16 in population 77). This pattern could suggest a separate glacial refugia in the Maritime Alps. Yet, at nuclear markers, these two populations were both assigned to cluster 1 and did not differ from neighbouring populations fixed for mitochondrial haplotype 16, suggesting that either divergence within the two nearby refugia was restricted to mitochondrial DNA or that there has been subsequent genetic homogenisation through gene flow in the region.

In Central Europe we found one nuclear cluster in the Sudetes Mountains (cluster 5), one in the Tatra Mountains (6) and one in the eastern and southern Carpathians [START_REF] Schmidt | Late and post-glacial history of meromictic Längsee (Austria), in respect to climate change and anthropogenic impact[END_REF]. In the Polish lowland nuclear assignment was less clear as clusters from the Sudetes and the Tatra Mountains co-occurred. In addition there were genotypes with intermediate assignments in tel-00880539, version 1 -6 Nov 2013 this part of the range. One mitochondrial DNA haplotype (number 8) was endemic to this area, though found in only one of the three sampled populations (population 44). This may indicate that Polish lowland populations originate from a different source than the Tatra and Sudetes populations. Alternatively, these populations could result from admixture between Tatra and Sudetes populations. To evaluate these hypotheses, new investigations are needed.

Refuges

To reconstruct refuges and migrational pathways, modern nuclear and mitochondrial DNA genetic structure can be used together with the fossil data. Early fossil evidence from the

LGM and the subsequent Heinrich event 1 suggests that at least six of the seven nuclear clusters originate from distinct refuges: cluster 1 from the Turin refuge, cluster 2 from the Venetian refuge, cluster 3 from a southern Carinthian refuge, cluster 5 from the Sudetes refuge, cluster 6 from the western Carpathians refuge and cluster 7 from the southern Carpathians refuge. No fossil data is available that points to a refuge corresponding to nuclear cluster 4. It seems likely that this cluster also originates from a separate refuge as the eastern Alpine margin provided favourable environments during the LGM where endemic herbaceous taxa associated to upper montane coniferous forests persisted [START_REF] Tribsch | In search for Pleistocene refugia for mountain plants: patterns of endemism and comparative phylogeography confirm palaeo-environmental evidence in the Eastern European Alps[END_REF].

The rare mitochondrial DNA haplotype found in the Maritime Alps seems to indicate the existence of another refuge that is also reflected in the early but limited fossil evidence found there before the Bølling/Allerød period at ~16.2 ka. Finally, both nuclear and mitochondrial data point to a distinct origin of populations from the Sudetes and from the Carpathians and hence corroborate evidence given by the fossils.

Colonization pathways

Fossils, nuclear and mitochondrial data indicate that recolonization of the southwestern Alps mainly involved source populations located in the Turin region. In addition there may have been some colonization from the presumed Maritime Alps refuge. For the central Swiss Alps forests. In the region located between the Lago Maggiore and Lake Garda, fossil and genetic data suggest a re-colonization from the southern Alpine foreland with the Venetian and Trentino-Alto-Adige region as the most important source area. Migration across the Alps might have followed the Adige-Inn valley. In fact, the Adige zone represents a second important contact zone [START_REF] Thiel-Egenter | Break zones in the distributions of alleles and species in alpine plants[END_REF]. This is testified by a genetic split of larch populations. Westwards to the Adige Valley, migrational pathways cannot be inferred in such a detail due to scarcity of the fossil data. However, migration in different directions from the refuge in southern Carinthia seems likely.

In Central Europe modern larch populations should originate mainly from upward shifts from refuges in the Sudetes, the northwestern Carpathians and the southern Carpathians. The lowland populations from central Poland are (slightly) genetically distinct from the Tatra and Sudetes populations and may originate from populations persisting north to the western Carpathians. However, more investigations will be necessary to conclude about this issue.

Climatic and anthropogenic impacts

The paleontological and genetic methodologies combined in this study allow evaluating climate and anthropogenic effects on history of European larch at multiple temporal and spatial scales and from different perspectives. The genetic approach allows historical insights but only from the perspective of persisting modern populations. It indicates that modern distribution of genetic lineages is explained on the one hand by climate (ancient genetic structure) and on the other hand by recent translocations (in superposition to the ancient genetic structure). To evaluate when anthropogenic changes became superimposed to climate changes, fossil data are necessary. For larch, fossils clearly show that populations were impacted by anthropogenic changes (e.g. grazing, burning, cutting) since the Bronze Age, as judged from shifts in pollen and macrofossil abundance in populations at different altitudes from different parts of the Alpine and Carpathian range (e.g. [START_REF] Feurdean | The usefulness of a long-term perspective in assessing current forest conservation management in the Apuseni Natural Park, Romania[END_REF][START_REF] Kaltenrieder | Zur Langzeitökologie des Lärchen-Arvengürtels in den südlichen Walliser Alpen[END_REF]Schneider & Tobolski 1985). In the Swiss Alps for instance stable subalpine forests with larch became replaced by meadows and shrublands due to contemporaneous climatic and anthropogenic changes whereas in a low altitudinal Italian site larch became favoured when cultivation of Juglans and Castanea started, confirming the predominant anthropogenic influence. In some cases fossil data can even confirm and complement genetic evidence of recent translocations by providing a time scale. In Slovakia tel-00880539, version 1 -6 Nov 2013 and the Czech Republic for instance, larch fossils become abundant 500 years ago in several low elevation sites clearly located beyond its natural range (Fig. 19). To date, inferring such time frame using genetic data alone is not possible.

Important processes that can only be investigated using a genetic perspective are introgression and admixture, which are increasingly endangering native plant and animal populations worldwide [START_REF] Allendorf | The problems with hybrids: setting conservation guidelines[END_REF]. In European larch human-induced admixture has been shown to be a frequent phenomenon, in particular in Slovakia, the Czech Republic and Poland. In some populations, including populations of the red list variant "polonica", admixture rates are alarming. Furthermore, as larch provenances differ in sensitivity towards larch canker, translocation of maladapted provenances can lead to population decline (Münch 1936). For instance, in France, larch canker has become a serious problem in larch plantations, both within and beyond the natural distribution range. In a related study, I applied the markers developed in chapter 3 to trace the origin of planted larch populations. I could show that the provenances that were most sensitive originate from the western Alps, which is in agreement with indications given by larch provenance tests (see Appendix n°1). This demonstrates that the markers developed in this study, along with the range-wide reference sample, can be used to answer practical questions in forestry.

These results show that to understand anthropogenic impact on tree populations, both fossil and genetic data are necessary. Fossil data allow to go further back in time and to understand the timing of human influences whereas genetic data allow studying translocation events in more detail (source, relative abundance and degree of admixture). Inference on the individual impact of specific long-term and short-term climate oscillations cannot be made based on modern genetics as it is not possible to correlate genetic changes to climate changes on the timescale of millennia. In fact, a lot of information on climate impact potentially registered in DNA has become lost due to subsequent population extinctions. Alongside with these intrinsic constraints, there are technical and methodological gaps in terms of genetic data acquisition and genetic data analysis. Fossils allowed us to precisely trace Larix responses to long-term trends and short-term climate events and revealed multiple range changes as well as shifts in species local abundance. The fact that six out of seven refuges could be identified (Schober, 1985). [START_REF] Sylvestre-Guinot | Possibilités d'appréciation de la sensibilité du genre Larix au Lachnellula willkommii (Hartig) Dennis par inoculations artificielles[END_REF]

Matériel et méthodes

Choix des peuplements :

En 2012, les recherches de provenances à l'aide de marqueurs microsatellites ont été réalisées sur 192 arbres provenant de 39 peuplements, choisis essentiellement parmi ceux pour lesquels nous possédions des mentions "administratives" de provenance. L'annexe 3 fournit le détail du nombre d'arbres échantillonnés par peuplement et la figure 1

:

  Paläoökologie, Populationsgenetik, Konifere, letzter Interglazial/Glazialzyklus, Klimawandel, Translokationen tel-00880539, version 1 -6 Nov 2013 L'histoire du mélèze d'Europe (Larix decidua Mill.)

  6 ka l'histoire de l'espèce dépendait essentiellement des facteurs climatiques et que par la suite une combinaison de facteurs climatiques et anthropiques doit être prise en compte. Les réponses de l'espèce à des changements climatiques (à long terme, ou à court terme) engendrant des changements d'aire et d'abondance sont uniquement observés grâce aux fossiles. Les résultats paléoécologiques soulignent l'aspect dynamique de l'aire, un élément important à considérer dans la gestion des forêts actuelles et futures. Cette thèse montre que les données fossiles et génétiques doivent être combinées pour mieux comprendre les impacts anthropiques et climatiques dans l'histoire d'une espèce. Vu la précision et la richesse des détails fournis par les deux approches, cette étude peut être considérée comme une nouvelle étape dans la reconstruction de l'histoire d'une espèce forestière à l'échelle de l'ensemble d'une aire de répartition. Elle met également à disposition des outils et des informations d'importance majeure pour d'autres champs de recherche et pour la foresterie. tel-00880539, version 1 -6 Nov 2013 Introduction .......................................................................................................................... tel-00880539, version 1 -6 Nov 2013

Figure 1

 1 Figure 1 Larix decidua in the Central Swiss Alps ...................................................................

Figure 2

 2 Figure 2 Distribution map of Larix decidua.............................................................................

Figure 3

 3 Figure 3 Binning, profiles and marker ranges of SSR multiplex 1 ..........................................

Figure 4

 4 Figure 4 Binning, profiles and marker ranges of SSR multiplex 2 ..........................................

Figure 5

 5 Figure 5 Minimum spanning network and distribution of combined mt haplotypes ...............

Figure 6

 6 Figure 6 Neighbour joining tree and distribution of the seven SSR clusters ...........................

Figure 7

 7 Figure 7 Distribution of opposite group purebred and admixed individuals ...........................

Figure 8

 8 Figure 8 Admixture in populations with/without indication for recent translocation ..............

Figure 9

 9 Figure 9 Distribution of first-generation migrants ...................................................................

Figure 10

 10 Figure 10 Abrupt climate events since 123 ka .........................................................................

Figure 11

 11 Figure 11 Map of the 1026 fossil sites investigated in this study ............................................

Figure 12

 12 Figure 12 Fossil distribution of larch from 130 -78.2 ka ........................................................

Figure 13

 13 Figure 13 Fossil distribution of larch from 73.5 -23.5 ka .......................................................

Figure 14 Fossil

 14 Figure 14 Fossil distribution of larch MIS 2 -MIS 1 ..............................................................

Figure 15

 15 Figure 15 Fossil distribution of larch from 12.8 -9.5 ka .......................................................

Figure 16

 16 Figure 16 Fossil distribution of larch from 9.5 -6.5 ka .........................................................

Figure 17

 17 Figure 17 Fossil distribution of larch from 6.5 -3.5 ka .........................................................

Figure 18

 18 Figure 18 Fossil distribution of larch from 3.5 -0.5 ka .........................................................

Figure 19

 19 Figure 19 Fossil distribution of larch during the last 500 years .............................................

Figure 20

 20 Figure 20 Studied modern larch populations and names of geographic locations .................

Figure 21

 21 Figure 21 Ancient genetic structure and colonization pathways. ...........................................

Figure S 1

 1 Figure S 1 ∆k statistics .............................................................................................................

Figure S 2

 2 Figure S 2 Individual bar plots .................................................................................................

Figure S 3

 3 Figure S 3 Number of observed versus theoretical translocation events .................................

Figure S 4

 4 Figure S 4 Detections GENECLASS -detections STRUCTURE ...................................................

Figure S 5

 5 Figure S 5 Genetic composition of presumed natural and introduced material. ......................

  7 ka reported in the literature .............................................. List 1 Larix pollen sites from databases ................................................................................. List 2 Larix stomata sites from the EPD ................................................................................ tel-00880539, version 1 -6 Nov 2013 CHAPTER 1: General introduction and acknowledgements tel-00880539, version 1 -6 Nov 2013

  Prof. Dr. Thomas Litt and carried out at the Steinmann Institute for Geology, Paleontology and Mineralogy of the University of Bonn (Germany). The population genetic part was supervised by Dr. Rémy Petit and Dr. Sophie Gerber and performed in the BIOGECO (Biodiversity, Genes and Communities) research unit affiliated to the University of Bordeaux and INRA (Institut National de la Recherche Agronomique). This allowed taking advantage of the expertises found at the two institutions, a precondition of this PhD. In addition, I collaborated with Dr. Sascha Liepelt and Prof. Dr. Birgit Ziegenhagen (University of Marburg, Germany) our partners in the DFG project. In parallel I contributed to a project on larch canker that was directed by Dr. Dominique Piou and Dr. Cécile Robin. In addition, I had the possibility to discuss my research with Prof. Dr. Maria Fernanda Sánchez-Goñi, directing the laboratory of paleoclimatology and marine paleoenvironment affiliated to the EPHE (Ecole Pratique des Hautes Etudes) and integrated in the EPOC (Environnements et Paléoenvironnements Océaniques et Continentaux) research unit of the University of Bordeaux 1.

  They will have to be significantly reworked before submission. All chapters benefitted from tel-00880539, version 1 -6 Nov 2013 the support of my supervisors and colleagues. In the following I document their contributions as an acknowledgement of their work and to clarify my own work. Chapter 2 I compiled the literature and wrote the manuscript. It was revised by Rémy Petit. For comments and complementary information I thank Luc Pâques, Berthold Heinze and Barbara Fussi. Chapter 3 I conceived the study, performed the experiments, produced and analyzed the data and wrote the manuscript. I got scientific advice during conception and writing from Rémy Petit and Sophie Gerber. Genotyping was performed in the Genome-Transcriptome facility of the Functional Genomic Centre of Bordeaux. I thank Franck Salin, Sarah Monllor, Christophe Boury, Philippe Chaumeil and Camille Lepoittevin as well as Andres Buser (ecogenics GmbH) for support and assistance during genotyping and primer design. For assistance in the various fields of this work I thank Vanina Guérin, Patrick Léger, Guillaume Lalanne-Tisné, Anne Laure Bouillot, Erwan Guichoux, Lélia Lagache, Olivier Lepais and Maxime Nardin. For support with plant material and assistance during sampling I thank Luc Pâques, Jutta Mäsgen, Laurent Bouffier, Frédéric Lagane, Christoph Sperisen, Yuri Hayda, Ihor Neyko, Guy Bour, Czeslaw Koziol, Philippe Rozenberg, Leopoldo Sanchez, Frédéric Millier, Jean-Paul Charpentier, Owe Martinsson, Lars Karlman, Hermann Spellmann (NW-FVA), Keiya Isoda, Nathalie Isabel, Marie-Claude Gros-Louis, the Ukrainian Research Institute for Moutain forestry, Ivano-Frankivsk, the Polish Osusznica Foerst Institute (Nadleśnictwo Osusznica), and the Polish Maskulińskie Forest Institute (Nadleśnictwo Maskulińskie). Chapter 4 I conceived the study assisted by Rémy Petit. I performed the experiments and produced the nuclear data. Sascha Liepelt produced the mtDNA data. I analyzed the data and wrote successive drafts which I progressively improved with the help of Rémy Petit. I discussed the work at different stages with Sophie Gerber. Genotyping was performed in the Genome-

For

  constant encouragement and presence over long and short distance I thank my family and my friends. Herzlichen Dank an euch, Hedwig, Michael, Nadine, Richard, Annette, Laura, Mathis, Silvia, Frank, Jutta, Michael, Jolam, Anne, Gabi, Sascha, Marga und Beate. Merci beaucoup à vous, Frédo, Laurent, Vanina, Emanuel, Stéphanie, François, Sophie, Carol, Claire, Patrice, Olivier, Laure et Virgil.. tel-00880539, version 1 www.trees4future.eu tel-00880539, version 1 -6 Nov 2013

Figure 1

 1 Figure 1 Larix decidua in the Central Swiss Alps Figure 1 Larix decidua in the Central Swiss Alps [photograph: C. Sperisen, 2010-10, with permission]

Figure 2

 2 Figure 2 Distribution map of Larix decidua

Figure 2

 2 Figure 2 Distribution map of Larix decidua showing the current natural distribution. [Modern distribution data compiled by E. Welk, AG Chorology, Geobotany Department, University Halle, based on map 21b in Meusel et al. (1965); modified in central Poland]

Figure 3 Figure 3 Figure 4 Figure 4

 3344 Figure 3 Binning, profiles and marker ranges of SSR multiplex 1 Figure 3 Binning, profiles and marker ranges of SSR multiplex 1. (a) Allelograms based on 413 individuals from across the range. The running allele number corresponds to the position of the allele in the list of allele raw sizes ranked in increasing order. (b) Example of an individual electropherograms. (c) Range sizes based on the same sample as in (a). In (b), arrows point to alleles at each locus, orange peaks correspond to fragments of the internal size standard LIZ600, and numbers above these peaks indicate fragment sizes (base pairs).

Figure 5 Figure 5 Figure 6

 556 Figure 5 Minimum spanning network and distribution of combined mt haplotypes Figure 5 Minimum spanning network (A) and distribution of the combined mitochondrial haplotypes (atpA and UBC460) before (B) and after (C) systematic translocation removal. In (A) circles represent haplotypes labelled by their codes and scaled to their frequencies. The purple pie chart summarizes haplotypes caused by minisatellite variation. Unlabelled circles symbolize predicted haplotypes that were not observed. Branches correspond to single mutations regardless of their length. In (B) and (C) circles represent the haplotype composition of populations (~8 individuals/population). Labels are population codes.

Figure 7

 7 Figure 7 Distribution of opposite group purebred and admixed individuals

Figure 7

 7 Figure 7 Distribution of opposite group purebred and regionally admixed individuals based on STRUCTURE assignments

Figure 8

 8 Figure 8 Admixture in populations (A) with and (B) without indication for recent translocation in Central Europe. Bars represent individual q-values, in grey, group 1, in white group 2.

Fig. 9 Fig. 9

 99 Fig. 9 Distribution of first-generation migrantsFig. 9 Distribution of first-generation migrants detected by GENECLASS. Spatial classification based on the likelihood calculations.

  high proportion of admixed compared to purely exotic genotypes and their widespread distribution suggest indeed that at least one generation has passed following the first introductions. Moreover, translocations typically involved multiple geographic sources with some genetic clusters being overrepresented, especially clusters 2 and 5. This result is tel-00880539, version 1 -6 Nov 2013 supported by historical information indicating that larch seeds from Tyrol (a region located in the Italian and Austrian parts of the Alps where cluster 2 is dominant) and the Sudetes (where cluster 5 is dominant) have preferentially been used in plantations(Münch 1936). Hence, our results shed new light on the "Lärchenmanie" by uncovering its irreversible long-term consequences through a modification of the genetic structure of the species, including areas where threatened gene pools have been recognized (e.g. "variety polonica" listed on the IUCN Red List).

Figure S 1 ∆k statistics Figure S 2 Figure S 2

 statistics22 Figure S 1 ∆k statistics

Figure S 3 Figure S 3 Figure

 33 Figure S 3 Number of observed versus theoretical translocation events

Figure S

 S Figure S Genetic composition of presumed natural and introduced material. Cluster colours are the same as in Fig. 6.

Figure 10

 10 Figure 10 Abrupt climate events since 123 ka.

Figure 10

 10 Figure 10 Abrupt climate events since 123 ka (a) Dansgaard-Oeschger events in the NGRIP ice core [after Wolff et al. 2010]; (b) Heinrich Events in the MD99-2331 core from the North Atlantic,

  cited in the text. Grey shaded bars indicate periods of important forest advances at latitudes >40°N.

  Duration and intensity of D-O events were variable. During MIS 5 they were particularly long. During MIS 4 occurred the strongest warming of the last glacial cycle(D-O 19, 16°C,

  -S 13, List 1, List 2, List 3, Supporting information). This compilation is based on newly assembled sites from the literature (207 sites including 169 Larix sites), ALPADABA sites (156/105), PALYCZ sites (114/30) and complementary EPD sites (549/51).

Figure 11

 11 Figure 11 Map of the 1026 fossil sites investigated in this study * modern distribution data compiled by E. Welk, AG Chorology, Geobotany Department, University Halle, based on map 21b in Meusel et al. (1965); modified in central Poland

(Figure 12

 12 Figure 12 Fossil distribution of larch from 130 -78.2 ka

  , Supporting information). Larch fossils are found in the Alpine region and the north-central European lowlands and also in the Vosges Mountains. In the north-central European lowlands fossil abundance indicates a distributional maximum of larch.

Figure 13

 13 Figure 13 Fossil distribution of larch from 73.5 -23.5 ka

Fig. 14 Figure 14

 1414 Fig. 14 Fossil distribution of larch MIS 2 -MIS 1 Figure 14 Fossil distribution of larch from 23.5 -12.8 ka

  , Supporting information). 35 of them document Larix occurrence since at least 18 ka. Fossils are abundant in the southern Alpine and Carpathian regions and are also found in a few sites of the Polish lowland as well as in the Pannonian basin. In the Alps regions of increased fossil abundance alternate with regions of reduced abundance in west-east direction, pointing to several localities were larch populations could persist (Turin region, Venetian region, South Carinthian region). Evidence preferentially tel-00880539, version 1 -6 Nov 2013

Figure 15

 15 Figure 15 Fossil distribution of larch from 12.8 -9.5 ka

Figure 16 Figure 17 Figure 18 Figure 19

 16171819 Figure 16Fossil distribution of larch from 9.5 -6.5 ka

-

  The second Weichselian stadial was similar to the first and larch should have persisted at the same places. -During the second early Weichselian interstadial larch expanded again in the northcentral European lowlands and in the Alps. Compared to the first interstadial forest development was less pronounced. At the end of this interstadial forests finally declined in the north-central European lowlands. -In the Alpine region there was a conspicuous advance of larch at mid-altitudes during the third early Weichselian interstadial. MIS -MIS 4 was one of the most severe climatic episode since the last 130 ka, causing major extinctions of larch populations and greatly restricting population sizes. MIS -During MIS 3 expansion of larch was favoured by D-O events 12 and 14 in the northern Alpine region. Distribution within this period was wide, covering the Alpine region, the Carpathian region, the Pannonian Plain and the Polish lowlands. Communities including larch were diverse (boreal forests, hemiboreal forests, forest tundra, forest steppes, tundra or steppes) MIS -In the pre-LGM part of MIS 2 the range of larch was very dynamic. The species was favoured by Heinrich event 2 as evidenced in the Alps and the Pannonian basin, whereas it was disfavoured by D-O 2 and 3 as seen in the Pannonian basin. -During the LGM and after, larch persisted in the Alpine and the Carpathian foothills as well as in the Pannonian basin. The following areas can be considered as LGMrefuges: Turin region, Maritime Alps (?), Venetian region, southern Carinthia, Sudetes, western Carpathians (Tatra Mts) and southern Carpathians. -During early deglaciation larch recolonized rapidly territories recently freed from the ice and built light pioneer forest at low elevations. Major forest development seemed to have been constrained by Heinrich event 1 (which was cooler than the LGM in western Europe) tel-00880539, version 1 -6 Nov 2013 MIS 1 -The Bølling/Allerød interstadial (D-O 1) triggered a rapid spread of larch to higher elevations and across the Polish plain leading to a wide distribution range. However, the tree line did not reach the level observed in the Holocene. -The Younger Dryas caused heterogeneous responses ranging from clear decline at high elevations, no significant change or expansion at low elevation. -The rapid temperature rise at the beginning of the Holocene triggered a rapid colonization of high elevation mountain ranges. Subalpine larch forests became established during the first two millennia of the Holocene and appeared to be in dynamic equilibrium with climate. Low elevation populations were successively replaced by other species. The species decreased until its modern distribution range was reached at ~ 5 ka. -Anthropogenic impacts on larch populations became important at the Bronze Age and remained high until Modern Times. Human impacts were varied, ranging from extinctions close to the treeline and increased abundance and colonization of new areas, which led to disequilibrium between larch populations and climate. Recent land abandonment by agriculture should result in continuing larch expansion.Abrupt climate events that (may) have influenced the history of larch the history of larch since the last interglacial shows that the species has persisted during the last 130,000 years close to its modern distribution range while at the same time reacting rapidly to short episodes of climate change. This illustrates the flexibility of larch towards abrupt climate change in the long-term. In the short-term, anthropogenic changes resulted in disequilibrium between larch distribution and climate, with new habitats artificially created quickly occupied by the species and artificial admixture of sometimes distant and divergent populations (see chapter 4). This specificity should be taken into account in future attempts to estimate how the species will respond to forecasted temperature increase.tel-00880539, version 1 -6 Nov 2013Supporting Information

CHAPTER 6 :

 6 Synthesis and perspectives tel-00880539, version 1 -6 Nov 2013In my PhD I precisely documented the consequences of past climate and anthropogenic changes in European larch populations working at multiple temporal and spatial scales and from different perspectives. Before this project information on the history of Larix decidua was very limited due to a lack of high resolution genetic data and of a detailed fossil data compilation. A thorough literature survey on existing genetic markers in European larch and considerations of future needs in larch conservation monitoring and traceability motivated the design of new nuclear microsatellites (Chapter 2 and 3). The highly informative markers I obtained could be applied successfully on a range-wide sample of 45 modern Larix populations. In the genetic analysis I first concentrated on recent plantation history. I could establish a baseline for future studies dealing with translocations (Chapter 4) and reconstruct the ancient genetic structure to be compared with paleontological evidence. In the paleontological part of my work I focused on a detailed range-wide and chronologically precise compilation that could be correlated to past climate and anthropogenic events, albeit I could only briefly address the latter point (Chapter 5). The wealth of precise genetic and paleobotanical data provides a sound basis to discuss the origin of ancient genetic structure of modern Larix decidua populations and to evaluate past climate and anthropogenic impact.

Figure 20 Figure 20

 2020 Figure 20 Studied modern larch populations and names of geographic locations Figure 20 Studied modern larch populations and names of geographic locations cited in the text

  Figure 21 Ancient genetic structure and colonization pathways.

Figure 21

 21 Figure 21 Ancient genetic structure at nuclear microsatellites and mtDNA, and colonization pathways. Red symbols represent early fossil evidence (23.5 -14.7 ka). Black dots correspond to studied modern populations. Haplotypes with an asterisk are minisatellite variants. Dashed line symbolizes introgression of mtDNA.

  with fossils confirms the high quality of the fossil compilation. The detailed historical inferences on recent and ancient history of European larch based on combined and complementary genetic and paleontological results can be seen as a new step in reconstructing tree history. tel-00880539, version 1 -6 Nov 2013 Perspectives Using modern population samples and fossil data on finer spatial and temporal scales will advance our knowledge on processes related to translocations, an important task that has been neglected so far. The improved understanding of European larch history and the accurate description of its genetic structure at presumably neutral molecular markers provide a baseline for investigations on adaptation and selection in the context of future climate change. It can also represent a starting point for forthcoming palaeogenetic studies that could focus on the genetic impact of rapid past climate events. tel-00880539, version 1 -6 Nov 2013 L'enquête lancée par le DSF en 2012 pour caractériser la fréquence et la sévérité des attaques de chancre dans 55 peuplements de mélèze du Massif Central n'a pas permis de mettre en évidence une différence de sensibilité entre les reboisements réalisés avec des provenances Sudètes ou d'Europe de l'Est et ceux réalisés avec des provenances ouest-européennes. Cette absence d'effet est en contradiction avec les études conduites en Europe depuis 1944 sur la variabilité naturelle du mélèze d'Europe (1ère et 2ème expériences internationales IUFRO) qui ont confirmé que les populations d'Europe de l'ouest sont plus sensibles à la maladie que celles d'Europe centrale

  ont par ailleurs démontré expérimentalement que les provenances alpines ont un moins bon comportement à l'inoculation artificielle que celles d'Europe de l'Est. Pour expliquer cette contradiction, nous avons fait l'hypothèse que les certificats de provenance recueillis par les gestionnaires avant chaque reboisement et conservés dans les archives pouvaient comporter des erreurs et qu'ils ne correspondaient pas forcément à l'origine exacte du matériel génétique utilisé. Pour tester cette hypothèse, nous avons utilisé les marqueurs microsatellites spécifiques du Mélèze d'Europe mis au point par Wagner et al (2012), qui permettent de différencier 7 grandes régions de provenance à l'intérieur de l'aire naturelle de Larix decidua. (Annexe 1). Les premiers résultats encore provisoires sont présentés ci après.

Figure 1 -

 1 Figure 1 -Répartition géographique des peuplements de Mélèze dont l'origine génétique a été recherchée par marqueurs.

Figure 2 .Figure 3 :Figure 4 .

 234 Figure 2. Distribution comparée des 192 mélèzes en fonction de leur origine (tous peuplements confondus). ALP : origine Alpes, CE : origine Europe centrale, NA : non documenté ; I : individus intermédiaires.

Figure 5 :

 5 Figure 5 : Classification croisée de l'origine des 39 peuplements de mélèze, informations déclarées su

Table 1

 1 Organelle markers used in Larix decidua ...................................................................

Table 2

 2 Geographical origin of the 18 Larix decidua populations for SSR design .................

Table 3

 3 Characteristics of SSR multiplexes 1 and 2 ................................................................

Table 4

 4 Results of transferability tests in six other Larix species ............................................

Table 5

 5 Counts of mito-nuclear genotype combinations .........................................................

Table S 1

 S Simplex test results for final markers ......................................................................

Table S 2

 S Sampling information of the 45 studied Larix decidua populations ........................

Table S 3

 S ∆k statistics ..............................................................................................................

Table S 4

 S Simulation results .....................................................................................................

Table S 5

 S F IS before and after systematic translocation removal .............................................

Table S 6

 S Fossil sites from 130 -112 ka reported in the literature ........................................

Table S 7

 S Fossil sites from 109 -87 ka reported in the literature ..........................................

Table S 8

 S Fossil from 83 -78.2 ka reported in the literature .................................................

Table S 9

 S Fossil sites from 73.5 -59.4 ka reported in the literature ......................................

Table S 10

 S Fossil sites from 59.4 -27.8 ka reported in the literature ....................................

Table S 11

 S Fossil sites from 27.8 -19 ka reported in the literature .......................................

Table S 12

 S Fossil sites from 19 -14.7 ka reported in the literature .......................................

Table S 13

 S Fossil sites since 14.

Table 1

 1 Organelle markers used in Larix decidua

	Fragment	Type	Analysis method	Spatial scale	Scope	Reference
	TF	Chloroplast	PCR RFLP	Northern hemisphere	Larix phylogeny	Semerikov & Lascoux 2003,
						Semerikov et al. 2003
		Chloroplast	PCR RFLP	Northern hemisphere	Larix phylogeny	Semerikov & Lascoux 2003,
	rpl20trnW					Semerikov et al. 2003
	rpl20trnW	Chloroplast	PCR RFLP	Northern hemisphere	Larix phylogeny	Semerikov & Lascoux 2003,
						Semerikov et al. 2003
	rpl20trnW	Chloroplast	PCR RFLP	Northern hemisphere	Larix phylogeny	Semerikov & Lascoux 2003,
						Semerikov et al. 2003
	trnLV	Chloroplast	PCR RFLP	Northern hemisphere	Larix phylogeny	Semerikov & Lascoux 2003,
						Semerikov et al. 2003
	CS	Chloroplast	PCR RFLP	Northern hemisphere	Larix phylogeny	Semerikov & Lascoux 2003,
						Semerikov et al. 2003
	psbD	Chloroplast	PCR RFLP	Northern hemisphere	Larix phylogeny	Semerikov & Lascoux 2003,
						Semerikov et al. 2003
	Pt9383	Chloroplast	Microsatellite	Northern hemisphere	Larix phylogeny	Semerikov & Lascoux 2003,
						Semerikov et al. 2003
	Pt26081	Chloroplast	Microsatellite	Northern hemisphere	Larix phylogeny	Semerikov & Lascoux 2003,
						Semerikov et al. 2003
	Pt30204	Chloroplast	Microsatellite	Northern hemisphere	Larix phylogeny	Semerikov & Lascoux 2003,
						Semerikov et al. 2003
	ll	Chloroplast	PCR RFLP	Eurasia	Species	Acheré et al. 2004
				L. decidua, L.kaempferi	discrimination
	trnT-trnF	Chloroplast	Sequence	Northern hemisphere	Larix phylogeny	Wei and Wang 2003
	matK	Chloroplast	Sequence	Northern hemisphere	Species	Gros-Louis et al. 2005
				L. decidua, L.kaempferi	discrimination
				L. sibirica, L. laricina
	trnL	Chloroplast	Sequence	Northern hemisphere	Species	Gros-Louis et al. 2005
				L. decidua, L.kaempferi	discrimination
				L. sibirica, L. laricina
	trnT-trnL	Chloroplast	Sequence	Northern hemisphere	Species	Gros-Louis et al. 2005
				L. decidua, L.kaempferi	discrimination
				L. sibirica, L. laricina
	trnL-trnF	Chloroplast	Sequence	Northern hemisphere	Species	Gros-Louis et al. 2005
				L. decidua, L.kaempferi	discrimination
				L. sibirica, L. laricina
	nad5-1/2	Mitochondrial	PCR RFLP	Northern hemisphere	Larix phylogeny	Semerikov & Lascoux 2003,
					Postglacial	Semerikov et al. 2003
					history Siberia
	nad4-3c/4r	Mitochondrial	PCR RFLP	Northern hemisphere	Larix phylogeny	Semerikov & Lascoux 2003,
					Postglacial	Semerikov et al. 2003
					history Siberia

Table 1 :

 1 Organelle markers used in Larix decidua tel-00880539, version 1 -6 Nov 2013

	Fragment	Type	Analysis method	Spatial scale	Scope	Reference
	UBC460	Mitochondrial	PCR RFLP	Northern hemisphere	Larix phylogeny	Semerikov et al. 2006
					Postglacial	Semerikov et al. 2007
					history Siberia
		Mitochondrial	PCR RFLP	Northern hemisphere	Larix phylogeny	Semerikov et al. 2006
	R11				Postglacial	Semerikov et al. 2007
					history Siberia
	C8	Mitochondrial	PCR RFLP	Northern hemisphere	Larix phylogeny	Semerikov et al. 2006
					Postglacial	Semerikov et al. 2007
					history Siberia
	F13	Mitochondrial	Direct PCR	Eurasia	Species	Acheré et al. 2004
				L. decidua, L.kaempferi	discrimination
	Cox1-1	Mitochondrial	Sequence	Northern hemisphere	Species	Gros-Louis et al. 2005
				L. decidua, L.kaempferi	discrimination
				L. sibirica, L. laricina
	matR-1	Mitochondrial	Sequence	Northern hemisphere	Species	Gros-Louis et al. 2005
				L. decidua, L.kaempferi	discrimination
				L. sibirica, L. laricina
	nad1-b/c	Mitochondrial	Sequence	Northern hemisphere	Species	Gros-Louis et al. 2005
				L. decidua, L.kaempferi	discrimination
				L. sibirica, L. laricina
	nad3-1	Mitochondrial	Sequence	Northern hemisphere

L. decidua, L.kaempferi L. sibirica, L. laricina Species discrimination Gros-Louis et al. 2005 nad5-1 Mitochondrial Sequence Northern hemisphere L. decidua, L.kaempferi L. sibirica, L. laricina Species discrimination Gros-Louis et al. 2005 tel-00880539, version 1 -6 Nov 2013 CHAPTER 3

: Two highly informative dinucleotide SSR multiplexes for European larch Published 2012 in Molecular Ecology Resources

tel-00880539, version 1 -6 Nov 2013

Table 2

 2 Geographical origin of the 18 Larix decidua populations for SSR design

	Pop ID Provenance	Country	Lat	Long	Altitude	Trial name	Nb analyzed
								individuals
	4	Fernpass	Austria	47.37	10.90 1150	Münden	24
	8	Semmering	Austria	47.63	15.77 1200	Riedesel	20
	9	Lammerau	Austria	48.11	15.93 610	Münden	21
	10	Neulengbach	Austria	48.05	15.93 560	Winnefeld	21
	21	Pragelato	Italy	45.02	4.93 1900	Riedesel	22
	24	Briançon/Montgenèvre France	44.93	6.72 1730	Riedesel	22
	39	Zabřeh-Dubicko	Czech Republic	49.83	16.97 400	Riedesel	24
	40	Ruda	Czech Republic	49.98	16.90 480	Riedesel	24
	42	Góra Chełmova	Poland	50.80	21.10 347	Sellhorn	24
	43	Bliźyn	Poland	51.07	20.73 330	Sellhorn	24
	72	Rekowo	Poland	54.08	17.46 184	in situ	20
	73	Ruciane Nida	Poland	53.64	21.54 132	in situ	24
	79	Brusturjany	Ukraine	48.42	24.02 1100	in situ	24
	81	Vallée de la Tinée	France	44.09	7.09 1070	in situ	24
	82	Sils Maria	Switzerland	46.43	9.77 2000	in situ	24
	83	Zinal	Switzerland	46.11	7.64 2000	in situ	23
	84	Sinaia Forest	Romania	45.33	25.50 1500	in situ	24
	85	Voineasa Forest	Romania	45.37	23.93 1000	in situ	24

Table 2 .

 2 The cycling conditions for the two multiplexes differed only in the number of PCR cycles and were as follows: an initial step at For subsequent genotype scoring and accurate allele binning based on raw sizes(Guichoux et al. 2011a), we used STRand (http://www.vgl.ucdavis.edu/informatics/download_strand.php)

	tel-00880539, version 1 -6 Nov 2013
	95°C for 15min; then 35 (multiplex1)/30 (multiplex 2) cycles at 94°C for 30s, 56°C for 1min
	and 72°C for 60s; and a final incubation at 60°C for 30min. PCR products were separated on
	3% agarose gels stained with GelRED (Biotium, USA) and diluted 18 times in pure water for
	genotyping with the internal lane size standard LIZ600 on an ABI 3730 (Applied Biosystems,

USA). This optimized protocol was communicated to a collaborating laboratory

(INRA Orléans, France)

to test its robustness. We also tested transferability on the six other larch species from Asia and North America. across different laboratories. This enables accurate and easy data exchange and combined data analyses. A second estimate of error rate was obtained by comparing scoring across readers at a subset of 264 individuals. Two types of errors were distinguished. Type A corresponds to cases where reader 1 calls a genotype as heterozygous and reader 2 as homozygous or vice versa. Type B corresponds to cases where a wrong allele is called by one of the readers. These error types can be corrected after being identified. There can be other kinds of incoherencies between readers, for instance if reader 1 calls a genotype and reader 2 sets missing data or if readers are calling different genotypes due to profile ambiguities which, in contrast to type A and B errors, cannot be corrected and eventually have to be set as missing data. Total allele number (nA), observed (H O ) and expected heterozygosity (H E ), inbreeding index (F IS ) and fixation index (F ST ) were estimated based on 413 individuals from 18 populations from across the range with 20-24 individuals per population (Table

2

) using GENEPOP

[START_REF] Raymond | GENEPOP (Version 1.2): Population Genetics Software for Exact Tests and Ecumenicism[END_REF][START_REF] Rousset | genepop'007: a complete re-implementation of the genepop software for Windows and Linux[END_REF]

) and GENEALEX

(Peakall & Smouse 2006)

. Presence of null alleles was checked on a subset of 12 populations that had a minimum of 19 individuals without missing data using

MICRO-CHECKER v.2.2.0.3 (Van Oosterhout et al. 2006)

. tel-00880539, version 1 -6 Nov 2013

Table 3

 3 Characteristics of SSR multiplexes 1 and 2

	Locus*	Reference	Primer sequences (5´-3´)	Motif	Dye	[C] 1	Size (bp)	NA	HO	HE	FIS	FST	mism †
	Multiplex 1
	Ld31	this study	F: TTGAACTAGGGAGATCCGGC	(AC)18	FAM	2.0	104-147	23	0.751	0.776	0.053	0.074
			R: AATAAAATAGCATTCCATGTGTAGC
	bcLK211	Isoda &	F: CCATTCTCCATAGGTTCATTG	(CT)16	FAM	2.5	174-242	28	0.746	0.745	0.037	0.052
		Watanabe, 2006	R: ATGCTCCTTACTAAGTCAGATACAC
	Ld30	this study	F: TTGTAGGTGTGTATGAAAGTTCTG	(AC)18	VIC	1.68	100-138	17	0.674	0.730	0.100	0.133
			R: TGCCACTCTATTTCCTTAATGCC
	bcLK228	Isoda &	F: CCCTAACCCTAGAATCCAATAA	(AG)18	VIC	0.6	165-215	15	0.869	0.837	-0.013	0.064
		Watanabe, 2006	R: GAGGAAGGCGACAAGTCATT
	Ld50	this study	F: GAAGGCGACTTTACATGCCC	(CA)18	PET	2.2	157-205	19	0.729	0.761	0.066	0.069
			R: TCCATCTTTATGTCTCTTCCATGC
	bcLK189	Isoda &	F: ACCATACGCATACCCAATAGA	(AG)17AT(AG)6	NED	1.2	142-172	15	0.733	0.789	0.096	0.074
		Watanabe, 2006	R: AGTTTTCCTTTCCCACACAAT
	bcLK253	Isoda &	F: AACACCATAGTGCAATGTGC	(AG)17	NED	1.1	195-227	17	0.821	0.806	0.006	0.071
		Watanabe, 2006	R: TCCTCTTGTTGATGCCACTT
	Multiplex 2
	Ld58	this study	F: AATGGCAAGAGCAGCAATCC	(AC)15	FAM	2.1	131-183	25	0.775	0.815	0.077	0.073
			R: TCCAGGAATGATTTATCGAGAGC
	Ld45	this study	F: TGTGGGAGGTATAGCTTGGC	(CA)13	FAM	2.25	198-216	12	0.653	0.723	0.121	0.092
			R: AGTAGGATGGAATGATGGAAACAC
	Ld42	this study	F: TCGTATGCATTGTCCAAATTTCC	(TG)14	VIC	1.8	167-191	9	0.551	0.590	0.084	0.134
			R: TCCAAGTGAGGTCACACGAG
	bcLK263	Isoda &	F: CGATTGGTATAGTGGTCATTGT	(TC)20	PET	3.2	185-259	36	0.801	0.868	0.099	0.056
		Watanabe, 2006	R: CCATCATACCTTCTTGAAGAG
	Ld101	this study	F: ACACCAAGGACTCTCTGACTAC	(AC)12	NED	1.3	179-215	15	0.382	0.395	0.057	0.080
			R: GGTGATTCCAGAAGCAGGTG
	Ld56	this study	F: AGCCATCGTGGTTCTTCTTTG	(AC)16	NED	1.6	219-247	14	0.749	0.769	0.049	0.099
			R: CTTGTAACTGTGCACCCACC
	[C]: primer concentration in the primer premix [µM], N

A : number of alleles, H O : observed heterozygosity, H E : expected heterozygosity, F IS : inbreeding index, F ST : fixation index, *Sequence codes for primers: Ld31: lardec012611, Ld30: lardec001529, Ld50: lardec022835, Ld58: lardec022359, Ld45: lardec024823, Ld42: lardec023929, Ld101: lardec025807, Ld56: lardec023228. †number of Mendelian inconsistencies identified by comparing the genotype of the six half-sib families and their mother

Table 3

 3 Characteristics of multiplexes 1 and 2 based on 413 Larix decidua samples from across its distribution range

	tel-00880539, version 1 -6 Nov 2013

  Nov 2013 homozygote-classes. The analysis of a subsample of 12 populations pointed to 13 cases out of 156 population/marker combinations suggesting the presence of null alleles, involving 7 of the 13 loci (TableS2, Supporting information). The average null allele frequency across all loci per population ranged between 2.4% and 6.6%. As shown in simulation studies of

	Chapuis & Estoup (2007), null alleles with frequencies between 5-8% should have only
	reduced effects on classical estimates of population differentiation. Hence, the genotyping
	results could be used directly with no need of data correction for null allele genotypes.
	However, as recommended by Oddou-Muratorio et al. (2009), correction of null allele
	genotypes remains of interest for some other analyses. This can be done by using the
	correction options implemented in different software (e.g. MICRO-CHECKER).

tel-00880539, version 1 -6

Transferability of the markers was tested in six other Larix species and led to heterogeneous results, although new alleles have been identified in all species (Table

4

). Note that allelic richness estimates for the different species are not directly comparable as we used different sample sizes. In cases of transfer problems we differentiated four different kinds which are given in the table (a-d) and explained in its footnote. While our datasets did not allow detailed conclusions about success of transferability according to phylogenetic relationships, transfer rate was lower for L. sibirica. According to nuclear phylogenies based on AFLP and ITS data

[START_REF] Gernandt | Internal transcribed spacer region evolution in Larix and Pseudotsuga (Pinaceae)[END_REF] Semerikov et al. 2003)

, this species forms a clade with L. decidua and the other Eurasian species. However, other phylogenies based on chloroplast data separate L. sibirica from all other Larix species, in agreement with our data

[START_REF] Qian | Genetic relationships among larch species based on analysis of restriction fragment variation for chloroplast DNA[END_REF] Semerikov et al. 2003)

. tel-00880539, version 1 -6 Nov 2013

Table 4

 4 Results of transferability tests in six other Larix species

	Locus	L. kaempferi*	L. gmelinii*	L. sibirica*	L. laricina †	L. lyallii †	L. occidentalis †
		(12) ‡	(12) ‡	(21) ‡	(10) ‡	(4) ‡	(4) ‡
	Multiplex 1
	Ld31	8	8	5 ¶	5	2	2
	bcLK211	4	4	4	5	2	4
	Ld30	- §	- §	- §	- §	3	- §
	bcLK228	3	7	9	8	4	- † †
	Ld50	12	8	3 ¶	- §	- §	- §
	bcLK189	6	8	6 ¶	- §	2	- §
	bcLK253	4	3	3	8	4	3
	Multiplex 2
	Ld58	-**	- §	6	5	3	3
	Ld45	-**	11	2 ¶	3	1	4
	Ld42	6	7	- §	5	3	6
	bcLK263	5	3	15	12	7	6
	Ld101	7	12	9	2	2	3
	Ld56	9	9	6	2	2	8
	*Eurasian species.
	†American species.
	‡Number of tested individuals in brackets.
	§No amplification.
	¶Amplification in only a subset of individuals.

**Only one locus (either Ld58 or Ld45) was amplified but it was not clear which one. † † Profile ambiguities.

Table 4

 4 Results of transferability in six other Larix species. Numbers correspond to allele counts.

	tel-00880539, version 1 -6 Nov 2013

Table S 1

 S Simplex test results for final markers

						Test 1 Polymorphism		Test 2 Null alleles
	Marker	Sequence name	Repeat Type	Nb Repeat Units	Quality of profile	Nb alleles	Nb individuals	Candidate for validation	Test for further validation	Nb alleles	Nb incoherencies
	Ld_31	lardec012611	(AC)	18	ok	5	7	Y	progeny test	11
	Ld_50	lardec022835	(CA)	18	ok	6	8	Y	progeny test	14
	bcLK211	bcLK211	(CT)	16	ok	10	7	Y	progeny test	17
	bcLK189	bcLK189	(AG) 17 AT(AG) 6	17+6	ok	7	7	Y	progeny test	13
	bcLK228	bcLK228	(AG)	18	ok	7	7	Y	progeny test	16
	Ld_58	lardec022359	(AC)	15	ok	7	7	Y	progeny test	15
	Ld_56	lardec023228	(AC)	16	ok	8	7	Y	progeny test	12
	bcLK253	bcLK253	(AG)	17	ok	9	7	Y	progeny test	11
	bcLK263	bcLK263	(TC)	20	ok	10	7	Y	progeny test	14
	Ld_42	lardec023929	(TG)	14	ok	5	7	Y	progeny test	8
	Ld_45	lardec024823	(CA)	13	ok	6	7	Y	progeny test	7
	Ld_30	lardec001529	(AC)	18	ok	5	7	Y	progeny test	8
	Ld_101	lardec025807	(AC)	12	ok	4	5	Y	progeny test	7

Table S2

 S2

MICRO-CHECKER results (see online resources of Molecular Ecology DOI 10.1111/j.1755-0998.2012.03139.x) tel-00880539, version 1 -6 Nov 2013

Table 5

 5 Counts of mito-nuclear genotype combinations for different spatial scales (gr = group)

	tel-00880539, version 1 -6 Nov 2013

Tab. 5 Counts of mito-nuclear genotype combinations

Table S 2

 S Sampling information of the 45 studied Larix decidua populations

	ID		Lat dd	Lon dd	Alt	Provenance	Country	Trial location	Nb SSR	Nb mt
		1	47.33	14.53	1350 Moederbrugg	AT	Münden		8
		2	47.20	10.67	1100 Schoenwies	AT	Münden		8
		3	46.87	13.35	900 Muehldorf	AT	Riedesel		8
		4	47.37	10.90	1150 Fernpass	AT	Münden		8
		6	47.83	15.15	800 Langau 45	AT	Münden		7
		8	47.63	15.77	1200 Semmering	AT	Riedesel		10
		9	48.05	15.93	610 Lammerau	AT	Münden		9
		10	48.12	15.93	560 Neulengbach	AT	Winnefeld		9
		11	47.50	16.03	1000 Wechselgebiet	AT	Münden		9
		15	46.98	11.97	1100 Bruneck	IT	Münden		10
		16	46.32	11.45	1200 Cavalese	IT	Riedesel		10
		18	46.07	11.32	600 Tenna	IT	Münden, Riedesel		7
	tel-00880539, version 1 -6 Nov 2013	21 23 26 27 39 40 42 43 44 47 49 50 51 53 56 58	45.02 44.78 44.87 46.17 49.83 49.98 50.80 51.07 51.83 47.47 50.05 50.10 49.00 49.13 47.80 46.82	6.93 6.90 6.65 7.83 16.97 16.90 21.10 20.73 20.75 13.10 17.55 17.57 19.88 20.18 13.30 15.17	1900 Pragelato 1560 Embrun, Aiguilles 1400 Briancon, de Villard FR IT FR 1550 St Niklaus CH 400 Zabreh-Dubicko CZ 480 Ruda nad Moravou CZ 347 Gora Chelmova PL 330 Blizyn PL 180 Mala Wies PL 830 Bluehnbachtal AT 550 Krnov CZ 450 Krnov CZ 800 Cierny Vah CZ 1200 Smokovec CZ 700 Fuschlsee AT 800 Deutschlandsberg AT	Riedesel Riedesel Riedesel Riedesel Riedesel Riedesel Sellhorn Sellhorn Riedesel Spießingsho Riedesel Münden Münden Riedesel, Spießingshol Oerrel Münden		9 8 8 8 8 9 9 8 8 8 9 9 7 12 8 9
		59	49.12	20.82	830 Brezovicka	CZ	Münden		10
		66	49.00	19.88	750 Cierny	CZ	Münden		9
		67	48.85	19.12	850 Stare Hory	CZ	Münden, Oerrel		9
		68	48.92	20.08	1400 Liptovska	CZ	Rotenburg		8
		72	54.08	17.46	184 Rekovo	PL	in situ		10
		73	53.64	21.54	132 Ruciane-Nida	PL	in situ		9
		76	44.92	6.72 1500	Briançon,	FR	in situ		8
						Montgenèvre
		77	44.86	6.64 1500	Briançon, de Villard FR	in situ		8
		78	48.65	24.38 423	Solotwyn	UK	in situ		-
		79	48.42	24.02	1100 Brusturjany	UK	in situ		8
		80	48.05	24.18 500	Rahiv	UK	in situ		-
		81	44.09	7.09	1070 Vallée de la Tinée	FR	in situ		7
		82	46.43	9.77	2000 Sils Maria	CH	in situ		8
		83	46.11	7.64	1800 Zinal	CH	in situ		6
		84	45.33	25.50	1500 Sinaia Forest	RO	in situ		9
						District
		85	45.37	23.93	1000 Voineasa Forest	RO	in situ		8
						District
		86	46.92	25.90	1500 Bicaz	RO	in situ		6

Table S 3

 S ∆k statistics

	K	LnP(D)	L(K) sd	delta K
	2	-57237.39 2.13	369.26
	3	-55646.24 2.17	530.14
	4	-55204.74 1335.02	0.19
	5	-55010.99 2805.84	0.35
	6	-53846.35 186.71	4.16
	7	-53458.51 18.51	9.28
	8	-53242.43 117.09	0.63
	9	-53100.08 97.94	0.45

Table S 4

 S Simulation results

	scenario	purebreds opposite group	regionally admixed
	cluster 1 panmixia	0	0.013
	cluster 2 panmixia	0	0.024
	cluster 3 panmixia	0	0.023
	cluster 4 panmixia	0	0.046
	cluster 5 panmixia	0	0.054
	cluster 6 panmixia	0	0.111
	cluster 7 panmixia	0	0.059
	group 1 panmixia	0	0.133
	group 2 panmixia	0	0.123

Table S 5

 S F IS before and after systematic translocation removal

		POP FIS per population POP Fis per population Introduction g78 g58	f58 g80	g6	g42	f6	g43	g8	f8 f43	g9 g44	f9	f44	g10	g86	f10	g11 f86	f11 g84		g79 f84	g39	g85	f39	f85	g40	f40 g73
	tel-00880539, version 1 -6 Nov 2013 tel-00880539, version 1 -6 Nov 2013 tel-00880539, version 1 -6 Nov 2013	POP* bcLK25 bcLK21 bcLK25 bcLK21 High resolution records documenting climate history open new perspectives for investigating g81 f81 g77 f77 g26 f26 g76 f76 g21 f21 g23 -0.006 -0.032 -0.02 -0.038 0.065 -0.014 -0.048 -0.036 -0.083 -0.164 0.031 0.016 0.064 0.017 -0.146 -0.168 -0.184 -0.195 0.138 0.084 0.004 0.102 -0.075 0.087 0.06 0.029 -0.058 -0.069 0.278 0.021 0.034 0.046 0.03 -0.08 -0.117 -0.115 FIS per population bcLK25 -0.054 -0.057 0.002 0.002 0.156 0.199 0.007 0.007 0.12 -0.019 bcLK21 -0.01 -0.091 0.086 0.086 -0.097 -0.172 0.095 0.104 0.115 0.099 0.016 0.02 bcLK22 -0.191 -0.218 0.1 0.1 0.126 0.094 0.019 -0.005 -0.055 -0.178 0.067 Ld30 0.222 0.259 -0.091 -0.091 -0.066 -0.186 -0.019 0.001 0.17 0.026 -0.02 Ld31 -0.12 -0.091 -0.25 -0.25 -0.108 -0.171 -0.011 -0.007 0.185 0.096 -0.013 Ld50 0.321 0.278 0.193 0.193 0.018 -0.266 0.016 -0.041 -0.011 -0.215 0.018 bcLK18 -0.019 0.012 -0.209 -0.209 -0.202 -0.259 -0.048 -0.037 0.121 0.226 0.039 Ld101 -0.054 -0.057 0.098 0.098 0.181 0 0.003 0.017 0.101 -0.032 -0.054 Ld42 0.247 0.284 0.241 0.241 0.117 -0.179 -0.066 -0.029 0.164 -0.093 0.27 Ld56 -0.087 -0.089 -0.124 -0.124 -0.058 -0.125 0.068 0.083 0.048 0.057 0.095 Ld58 0.246 0.215 -0.075 -0.075 0.027 -0.085 -0.002 0.012 0.169 0.121 0.246 bcLK26 -0.014 -0.016 -0.058 -0.058 0.113 0.097 0.043 0.048 0.107 0.086 0.183 Ld45 0.021 -0.053 0.165 0.165 0.162 0.17 -0.082 -0.073 0.194 -0.009 0.045 All 0.034 0.02 -0.002 -0.002 0.024 -0.062 0.005 0.01 0.111 0.019 0.07 POP* g2 f2 g4 f4 g18 f18 g16 f16 g15 f15 g47 FIS per population bcLK25 0.055 -0.003 0.072 -0.069 0.045 0.039 -0.059 -0.114 -0.079 -0.04 0.079 bcLK21 -0.064 -0.095 -0.03 -0.094 0.062 0.018 0.14 -0.049 -0.024 -0.006 0.169 bcLK22 -0.205 -0.195 -0.01 -0.122 -0.035 -0.032 0.033 0.083 0.011 0.04 -0.061 Ld30 -0.021 0 0.031 -0.205 -0.026 -0.211 0.013 0.061 -0.144 -0.19 0.213 Ld31 0.214 0.193 -0.122 -0.193 -0.147 -0.199 -0.03 0.006 -0.032 -0.08 0.021 Ld50 -0.096 -0.075 0.052 0.018 0.071 0.086 -0.026 -0.263 -0.224 -0.382 0.034 bcLK18 0.019 -0.061 -0.078 -0.051 0.012 -0.078 0.069 -0.034 0.089 -0.026 0.075 Ld101 -0.045 -0.066 -0.077 -0.067 0.582 0.458 -0.177 -0.102 0.034 0.028 -0.053 Ld42 -0.136 -0.151 0.121 0.026 0.223 0.39 0.188 -0.077 -0.179 -0.151 0.145 Ld56 -0.085 -0.119 -0.114 -0.167 -0.007 -0.158 0.064 0 0.015 0.04 -0.148 Ld58 0.093 0.031 0.085 0.072 0.084 0.082 0.076 0.109 -0.042 -0.021 0.282 bcLK26 -0.056 -0.052 0.201 0.182 0.151 -0.009 0.093 -0.047 -0.167 -0.189 -0.064 Ld45 -0.177 -0.164 0.019 0.072 0.054 -0.091 0.083 -0.06 0.063 0.107 -0.09 * g: global sample, f: final sample after removal; Fis was not recalculated if >50% of the individuals had been removed (populations 42, 51) f23 g83 f83 g27 f27 g82 f82 0.08 0.016 0.011 -0.087 -0.025 0.011 -0.082 0.243 0.327 0.282 0.038 -0.036 -0.16 -0.117 -0.07 0.004 0.015 -0.006 -0.028 0.013 -0.139 0.287 0.259 -0.038 -0.015 0.085 0 0.156 0.156 -0.062 -0.143 -0.078 -0.073 -0.09 -0.074 -0.074 0.078 0.012 -0.048 -0.085 0.027 -0.003 -0.003 -0.217 -0.313 -0.121 0.018 -0.221 -0.221 -0.054 -0.188 0.25 -0.022 0.068 0.068 -0.228 -0.255 -0.018 0.004 0.134 0.134 0.159 0.126 0.02 -0.04 0.059 0.059 -0.143 -0.222 0.317 -0.008 0.056 0.056 0.068 0.12 -0.026 0.259 0.003 0.003 -0.082 -0.063 0.062 0.152 -0.166 -0.166 -0.123 -0.173 0.021 0.019 0.197 0.049 0.049 0.077 0.13 0.127 0.164 0.167 0.162 0.162 0.086 0.113 0.021 0.025 0.021 0.188 0.188 -0.004 -0.005 0.045 0.066 0.05 0.029 0.029 -0.032 -0.066 0.046 0.029 f47 g56 f56 g3 f3 g1 f1 0.057 0.143 0.16 0.035 -0.036 0.046 0.027 0.044 0.073 0.091 0.034 0.025 -0.026 -0.106 -0.06 0.006 0.012 0.227 0.299 -0.198 -0.348 0.345 -0.062 -0.077 0.083 0.015 -0.073 -0.092 -0.014 -0.025 0.005 0.107 0.128 0.065 0.112 -0.003 -0.003 -0.02 0.18 0.02 0.132 0.081 0.111 0.079 0.056 0.261 0.164 0.133 0.125 -0.067 -0.116 -0.096 0.29 -0.014 -0.054 -0.054 0.034 -0.143 -0.151 0.096 -0.008 0.031 -0.02 -0.071 -0.156 -0.157 0.023 -0.032 0.156 0.081 0.304 -0.007 -0.073 -0.032 -0.024 0.109 0.106 -0.106 -0.016 -0.024 0.028 0.05 0.073 -0.221 -0.102 -0.083 0.428 0.434 -0.008 0.091 0.054 bcLK26 0.203 0.26 -0.042 -0.079 0.035 -0.057 0.032 0.074 -0.048 -0.058 0.037 0.089 -0.051 0.448 0.516 0.208 0.154 Ld45 -0.204 -0.195 -0.009 -0.026 -0.037 -0.071 0.229 0.118 0.287 0.296 -0.008 -0.135 0.025 0.02 -0.091 0.041 0.002 All -0.001 -0.002 0.015 -0.005 -0.006 -0.03 0.038 -0.019 0.064 0.052 0.079 0.053 0.1 0.073 0.047 0.065 0.036 POP g49 f49 g50 f50 g72 F72 g66 f66 g67 f67 g68 f68 g51 g53 f53 g59 f59 FIS per population bcLK25 -0.153 -0.156 -0.086 -0.039 -0.011 -0.062 0.109 0.036 -0.065 -0.078 -0.12 -0.133 -0.042 -0.047 -0.015 0.02 0.023 bcLK21 0.066 -0.012 0.036 -0.043 0.272 0.229 -0.011 0.022 -0.027 -0.037 0.002 0.056 -0.084 -0.106 -0.078 0.055 0.022 bcLK22 -0.098 -0.082 0.003 -0.021 -0.038 -0.106 -0.073 -0.139 0.148 0.224 0.048 0.082 -0.006 -0.011 -0.07 -0.07 -0.068 Ld30 -0.121 -0.129 0.077 -0.021 -0.072 -0.114 -0.175 -0.148 -0.082 -0.103 0.075 0.021 0.029 0.228 0.27 -0.07 -0.134 Ld31 -0.094 -0.095 0.045 0.092 0.11 0.13 0.19 0.044 0.061 0.067 0.051 0.081 -0.021 0.062 -0.071 -0.006 0.08 Ld50 -0.004 0.025 0.058 0.015 0.173 0.16 0.225 0.366 0.031 -0.012 0.199 0.199 0.011 0.189 0.223 0.1 -0.099 bcLK18 0.073 0.02 0.112 0.091 0.019 0.018 -0.034 -0.12 0.035 -0.011 0.01 0.036 -0.025 -0.044 0.002 -0.001 -0.101 Ld101 0.012 0.089 0.021 -0.022 0.017 0.142 -0.138 -0.036 0.429 0.521 -0.162 -0.145 -0.034 0.066 0.084 0.11 0.262 Ld42 0.063 -0.006 0.129 0.129 -0.071 0.015 0.284 0.392 0.01 -0.068 0.207 0.171 -0.106 0.134 0.214 0.241 0.273 Ld56 0.106 0.098 -0.099 -0.244 0.203 0.216 0.292 0.514 0.36 0.202 0.027 0.004 0.136 0.179 0.252 0.153 0.098 Ld58 0.174 0.163 0.002 0.026 0.08 0.008 0.021 0.077 0.019 -0.026 -0.052 -0.032 0.013 0.074 0.133 0.08 -0.021 bcLK26 0.554 0.524 0.299 0.293 0.411 0.479 0.345 0.568 0.114 0.005 0.232 0.277 0.084 0.08 0.05 0.19 0.282 Ld45 -0.193 -0.291 0.146 0.269 -0.093 -0.13 0.108 0.2 -0.026 -0.011 -0.012 0.03 0.094 0.011 0.121 0.181 0.287 All 0.037 0.016 0.058 0.043 0.088 0.077 0.098 0.149 0.071 0.04 0.046 0.059 0.009 0.062 0.085 0.073 0.063 bcLK26 0.026 0.283 0.154 0.111 0.241 -0.045 -0.051 -0.118 -0.115 0.331 0.212 -0.015 -0.021 Ld45 0.006 -0.017 0.041 0.195 0.223 -0.056 -0.098 0.068 -0.162 0.432 0.424 0.207 0.137 0.121 All 0.007 0.089 0.068 0.134 0.06 -0.015 -0.033 0.056 0.03 0.137 0.139 0.062 0.062 0.023 * g: global sample, f: final sample after removal; Fis was not recalculated if >50% of the individuals had been removed (populations 42, 51) 0.016 -0.157 Ld58 -0.004 0.026 -0.052 -0.112 0.051 0.054 0.067 -0.063 -0.138 -0.138 0.048 -0.074 0.321 0.17 0.287 0.048 0.051 Ld58 0.128 0.058 -0.003 0.03 -0.038 -0.043 -0.037 0.018 -0.04 -0.017 -0.041 0.028 0.104 0.208 -0.025 Ld56 -0.001 -0.007 -0.074 -0.088 -0.219 -0.204 0.06 -0.047 -0.072 -0.061 0.162 0.136 0.196 0.01 -0.068 0.19 0.193 Ld56 -0.045 0.052 0.051 0.382 0.275 0.036 0.013 0.248 0.11 0.183 0.226 0.085 0.096 0.01 0.299 Ld42 0.142 -0.109 0.023 -0.027 0.217 0.227 0.366 0.328 0.018 -0.063 0.16 0.018 -0.139 0.108 -0.013 -0.024 -0.167 Ld42 -0.029 -0.015 0.104 0.243 0.125 -0.195 -0.278 0.079 0.032 -0.12 -0.164 0.014 0.059 0.093 -0.098 Ld101 0.363 0.431 -0.04 -0.027 -0.023 0 -0.128 -0.172 -0.022 -0.026 0.25 0.474 0.29 0.108 0.184 0.108 0.138 Ld101 -0.228 0.082 0.38 0.092 -0.213 -0.157 -0.25 -0.136 -0.143 0.135 0 -0.038 0.005 0.1 0.009 bcLK18 -0.235 -0.226 0.134 0.203 0.135 0.078 0.203 0.084 0.007 0.007 -0.023 -0.054 0.12 0.018 -0.108 0.148 0.183 bcLK18 0.095 0.2 -0.027 0.111 0.169 -0.013 -0.055 0.082 0.065 0.28 0.337 0.08 0.197 -0.084 0.224 Ld50 0.065 0.085 0.025 0.007 -0.184 -0.152 0.088 -0.013 0.009 0.043 0.087 0.202 0.14 0.169 0.12 -0.064 -0.113 Ld50 -0.044 0.12 0.233 0.155 -0.025 0.042 0.002 0.125 0.141 -0.107 -0.094 0.099 0.131 -0.087 -0.115 bcLK22 -0.11 -0.106 -0.008 -0.027 -0.072 -0.059 -0.051 -0.046 0.254 0.297 -0.017 -0.109 -0.072 -0.034 -0.162 0.09 0.036 Ld30 -0.035 -0.056 0.07 0.094 0.025 0.053 -0.064 -0.148 0.246 0.255 0.036 -0.061 0.254 0.062 0.26 0.1 0.05 Ld31 -0.008 0.04 0.085 0.027 0.049 -0.021 -0.044 -0.118 0.179 0.133 0.111 0.262 -0.002 -0.129 -0.309 0.111 0.082 bcLK22 0.001 0.121 0.093 0.111 0.058 -0.01 0.005 0.061 0.07 -0.128 -0.146 -0.041 -0.092 -0.045 Ld30 -0.042 0.153 0.021 0.155 -0.174 0.279 0.375 0.204 0.239 0.185 0.353 0.192 0.148 -0.062 Ld31 -0.143 -0.062 0.064 0.049 -0.052 -0.073 -0.06 0.302 0.258 0.253 0.259 0.217 0.127 -0.04 forests dynamics
		All	-0.032	-0.053	0.022	-0.03		0.058	-0.007	0.042	-0.03		-0.051	-0.066	0.056	0.032	-0.011	-0.014		0.125		0.089	0.036	0.014
		* g: global sample, f: final sample after removal; Fis was not recalculated if >50% of the individuals had been removed (populations 42, 51)

Table S 6

 S Fossil sites from 130 -112 ka reported in the literature

	Site	Latdd Londd	Alt m asl Country	M*	P*	Reference
	Amersfoort 3	52.16	5.39	<100	NL	X	0	Zagwijn (1961)
	Aschenhütte	51.68 10.30	240	DE	0	1	Ricken & Grüger (1988)
	Site Azzano Decimo Amersfoort 3 Barendorf 2 and 3 Aschenhütte Brørup Azzano Decimo Dziernakowo Barendorf 2 and 3 Füramoos Brørup Gondiswil Dziernakowo Gröbern Füramoos Groß Todtshorn	Latdd Londd 45.89 12.71 52.16 5.39 53.23 10.52 51.68 10.30 55.48 9.02 45.89 12.71 53.10 23.62 53.23 10.52 47.98 9.88 55.48 9.02 47.15 7.87 53.10 23.62 51.69 12.46 47.98 9.88 53.21 9.78	Alt m asl Country 10 IT <100 NL 74 DE 240 DE <100 DK 10 IT 164 PL 74 DE 662 DE <100 DK 640 CH 164 PL 107 DE 662 DE NA DE	M* X X X 0 0 X X X X 0 0 X 0 X 0	P* 0 0 1 0 1 0 1 0 1 0 1 0 1 0 1	Pini et al. (2009) Reference Freund et al. (1997) Zagwijn (1961) Andersen (1961) Ricken & Grüger (1988) Kupryjanowicz (2008) Pini et al. (2009) Müller (2000, 2003) Freund et al. (1997) Wegmüller (1992) Andersen (1961) Litt (1990, 1994), Mai (1990a) Kupryjanowicz (2008) Müller (2000, 2003) Caspers (1997), Freund (1997)
	Gondiswil Horoszki Duze	47.15 52.26 23.01 7.87	640 180	CH PL	0 1	1 1	Wegmüller (1992) Granoszewski (2003)
	Gröbern Hunteburg	51.69 52.44	12.46 8.28	107 <100	DE DE	0 0	0 1	Litt (1990, 1994), Mai (1990) Hahne (1994b)
	Groß Todtshorn Ioannina	53.21 39.75 20.85 9.78	<100 470	DE GR	0 X	0 0	Caspers (1997), Freund (1997a) Tzedakis (2000), Tzedakis et al. (2002)
	Horoszki Duze Jammertal	52.26 48.10	23.01 9.73	180 578	PL DE	1 X	1 1	Granoszewski (2003 Müller (2000, 2001)
	Ioannina Keller	39.75 54.08	20.85 9.43	470 <100	GR DE	X X	0 1	Tzedakis (2000), Tzedakis et al. (2002) Menke (1970)
	Jammertal Kittlitz	48.10 51.84 13.93 9.73	578 170	DE DE	X X	1 1	Müller (2001, 2000) Erd (1973)
	Keller La Grande Pile	54.08 47.73	9.43 6.50	<100 330	DE FR	X X	0 1	Menke (1970) Woillard (1975, 1978),
	Kittlitz	51.84	13.93	170	DE	X	1	Erd (1973) de Beaulieu & Reille (1992a)
	La Grande Pile Lac du Bouchet (D)	47.73 44.92	6.50 3.78	330 1200	FR FR	X X	0 0	Woillard (1975, 1978), Reille & de Beaulieu (1990)
	Lago Grande di Monticchio	40.93 15.58	656	IT	X	0	de Beaulieu & Reille (1992a) Allen et al. (1999)
	Lac du Bouchet (D) Lake Fimon	44.92 45.47 11.53 3.78	1200 23	FR IT	X X	0 1	Reille & de Beaulieu (1990) Pini et al. (2010)
	Lago Grande di Monticchio Les Echets	40.93 45.87	15.58 4.92	656 267	IT FR	X X	0 0	Allen et al. (1999) de Beaulieu & Reille (1984, 1989)
	Lake Fimon Lunteren	45.47 52.09	11.53 5.62	23 <100	IT NL	X X	1 0	Pini et al. (2010) Zagwijn (1961)
	Les Echets MD04-2845	45.87 45.35 -5.22 4.92	267 -4100	FR NA	X X	0 0	de Beaulieu & Reille (1984, 1989) Sánchez Goñi et al. (2008)
	Lunteren MD95-2042	52.09 37.80 -10.17 5.62	<100 -3148	NL NA	X X	0 0	Zagwijn (1961) Sánchez Goñi et al. (1999)
	Margreteberg Mondsee	56.77 47.85 13.35 12.74	<100 540	SE AT	X 0	1 1	Påsse et al. (1988) Klaus (1975), Drescher-Schneider (2000),
	MD04-2845	45.35	-5.22	-4100	NA	X	0	Sánchez Goñi et al. (2008) Oeggl & Unterfrauner (2000)
	MD95-2042 Neheim-Hüsten	37.80 -10.17 51.45 7.97	-3148 <100	NA DE	X 0	0 0	Sánchez Goñi et al. (1999) Teunissen et al. (1972)
	Mondsee Neuenhausen-Veldhausen	47.85 52.52	13.35 6.99	540 <100	AT DE	0 X	1 1	Klaus (1975), Drescher-Schneider (2000), Freund (1997b)
	Odderade 5	54.14	9.19	<100	DE	0	1	Oeggl & Unterfrauner (2000) Averdieck (1967)
	Neheim-Hüsten Oerel	51.45 53.48	7.97 9.06	<100 <100	DE DE	N 0	0 1	Teunissen et al. (1972) Behre & Lade (1986), Behre et al. (2005)
	Neuenhausen-Veldhausen Osterwanna I and II	52.52 53.74	6.99 8.80	<100 <100	DE DE	X X	0 1	Freund (1997b) Behre (1974)
	Odderade 5 Padul	54.14 37.00 -3.07 9.19	<100 785	DE ES	0 X	0 0	Averdieck (1967) Pons & Reille (1988)
	Oerel Quakenbrück	53.48 52.67	9.06 7.96	<100 <100	DE DE	0 0	0 1	Behre & Lade (1986), Behre et al. (2005) Hahne et al. (1994a)
	Osterwanna I and II Rederstall I and II	53.74 54.23	8.80 9.21	<100 <100	DE DE	X X	0 1	Behre (1974) Menke & Tynni (1984)
	Quakenbrück Ribains	52.67 44.84	7.96 3.82	<100 1192	DE FR	0 X	0 0	Hahne et al. (1994a) de Beaulieu & Reille (1992b)
	Rederstall I and II Samerberg 1973	54.23 47.75 12.20 9.21	<100 612	DE DE	X 1	1 1	Menke & Tynni (1984) Grüger (1979)
	Ribains Sokli	44.84 67.08 29.18 3.82	1192 220	FR FI	X X	0 1	de Beaulieu & Reille (1992b) Helmens et al. (2000, 2007),
	Samerberg 1973	47.75	12.20	612	DE	1	1	Grüger (1979) Engels et al. (2010)
	Sokli Solniki	67.08 53.50 23.20 29.18	220 143	FI PL	X X	1 1	Helmens et al. (2000, 2007), Kupryjanowicz (2008)
	Stenberget	55.52 13.55	<100	SE	X	1	Engels et al. (2010) Berglund & Lagerlund (1981)
	Solniki Tenagi Philippon II	53.50 41.02 24.03 23.20	143 40	PL GR	X X	1 0	Kupryjanowicz (2008) Wijmstra (1969)
	Stenberget Valle di Castiglione	55.52 41.88 12.76 13.55	<100 44	SE IT	X X	1 0	Berglund & Lagerlund (1981) Follieri et al. (1988, 1998)
	Tenagi Philippon II Wladyslawow	41.02 52.13 18.47 24.03	40 <100	GR PL	X 0	0 1	Wijmstra (1969) Tobolski (1986, 1991)
	Valle di Castiglione Zgierz-Rudunki	41.88 51.84 19.42 12.76	44 200	IT PL	X 0	0 1	Follieri et al. (1988, 1998) Jastzebska-Mamelka (1985)
	Wladyslawow	52.13	18.47	<100	PL	0	1	Tobolski (1986, 1991)
	Zgierz-Rudunki	51.84	19.42	200	PL	0	1	Jastzebska-Mamelka (1985)

*1: presence, 0: absence, X: not investigated; M: Macrofossils; P: Pollen tel-00880539, version 1 -6 Nov 2013 *1: presence, 0: absence, X: not investigated; M: Macrofossils, P: Pollen

Table S 7

 S Fossil sites from 109 -87 ka reported in the literature

	tel-00880539, version 1 -6 Nov 2013

Table S 8

 S Fossil from 83 -78.2 ka reported in the literature

		Site	Latdd	Londd	Alt m asl Country	M*	P*	Reference
		Azzano Decimo	45.89	12.71	10	IT	X	0	Pini et al. (2009)
		Dziernakowo	53.10	23.62	164	PL	X	1	Kupryjanowicz (2008)
		Füramoos	47.98	9.88	662	DE	X	1	Müller (2000, 2003)
		Gondiswil	47.15	7.87	640	CH	0	1	Wegmüller (1992)
		Gröbern	51.69	12.46	107	DE	1	1	Litt (1990, 1994), Mai (1990)
		Groß Todtshorn	53.21	9.78	NA	DE	0	1	Caspers (1997), Freund (1997a)
		Horoszki Duze	52.26	23.01	180	PL	1	1	Granoszewski (2003)
		Hunteburg	52.44	8.28	<100	DE	0	1	Hahne et al. (1994b)
		Ioannina	39.75	20.85	470	GR	X	0	Tzedakis (2000), Tzedakis et al. (2002)
		Jammertal	48.10	9.73	578	DE	X	1	Müller (2000, 2001)
		Kittlitz	51.84	13.93	170	DE	X	1	Erd (1973)
		La Grande Pile	47.73	6.50	330	FR	X	1	Woillard (1975, 1978),
									de Beaulieu & Reille (1992a)
		Lac du Bouchet (D)	44.92	3.78	1200	FR	X	0	Reille & de Beaulieu (1990)
		Lagaccione	42.57	42.57	355	IT	X	0	Follieri et al. (1998), Magri (1999)
	tel-00880539, version 1 -6 Nov 2013	Lago Grande di Monticchio Lake Fimon Les Echets MD04-2845 MD95-2042 Mondsee Neheim-Hüsten Odderade 5 ODP site 976 Oerel Padul Quakenbrück Rederstall I and II	40.93 45.47 45.87 45.35 37.80 47.85 51.45 54.14 36.20 53.48 37.00 52.67 54.23	15.58 11.53 4.92 -5.22 -10.17 13.35 7.97 9.19 -4.30 9.06 -3.07 7.96 9.21	656 23 267 -4100 -3148 540 <100 <100 -1108 <100 785 <100 <100	IT IT FR --AT DE DE DE ES DE DE	X X X X X 1 0 0 X 1 X 0 X	0 1 1 0 0 1 0 1 0 1 0 1 1	Allen et al. (1999) Pini et al. (2010) de Beaulieu & Reille (1984, 1989) Sánchez Goñi et al. (2008) Sánchez Goñi et al. (1999) Klaus (1975), Drescher-Schneider (2000), Oeggl & Unterfrauner (2000) Teunissen et al. (1972) Averdieck (1967) Combourieu-Nebout et al. (2002) Behre and Lade (1986), Behre et al. (2005) Pons & Reille (1988) Hahne et al. (1994a) Menke & Tynni (1984)
		Ribains	44.84	3.82	1192	FR	X	0	de Beaulieu & Reille (1992b)
		Samerberg 1973	47.75	12.20	612	DE	1	1	Grüger (1979)
		Sokli	67.08	29.18	220	FIN	X	0	Helmens et al. (2000, 2007),
									Engels et al. (2010)
		Tenagi Philippon II	41.02	24.03	40	GR	X	0	Wijmstra (1969)
		Valle di Castiglione	41.88	12.76	44	IT	X	0	Follieri et al. (1988, 1998)
		Wladyslawow	52.13	18.47	<100	PL	0	1	Tobolski (1986, 1991)
		Zgierz-Rudunki	51.84	19.42	200	PL	1	1	Jastzebska-Mamelka (1985)
		*1: presence, 0: absence, X: not investigated; M: Macrofossils, P: Pollen

Table S 9

 S Fossil sites from 73.5 -59.4 ka reported in the literature

		Site	Latdd Londd Alt m asl Country	M*	P*	Reference
		Abric Romaní	41.53	1.68	350	-	X	0	Burjachs & Julià (1994)
		Azzano Decimo	45.89	12.71	10	IT	X	0	Pini et al. (2009)
		Füramoos	47.98	9.88	662	DE	X	1	Müller (2000, 2003)
		Gondiswil	47.15	7.87	640	CH	0	1	Wegmüller (1992)
		Ioannina	39.75	20.85	470	GR	X	0	Tzedakis (2000), Tzedakis et al. (2002)
									Woillard (1975, 1978),
		La Grande Pile	47.73	6.50	330	F	X	0	de Beaulieu & Reille (1992a)
		Lac du Bouchet (D)	44.92	3.78	1200	F	X	0	Reille & de Beaulieu (1990)
		Lagaccione	42.57	42.57	355	IT	X	0	Follieri et al. (1998), Magri (1999)
		Lago Grande di Monticchio	40.93	15.58	656	IT	X	0	Allen et al. (1999)
		Lake Fimon	45.47	11.53	23	IT	X	1	Pini et al. (2010)
		Les Echets	45.87	4.92	267	F	X	1	de Beaulieu & Reille (1984, 1989)
		MD04-2845	45.35	-5.22	-4100	X	X	0	Sánchez Goñi et al. (2008)
		MD95-2039	40.58 -10.35	-3381	X	X	0	Roucoux et al. (2001)
		MD95-2042	37.80 -10.17	-3148	X	X	0	Sánchez Goñi et al. (1999)
	tel-00880539, version 1 -6 Nov 2013	Megali Limni ODP site 976 Oerel Padul Polanów Samborzecki Ribains Samerberg 1973 Sokli Stracciacappa Tenagi Philippon II Valle di Castiglione *1: presence, 0: absence, X: not investigated; M: Macrofossils, P: Pollen 39.10 26.32 323 X 36.20 -4.30 -1108 -53.48 9.06 <100 DE 37.00 -3.07 785 ES 50.63 21.67 165 PL 44.84 3.82 1192 F 47.75 12.20 612 DE 67.08 29.18 220 FI 42.13 12.32 220 X 41.02 24.03 40 GR 41.88 12.76 44 IT	X X 0 X X X 0 X X X X	0 0 0 0 1 0 0 0 0 0 0	Margari et al. (2007, 2009) Combourieu-Nebout et al. (2002) Behre & Lade (1986), Behre et al. (2005) Pons & Reille (1988) Komar et al. (2009) de Beaulieu & Reille (1992b) Grüger (1979) Helmens et al. (2000, 2007), Engels et al. (2010) Giardini (2007) Wijmstra (1969) Follieri et al. (1988, 1998)

Table S 10

 S Fossil sites from 59.4 -27.8 ka reported in the literature

		Site Site	Latdd Latdd Londd Alt m a.s.l. Reference Londd Alt m asl Country	M*	P*	Ch*	Reference
		Sokli Totenmoos	67.08 46.55	29.18 11.01	220 1718 Heiss et al. (2005) FI X	0	X	Helmens et al. (2000, 2007),
		Site Abric Romaní Azzano Decimo Baggagera Pian di Gembro Spadzista Street Simplon_Hobschensee Stracciacappa Palù bei Edolo Luci Stránska skàlà Taul Zanoguti	Latdd 41.53 45.89 45.74 46.20 50.05 46.25 42.13 46.18 46.27 49.18 45.33	Londd Alt m asl Country 1.68 350 -12.71 10 IT 9.39 260 IT 10.17 1350 Pini (2002), Vescovi et al. (2007) M* P* X 0 X 0 X 0 19.92 <100 PL X X 8.02 2017 Lang & Tobolski (1985) 12.32 220 -X 0 10.33 660 Gehring (1997) 25.75 1079 Tantau et al. (2003), Feurdean et al. (2007) Ch* X X 1 1 X 16.67 <100 CZ X X 1 22.80 1840 Farcas et al. (1999), Feurdean et al. (2007)	Reference Burjachs & Julià (1994) Pini et al. (2009) Engels et al. (2010) Musil (2003) Giardini (2007) Maspero (1996) Musil (2003), Damblon et al.
		Bialy Kościól Col di Val Bighera	50.72 46.28	17.03 10.31	180 2087 Gehring (1997) PL 1	1	X	Komar et al. (2009) (1996), Willis &
		Bohunice Passo del Tonale Tenagi Philippon II Kis-Mohos Tó Týn n. Bečvou Jezor-Jaworzno Alpe de Venosc Valle di Castiglione Autertal	49.17 46.31 41.02 48.41 49.51 50.24 44.99 41.88 46.86	16.58 10.58 24.03 20.41 17.62 19.20 6.12 12.76 13.93	<100 1883 Gehring (1997) CZ X 40 GR X 310 Willis et al. (1997), Willis et al. (2000) X 1 0 X 370 CZ X 1 X 255 Szczepanek & Stachowicz-Rybka (2004) 1644 Coûteaux (1962), Kral (1979) 44 IT X 0 X 1450 Fritz (1964), Kral (1979)	Svoboda & Svoboda (1985), Damblon et al. (1996), Musil (2003), van Andel (2004) Wijmstra (1969) Jankovská pers. comm. Willis & van Andel (2004) Follieri et al. (1988, 1998)
		Brzeźnica A Věstonice I Boecklweiher	50.10 48.87 47.64	21.48 16.63 12.96	130 255 610 Kral (1979) PL CZ	1 X	1 X	X 1	Mamakowa & Starkel (1974) Opravil (1994), Damblon et al.
		Brzeźnica B Bulhary Erd Brigels Edlbacher Moor Feichtauer Moor Füramoos Fusine Věstonice II Kendlmühlfilz	50.10 48.83 47.48 46.77 47.73 47.80 47.98 46.50 48.88 47.80	21.48 16.75 18.91 9.06 14.34 14.32 9.88 13.65 16.63 12.44	130 166 200 1520 Müller (1972) PL 1 CZ 1 HU X 610 Kral (1979) 1340 Kral (1979) 662 DE X 920 Kral (1982) 255 CZ X 525 Schmeidl (1977), Kral (1979) 1 1 X 1 X	X X 1 X 1	Mamakowa & Starkel (1974) Rybnikova & Rybnicek (1991) (1996), Damblon (1997), Slaviková-Veslá (1950), Musil (2003) Willis& van Andel (2004) Müller (2000, 2003) Mason et al. (1994), Opravil
	tel-00880539, version 1 -6 Nov 2013 tel-00880539, version 1 -6 Nov 2013 tel-00880539, version 1 -6 Nov 2013	Ioannina Istálloskö Jablůnka Kulna La Grande Pile Lac du Bouchet (D) Lagaccione Lago della costa Lago Grande di Monticchio Lake Banyoles Lake Fimon Lake Xinias Les Echets Seekarmoor Keutschachersee Laghetto di Somdogna Malga di Lussari Voka Willendorf --*1: presence, 0: absence, X: not investigated; M: Macrofossils, P: Pollen, Ch: Charcoals 39.75 20.85 470 GR X 48.16 20.42 330 HU X 49.38 17.95 350 CZ X 49.51 16.61 300 CZ X 47.73 6.50 330 FR X 44.92 3.78 1200 FR X 42.57 42.57 355 IT X 45.27 11.74 7 IT 1 40.93 15.58 656 IT X 42.13 2.75 173 -X 45.47 11.53 23 IT X 39.05 22.27 500 -X 45.87 4.92 267 FR X 47.25 13.39 1785 Kral (1979) 46.59 14.16 508 Kral (1979) 46.47 13.37 1442 Kral (1982) 46.50 13.55 1554 Kral (1982) 59.41 27.60 22 EE X 48.32 15.40 220 AU X 47.17 18.00 <100 HU X 48.33 21.25 200 HU X Moosham 47.66 13.79 1030 Kral (1979) Poelland 46.63 13.53 1050 Fritz (1973), Kral (1979) 0 X 1 X 0 0 0 1 0 0 1 0 1 1 X X X Pokljuka Sijec Julische Alpen 46.36 14.04 1170 Sercelj (1971), Kral (1979) Rödschitzer Moos 47.56 13.92 775 Kral (1979) Seemoos 47.08 13.78 1700 Bortenschlager (1964), Kral (1979) Segner 46.89 9.19 1880 Müller (1972), Kral (1979) Sur Oberhalbstein 46.52 9.63 1780 Heitz (1975), Kral (1979) Tourbière d'Hinter-Hoehi 47.17 9.16 1420 Hoffmann & Grobety (1968), Kral (1979) X 1 X 1 X X X X X X X X X X 1 1 1 Wansenmoos beim Zellhof 48.01 13.10 500 Krisai (1975), Kral (1979) Wiegenwald Stubachtal 47.22 12.61 1720 Kral (1979) Baumgartl 47.50 12.97 1720 Mayer (1966), Kral (1979) Cavazzo 46.37 13.04 270 Kral (1979) Cavazzo-Vuarbes 46.38 13.02 270 Kral (1982) Eben 47.42 13.39 850 Kral (1979) Egelsee ob Diemtigen 46.63 7.55 1000 Welten (1952), Kral (1979) Malga Varmost 46.51 12.59 1480 Kral (1982) Puergschachener Moor 47.58 14.33 632 Kral (1979) Schwimmend Moos 47.68 12.87 1370 Mayer (1966), Kral (1979)	Tzedakis (2000), Tzedakis et al. (2002) Musil (2003) Jankovska (2003), Jankovská & Pokroný (2008) Musil (2003) Woillard (1975, 1978), de Beaulieu & Reille (1992a) Reille & de Beaulieu (1990) Follieri et al. (1998), Magri (1999) Kaltenrieder et al. (2009) Allen et al. (1999) Pérez-Obiol & Julia (1994) Pini et al. (2010) Bottema (1979) de Beaulieu & Reille (1984, 1989) (1994), Damblon et al. (1996), Damblon (1997), Musil (2003), Willis & van Andel (2004) Bolikhovskaya & Molodkov (2007) Haesaerts et al. (1996), Damblon & Haesaerts (1997), Damblon (1997), Willis& van Andel (2004) Geyh et al. (1969), Willis & van Andel (2004) Krolopp (1977), Willis & van Andel (2004)
		MD04-2845 Weidfilz	45.35 47.78	-5.22 11.43	-4100 595 Gross (1965), Kral (1979) -X 0	X	Sánchez Goñi et al. (2008)
		MD95-2039 Lago Perso	40.58 44.91	-10.35 6.80	-3381 2000 Blarquez et al. (2010) -X	0	X	Roucoux et al. (2001)
		MD95-2042 Passo di Pramollo MD95-2043 Pieve Tesino Megali Limni Hirschbichl Lac du Loup	37.80 46.56 36.14 46.04 39.10 46.87 45.19	-10.17 13.28 -2.62 11.81 26.32 12.36 6.54	-3148 1551 Kral (1982) --1841 -1240 Kral (1980) 323 -2140 Oeggl & Wahlmüller (1994) X 0 X 0 X 0 2035 Blarquez et al. (2009)	X X X	Sánchez Goñi et al. (1999) Sánchez Goñi et al. (2002) Margari et al. (2007, 2009)
		Navarrés Lej da San Murezzan	39.10 46.49	-0.68 9.84	225 1768 Gobet et al. (2005) -X	0	X	Carrión & Van Geel (1999)
		ODP site 976 Gouillé Loéré	36.20 46.15	-4.30 7.36	-1108 2503 Tinner and Theurillat (2003) X 0	X	Combourieu-
		Lengi Egga	46.36	7.96	-2557 Tinner and Theurillat (2003)		Nebout et al. (2002)
		Padul	37.00	-3.07	785	ES	X	0	X	Pons & Reille (1988)
		Pavlov	48.87	16.67	270	CZ	X	1	1	Opravil (1994), Damblon et al.
										(1996), Damblon (1997)
		Polanów Samborzecki	50.63	21.67	165	PL	X	1	X	Komar et al. (2009)
		Prague-Podbaba	50.11	14.39	190	CZ	1	1	X	Jankovská & Pokroný (2008)
		Ribains	44.84	3.82	1192	FR	X	0	X	de Beaulieu & Reille (1992b)
		Riparo Fumane	45.56	10.92	350	IT	X	0	1	Maspero (1996)
		Šafárka	48.88	20.58	600	SK	1	1	X	Jankovská et al. (2002),
										Jankovská & Pokroný (2008)

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L'hypothèse de flux naturels de gènes entre le Tyrol et les Sudètes n'est pas à exclure totalement mais ils sont probablement très rares compte tenu de la distance et ils ne pourraient expliquer le nombre particulièrement important d'individus intermédiaires que l'on observe dans notre échantillon. En dernière hypothèse, on peut également penser à des erreurs de manipulation entre la récolte des graines et la vente des plants. Si elles ne sont pas totalement exclues, elles sont cependant peu vraisemblables à une aussi large échelle et n'auraient pas conduit à un aussi grand nombre de peuplements mélangés.

Development in Quaternary Science (eds. Sirocko F, Claussen M, Sanchez Goni M, Litt T), pp. 277-287. Elsevier. Münch E (1936) Das Lärchensterben. Forstwissenschaftliches Zentralblatt 58, 641-671. Musil R (2003) The Middle and Upper Palaeolithic game suite in Central and Southeastern Europe. In: Neanderthals and modern humans in the European Landscape during the Last Glaciation (eds. Andel TH, Davies SW), pp. 167-190. Cambridge Univ. Press, Cambridge. Naughton F, Sánchez Goñi MF, Desprat S, et al. (2007) Present-day and past (last 25000 years) marine pollen signal off western Iberia. Marine Micropaleontology 62, 91-114. Neville HM, Dunham JB (2011) Patterns of hybridization of nonnative cutthroat trout and hatchery rainbow trout with native redband trout in the Boise River, Idaho. North American Journal of Fisheries Management 31, 1163-1176. Nielsen EE, Bach LA, Kotlicki P (2006) Hybridlab (version 1.0): a program for generating simulated hybrids from population samples. Molecular ecology notes 6, 971-973. North GRIP Members (2004) High resolution climate record of the Northern Hemisphere reaching into the last interglacial period. Nature 431, 147-151. Oddou-Muratorio S, Vendramin G, Buiteveld J, Fady B (2009) Population estimators or progeny tests: what is the best method to assess null allele frequencies at SSR loci? Conservation Genetics 10, 1343-1347. Oeggl K (1992) Sediment-und Makrofossilanalysen aus dem Lanser See in Tirol (Austria): Ein Beitrag zur spätglazialen Bio-und Chronostratigraphie der Ostalpen. Flora 186, 317-339. Oeggl K, Unterfrauner H (2000) Die Pflanzenreste des Riss/Würm-Interglazials und des Würmglazials von Mondsee. Mitteilungen der Kommission fuer Quartärforschung der Österreichischen Akademie der Wissenschaften 12, 93-121. Oeggl K, Wahlmüller N (1994) Holozäne Vegetationsentwicklung an der Waldgrenze der Ostalpen: Die Plancklacke (2140m)/Sankt Jakob im Defreggen, Osttirol. Dissertationes Botanicae 234, 389-411. Opravil E (1994) Vegetation. In: Pavlov I -Excavation 1952-1983, Ch. V. ERAUL 66. The Dolní Vêstonice Studies 2 (ed. Svoboda J), pp. 177-180, Liège. Ortu E (2002) Reconstruction sur base pollinique de la dynamique de la végétation tardiglaciaire et Holocene dans les Alpes Maritimes italiennes, Université de Droit, d'Économie et des Sciences d'Aix-Marseille (Aix-Marseille III), Università degli studi di Pavia. Ortu E, David F, Caramiello R (2005) Effet des paramètres locaux sur le développement de la végétation dans le Vallon de St Anna di Vinadio (Alpes Maritimes; Italie). Ecoscience 12, 122-135. Ortu E, David F, Cramiello R (2003) Rôle de l'homme dans l'histoire de la végétation de la vallée Ellero (Alpes maritimes, Italie). C.R. Biologies 326, 631-637. Paetkau D, Slade R, Burden M, Estoup A (2004) Genetic assignment methods for the direct, real-time estimation of migration rate: a simulation-based exploration of accuracy and power. Molecular Ecology 13, 55-65. Paradis E, Claude J, Strimmer K (2004) APE: analyses of phylogenetics and evolution in R language. Bioinformatics 20, 289-290. Påsse T, Robertsson A-M, Miller U, Klingberg F (1988) A Late Pleistocene sequence at Margreteberg, southwestern Sweden. Boreas 17, 141-163. Peakall ROD, Smouse PE (2006) GENEALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Molecular ecology notes 6, 288-295. Pécsi M (1977) A hazai éurépai és az eurépai löszképzodmények paleogrográiai kutatása és összehasinlítása. Geonómia és Bányá 10, 183-221. tel-00880539, version 1 -6 Nov 2013

The Holocene fossil distribution indicates maximum abundance during the first two millennia followed by successive decline at low altitudinal sites caused by the spread of competing tree species . Modern species range restriction was reached at ~ 5ka.

At the beginning of the Holocene Larix rapidly moved upwards in response to an abrupt temperature rise of 4-6°C at ~11.5-11.4 ka causing a timberline shift of 800 m in 200-300 years (Tinner 2007). The establishment of larch subalpine forests has been studied very precisely and its timing has been estimated based on macrofossils from different high altitude sites in southern Switzerland and Northern Italy [START_REF] Gobet | Early-Holocene afforestation processes in the lower subalpine belt of the Central Swiss Alps as inferred from macrofossil and pollen records[END_REF]). In the Valais and the Lombardy larch establishment has been dated around 11.4-11.0 ka whereas in Upper Engadine it happened 300 years later at ~10.7 ka. For the other parts of the Alps less data is available making it difficult to precisely estimate larch forest establishment. However, for the Western Alps macrofossils and a continuous pollen curve from a high altitudinal site in the Aosta valley point to subalpine forest establishment between 11 and 10.5 ka, which is in agreement with another pollen record from the same valley [START_REF] Brugiapaglia | Evoluzione altitudinale spazio-temporale degli alberi durante GLI ultimi 12000 anni in valle d'Aosta[END_REF]. In other parts of the Western Alps, subalpine larch forests appeared later. In the French Savoy, macrofossil data confirm the presence of larch since the onset of the Holocene, but forests got established only later at ~ 8 ka [START_REF] Blarquez | Trees in the subalpine belt since 11 700 cal. BP: origin, expansion and alteration of the modern forest[END_REF]. For the Maritime Alps, no macrofossils were found and inferences are hampered by discontinuous pollen curves and differing signals among study sites (Ortu 2002), but there are indications that subalpine forests established at ~9.8 ka (Ortu et al. 2005). For the eastern Alpine region, data are also scarce but macrofossils from Eastern Tirol dated back to ~11.4 ka demonstrate the early arrival of the species (Oeggl & Wahlmüller 1994), even though subalpine forest development of the eastern parts of the Alps could have been delayed [START_REF] Bortenschlager | Beiträge zur Vegetationsgeschichte Tirols. Inneres Ötztal und unteres Inntal[END_REF][START_REF] Gobet | Early-Holocene afforestation processes in the lower subalpine belt of the Central Swiss Alps as inferred from macrofossil and pollen records[END_REF].

The combined record (macrofossils, stomata, pollen) from Lake Brazi (1740) in the southern Carpathians reveals that forest establishment was synchronous to that in the Central Alps (~11.2 ka). For other parts of Central Europe such precise correlations cannot be made due to the lack of adequate records. However, there is clear trend of upward shift of the treelines in the northwestern Carpathians, with decreasing pollen values at low elevation coinciding with increasing values at higher altitudinal sites [START_REF] Koperowa | The history of the Late-Glacial and Holocene vegetation in Nowy Targ Basin[END_REF]. In contrast, the distribution of larch in the Polish lowland north of the Carpathian area seems to have remained stable. Cette forte diversité non attendue peut probablement s'expliquer en prenant en compte les bouleversements introduits par l'homme dans les différentes parties de l'aire du mélèze et notamment dans la zone des Sudètes. En analysant plus finement la diversité génétique des peuplements naturels de cette zone, Wagner (in press) a pu démontrer qu'il y avait eu, depuis probablement le 19 ième siècle, de très nombreuses introductions de mélèze en provenance d'Allemagne ou du Tyrol autrichien. Il est dès lors vraisemblable qu'une partie des graines récoltées dans des peuplements "naturels" des Sudètes l'ait été en réalité dans des peuplements en grande partie contaminés par des introductions plus ou moins anciennes de mélèzes "alpins" faites à proximité, voire sur des peuplements issus de graines non autochtones.

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