L Parasite

Planeri

Partners

Je Remercie

Thomas Boehm

Chrystelle Merci À Violette

Julien Merci

Merci À Bonnie

Zoé

Mathieu, Olivier, Guillaume, Damien, Emilien, François, Laurent, Karl Nadine Paul

Margaux Et Maxime

Pablo

Justine Aziliz

Charlotte Simon

Mélanie Azélie

Audrey

Loïc

Maxime, Léon, Mae, Medhi, Thibaut Emilie Lucie

Azélie Clara

Louise Dylan

Merci

Maxime Simon

Laureen D'

DIVERGENCE GENOMIQUE ET SPECIATION ENTRE LA LAMPROIE DE RIVIERE -PARASITE (LAMPETRA FLUVIATILIS) ET LA LAMPROIE DE PLANER -NON

Et voilà. C'est l'aboutissement de trois ans et demi de travail. Je pense à peine reconnaitre la personne que j'étais en début de thèse tant plein de choses sont arrivées depuis… beaucoup de réflexions et de doutes, un confinement, mais aussi énormément de rencontres, des voyages, des moments de partage beaucoup de rires. J'ai la chance de pouvoir dire que j'ai été extrêmement bien entourées, que ce soit personnellement ou professionnellement tout au long de cette thèse. Je pense qu'il sera difficile de remercier tout le monde à la hauteur de ce qu'il mérite en ces quelques paragraphes. Toutes ses personnes, chacune à leur niveau, ont-elles aussi énormément contribuées à l'aboutissement de ce travail.

J'aimerais tout d'abord remercier mon directeur de thèse, Guillaume Evanno, pour m'avoir fait confiance et m'avoir permis de réaliser cette thèse. Merci de m'avoir supportée pendant plus de 4 ans (si on compte mon stage de M2) et d'avoir partagé tes connaissances et ta bienveillance tout au long de ce projet.

Introduction

La biodiversité que nous observons aujourd'hui sur Terre résulte d'une multitude d'événements d'extinction et d'apparition de nouvelles espèces. Par conséquent, la compréhension de la manière dont de nouvelles espèces émergent demeure l'une des questions fondamentales en biologie évolutive (The Marie Curie SPECIATION Network, 2012). Ce processus, appelé spéciation, peut conduire à la divergence d'une population en entités distinctes appelées "espèces" (Cook, 1908). L'évolution de cette divergence se déroule dans ce que l'on appelle un continuum de spéciation, qui n'est pas unilatéral (Stankowski & Ravinet, 2021). Ainsi, des populations qui ont commencé à diverger peuvent retourner à leur état initial si la divergence entre elles n'était que partielle. Plus le processus de spéciation progresse, plus l'isolement reproducteur entre les entités devient important en raison de l'accumulation de barrières à la reproduction. Lorsque l'isolement reproducteur est total, la reproduction n'est plus possible [START_REF] Salzburger | Speciation[END_REF] et les deux espèces sont considérées comme distinctes selon le « Biological Species Concept » (Mayr, 1942(Mayr, , 1996)).

Les barrières à la reproduction peuvent être classées en trois catégories : l'isolement précopulatoire, l'isolement prézygotique postcopulatoire, et l'isolement postzygotique. Les barrières précopulatoires empêchent ou réduisent le transfert de gamètes entre les espèces (Coyne & Orr, 2004). Ces barrières peuvent être de nature comportementale, mécanique, temporelle, ou liées à des habitats différents entre espèces (Grant, 1994;Marshall & Cooley, 2000;Martin & Willis, 2007;Smadja & Butlin, 2009;Uy et al., 2018). Les barrières prézygotiques postcopulatoires interviennent après le transfert de gamètes mais avant la fécondation (Coyne & Orr, 2004). Elles peuvent être dues à un isolement gamétique compétitif (Gregory & Howard, 1994) ou non (Yeates et al., 2013). Et enfin, les barrières postzygotiques se divisent en deux catégories : les barrières extrinsèques qui diminuent les performances des individus hybrides dans leur environnement (Via et al., 2000;Coyne & Orr, 2004) et les barrières intrinsèques qui causent des problèmes de développement ou de fertilité chez les individus hybrides (Coyne & Orr, 2004;Kost et al., 2016;Cooper et al., 2019). En général, c'est l'accumulation de plusieurs de ces barrières reproductives qui conduit à un isolement reproducteur complet, car la plupart, à l'exception de la non-viabilité des hybrides et de la stérilité des hybrides, ne permettent pas de bloquer complètement la reproduction.

L'identification des gènes impliqués dans l'isolement reproducteur (« gènes de spéciation ») est un sujet important de recherche en spéciation (Coyne & Orr, 2004;Orr, 2005;Rogers & Bernatchez, 2007).

Les interactions entre loci de populations ou d'espèces différentes peuvent conduire à des incompatibilités chez les hybrides qui sont responsables de la non viabilité ou de problèmes de fertilité chez ces hybrides. La première modélisation de l'évolution de ces incompatibilités provient du modèle de Dobzhansky-Müller (DM). Les interactions entre loci peuvent conduire à des phénomènes tels que la vigueur hybride (l'hybride va avoir une meilleure fitness que les individus de la populations parentales ; Wakchaure et al., 2015) ou encore la dépression hybride (les hybrides vont avoir une fitness réduite comparés aux individus des populations parentales ; Gharrett et al., 1999). Outre les effets spécifiques de certains gènes, le rôle des réarrangements chromosomiques dans la spéciation est de plus en plus étudié car de tels réarrangements comme les inversions chromosomiques peuvent bloquer la recombinaison dans de larges zones du génome et engendrer des barrières reproductives impliquées dans la spéciation [START_REF] Rieseberg | Chromosomal rearrangements and speciation[END_REF]Zhang et al., 2021).

Au cours de cette thèse, nous avons choisi d'utiliser les lamproies comme modèle biologique, notamment en raison de leur place clé dans l'arbre phylogénétique, à la base des vertébrés (Docker, 2015;Hedges et al., 2015). Les lamproies font parties des cyclostomes, qui ne possèdent ni mâchoires, ni tissus minéralisés. Elles disposent d'un disque buccal qui leur permet de s'accrocher à différentes surfaces dans leur environnement, notamment sur leurs proies pour certaines d'entre elles (Borowiec et al., 2021). On recense environ 18 espèces de lamproies parasites et 19 de lamproies non parasites (Renaud & Cochran, 2019). Parmi ces espèces, on retrouve plusieurs fois le cas de ce que l'on appelle des « paires d'espèces » (Docker, 2009). Ces paires d'espèces sont constituées d'une lamproie parasite et d'une lamproie non parasite étroitement apparentées, qui sont considérées soit comme des écotypes, soit comme des espèces distinctes (Docker, 2015).

En Europe, les lamproies du genre Lampetra sont représentées par une paire d'espèces : la lamproie fluviatile ou de rivière (Lampetra fluviatilis), qui est parasite d'autres espèces de poissons et anadrome, et de la lamproie de Planer (L. planeri), qui est non parasite et résidente en eau douce. Les deux espèces ont un stade larvaire très similaire où elles sont aveugles, microphages, et vivent dans le substrat des cours d'eau pendant trois à sept ans. À la fin du stade larvaire, les larves subissent une métamorphose au cours de laquelle apparaissent les yeux et le disque buccal caractéristique. De nombreux autres changements anatomiques et physiologiques ont lieu pendant cette période, notamment pour préparer le changement de régime alimentaire et la migration en mer des espèces de lamproies parasites et anadromes. C'est notamment le cas de la lamproie fluviatile qui, après la métamorphose, rejoint le milieu marin pour se nourrir pendant deux à trois ans avant de revenir en rivière pour se reproduire. Les lamproies de Planer quant à elles, ne se nourrissent plus après la métamorphose et n'effectuent pas de migration en mer. Leur reproduction se déroule juste après la métamorphose en rivière [START_REF] Kelly | A review of the ecology and distribution of three lamprey species, Lampetra fluviatilis (L.), Lampetra planeri (Bloch) and Petromyzon marinus (L.): A context for conservation and biodiversity considerations in Ireland[END_REF]Docker, 2015). Dans l'ouest de la France, on trouve de nombreuses rivières dans lesquelles ces espèces sont en sympatrie (Rougemont et al., 2017). En dépit d'histoires de vie différentes, ces deux espèces se reproduisent dans les mêmes habitats et, malgré l'existence d'un isolement reproducteur partiel dû à la taille (Beamish & Neville, 1992 ; L. planeri est beaucoup plus petite que L. fluviatilis), des accouplements entre les deux espèces (Lasne et al., 2010), et des hybrides ont été observés (Rougemont et al., 2015(Rougemont et al., , 2017)). Néanmoins, la viabilité et la fécondité de ces hybrides dans la nature ne sont pas connus.

Objectifs de la thèse

Dans le chapitre 2, nous avons pour objectif d'approfondir les connaissances sur la divergence génomique entre L. planeri et L. fluviatilis. Nous savons qu'il existe des SNPs (Single Nucleotide Polymorphism) discriminants les deux espèces mais nous ne savons pas comment ils sont répartis dans le génome, ni quelle est l'architecture de la divergence. Pour étudier les différences génomiques, nous avons réalisé un nouveau génome de référence de haute qualité ainsi qu'une carte de liaison et une analyse fonctionnelle des gènes dans les zones d'intérêt découvertes.

Dans le chapitre 3, notre but est de déterminer s'il existe des barrières prézygotiques postcopulatoires.

L'isolement reproducteur semblant faible, il est possible qu'un isolement gamétique se soit mis en place. Pour vérifier cela, nous avons comparé la qualité du sperme des mâles L. planeri et L. fluviatilis et réalisé des expériences de compétition spermatique pour voir s'il existe un choix cryptique de la femelle favorisant les mâles du même écotype qu'elle (i.e. un avantage au conspécifique).

Dans le chapitre 4, notre objectif est de voir s'il existe des barrières postzygotiques. Nous avons étudié la fréquence des hybrides en milieu naturel en génotypant 1292 individus échantillonnés dans la zone hybride de rivière de l'OIR (Normandie -France) entre 2010 et 2021. Notre hypothèse est qu'il existe un phénomène de dépression hybride car dans l'étude de Q. Rougemont (2017), la majorité des hybrides semblaient être des F1s et peu d'hybrides de seconde génération ou plus avaient été identifiés. Nous avons également modélisé différent scenario d'hybridation pour comprendre quels facteurs recréeraient des fréquences d'hybrides comparables à celles que l'on pourrait trouver dans la zone hybride de notre étude. Et pour finir, nous avons réalisé des croisements contrôlés afin de mesurer les performances reproductives des hybrides.

Chapitre 2 : Divergence génomique entre les écotypes de lamproies parasites et non-parasites Dans le Chapitre 2, nous nous sommes intéressés à la divergence génomique entre L. fluviatilis et L. planeri. Étant donné que la lamproie n'est pas encore reconnue comme une espèce modèle en biologie, les ressources génomiques sont limitées. Au début de la thèse, l'une des seules ressources disponibles était le génome de la lamproie marine (Petromyzon marinus) (Smith et al., 2013).

Cependant, les espèces de lamproies que nous étudions ici appartiennent au genre Lampetra. Nous avons donc entrepris de séquencer et d'assembler le génome d'une lamproie de Planer. Nous avons utilisé un séquençage de type Pacbio HiFi, suivi d'un assemblage avec le logiciel HiFiasm (Chin et al., 2016). Ensuite, nous avons réalisé une carte de liaison avec le logiciel LepMap3 (Rastas, 2017) Au total, nous avons sélectionné 40 SNPs diagnostiques ainsi que 149 SNPs non diagnostics aléatoirement répartis dans le génome. Les échantillons ont été génotypés grâce à la technique de multiplexage Hi-plex (Hammet et al., 2019). Grâce aux marqueurs diagnostiques et aux logiciels Structure (Pritchard et al., 2000) et Newhybrids (Anderson, 2008), nous avons classé les individus en 6 catégories différentes : pur L. fluviatilis, pur L. planeri, F1 avec phénotypes de L. planeri ou L. fluviatilis, et les hybrides de deuxième génération ou plus, appelés « admixed » L. planeri ou L. fluviatilis. Nous avons observé que les individus hybrides (F1 + Admixed) représentaient plus de 60% des individus, que ce soit chez les adultes ou les larves. Si la proportion d'hybrides avait été plus élevée chez les larves que chez les adultes, cela aurait pu indiquer qu'une barrière postzygotique était responsable d'une mortalité hybride plus importante par rapport aux individus purs mais ce n'est pas le cas ici (Templeton et al., 1986;Stelkens et al., 2015). Les proportions d'hybrides étaient similaires chez les mâles et les femelles excluant des incompatibilité hybrides liés au sexe (Haldane, 1922;Johnson & Lachance, 2012).

Pour mieux comprendre ce fort taux d'individus hybrides, nous avons examiné de plus près les deux groupes de liaison présentant une forte différenciation. L'analyse en composantes principales (ACP) des SNPs du groupe de liaison présentant la plus forte différenciation a révélé un schéma intrigant avec trois groupes distincts. La mesure de l'hétérozygotie dans chacun de ces trois groupes a montré une forte hétérozygotie chez les individus hybrides. Ces résultats sont indicatifs d'une inversion potentielle (Mérot, 2020) en accord avec les résultats du chapitre 2. Le taux important d'hybrides observés dans cette étude est possiblement lié avec de la vigueur hybride ou overdominance en lien avec l'inversion.

La seconde approche a été de faire des simulations d'une zone hybride entre L. planeri et L. fluviatilis.

Nous avons réalisé ces simulations à l'aide du logiciel NEMO (Guillaume & Rougemont, 2006) qui permet de modéliser les individus de plusieurs populations sur plusieurs générations. Trois modèles ont été implémentés dans ce logiciel pour permettre de reproduire le plus fidèlement possible le cycle de vie et les comportements des deux écotypes. Un modèle d'accouplement sélectif entre écotypes, un modèle d'incompatibilité hybride ainsi qu'un modèle de fécondité différentielle ont été implémentés selon les hypothèses formulées avec les données disponibles sur ces écotypes avant ma thèse. Le taux de F1s observé dans les simulations s'est révélé toujours faible que celui observé dans les données de l'Oir, suggérant que de nouveaux scénarios de simulations devraient être explorés et inclure notamment une forme de vigueur hybride ou overdominance.

Enfin, nous avons réalisé des fécondations in vitro entre des individus purs L. fluviatilis, des purs L. planeri ainsi que des hybrides identifiés avec un marqueur diagnostic (Souissi et al., 2022). La classification des hybrides a été ensuite affinée avec l'utilisation de 40 marqueurs diagnostics. Nos résultats n'ont pas révélé de barrières postzygotiques que ce soit sur le taux de fécondation ou sur d'autres paramètres durant le développement larvaire.

Conclusion

En conclusion, cette thèse nous a permis d'acquérir une meilleure compréhension de l'architecture de la divergence entre L. fluviatilis et L. planeri. Nous avons identifié deux îlots de différenciation, dont l'un présente potentiellement une inversion. La forte fréquence d'hybridation observée pourrait résulter d'un effet d'overdominance lié à cette inversion. L'overdominance est un avantage pour les hétérozygotes par rapport aux homozygotes. Il est possible que les hétérozygotes porteurs de l'inversion (les hybrides observés dans le chapitre 4) soient avantagés en raison de l'important brassage génétique entre L. planeri et L. fluviatilis, ce qui aurait conduit à l'accumulation de mutations délétères et à une pléiotropie antagoniste. Les hétérozygotes seraient moins susceptibles de manifester ces mutations délétères et donc bénéficieraient d'un avantage sélectif. De plus, aucune barrière reproductive entre les espèces n'a pu être mise en évidence. Dans l'ensemble, ces résultats tendent à soutenir l'idée que se sont en réalité deux écotypes d'une même espèce. Les perspectives de ce travail consistent à comprendre comment ces écotypes parviennent à coexister malgré un taux d'hybridation aussi élevé. De nouvelles simulations pourront être réalisées en prenant en compte l'overdominance.

Il sera également important d'identifier les gènes différentiellement exprimés, en particulier dans la région de l'inversion, afin de mieux comprendre la divergence entre écotypes.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS

INTRODUCTION 1. SPECIES AND SPECIATION

Understanding how new species arise is one of the main aims of evolutionary biology. The current diversity of species can be explained by the balance between extinction and speciation, thus making speciation a central topic of research (The Marie Curie SPECIATION Network, 2012). Speciation, term proposed by (Cook, 1908), can lead a population to diverge and form two distinct entities called "species". However, since Darwin's On the origin of species (1859), biologists have not accorded on a definition of species and many species concepts have arisen (Coyne & Orr, 2004;[START_REF] Singh | Concepts of species and modes of speciation[END_REF]. This situation is called the "species problem" [START_REF] Salzburger | Speciation[END_REF]. At least 22 different species concepts exist [START_REF] Mayden | A hierarchy of species concepts: the denouement in the saga of the species problem[END_REF] and any change in a species concept directly impacts many important research fields [START_REF] Agapow | The impact of species concept on biodiversity studies[END_REF]. [START_REF] Brookfield | Genes, categories and species, the evolutionary and cognitive causes of the species problem[END_REF] stated that no set of experiments or observations could resolve which of these views is the right one and the debate started to be more philosophical than scientific. For evolutionary biologists, the Biological Species Concept (BSC, Mayr, 1942) is the most accepted and applied definition of species [START_REF] Salzburger | Speciation[END_REF] and directly addresses the question: "why do sympatric sexually reproducing organisms fall into discrete clusters?". The original definition of Mayr (1942) has been adjusted in 1995 due to difficulties in the classification of allopatric taxa (Coyne & Orr, 2004). Under that concept, species are considered to be natural populations that actually interbreed and are reproductively isolated from other such groups (Mayr, 1996). With this concept of species, we can highlight two possibilities to form a species: 1) their genetic differences preclude them from living in the same area and 2) that they inhabit the same area but their genetic differences make them unable to produce fertile hybrids. Therefore, under the BSC, hybridization and introgression should not exist and these phenomenon have been found to be more common than previously thought [START_REF] Mallet | Hybridization as an invasion of the genome[END_REF]. Another concept of species, the 'genic species concept' allows introgression while key genomic regions remain impermeable to gene flow [START_REF] Wu | The genic view of the process of speciation[END_REF]. It seems that the point at which sympatric populations should be called species is arbitrary. Even under BSC, hybridization is 'permissible' at very low rate at the condition that populations maintain their distinctness (Coyne & Orr, 2004). Speciation can also be defined as the evolution of reproductive isolation (RI) among populations [START_REF] Ravinet | Interpreting the genomic landscape of speciation: a road map for finding barriers to gene flow[END_REF]. This definition leads to the concept of a speciation continuum, which is not a recent idea as Darwin already talked about a gradual process (Stankowski & Ravinet, 2021). However, the term speciation continuum has only been used in few papers in the late 2000's and its use is gradually increasing. Stankowski & Ravinet (2021) proposed to define the speciation continuum as a continuum of reproductive isolation to clarify the term and make it concordant with the BSC. This definition implies that reproductive isolation is continuous and that speciation is a process rather than a discrete event. Therefore, to study speciation we need to understand what the barriers to gene flow are and how they arise. Reproductive isolation (RI) is defined as the reduction in potential gene flow between populations caused by isolating barriers. The speciation process is complete when RI = 1 (i.e. complete isolation). At that stage, the members of the new species do not interbred anymore with the ancestral species [START_REF] Salzburger | Speciation[END_REF]. The mechanisms preventing gene flow between species will be further detailed in the following section. Closely related population/species with some degree of ongoing gene flow can be considered as snapshots of the process of speciation. The understanding of speciation requires several pairs of populations, not necessarily at the same point of the speciation process, to understand how the process evolve (Stankowski & Ravinet, 2021). In addition, [START_REF] Lyell | Principles of Geology[END_REF] pointed out that "the present is a key to the past" but speciation takes time, typically millions of years (Hedges et al., 2015) and the context and evolution of divergence are not easily resolved due to the multitude of possible scenarios. Hybrid zones are the best places to study speciation [START_REF] Hewitt | Quaternary phylogeography: the roots of hybrid zones[END_REF] and how hybridisation / homogenisation of the genome act or not. To understand the speciation process we need to take into account every component that can lead to reproductive isolation as it is a multilevel process changing through space and time [START_REF] Abbott | Hybridization and speciation[END_REF]. Observed hybridization is potentially just a snapshot of a complex interaction that change overtime [START_REF] Abbott | Hybridization and speciation[END_REF]. The establishment of a genetic barrier depend of the level of gene flow between populations. Depending on the context, gene flow can homogenise the genome [START_REF] Nosil | Speciation with gene flow could be common[END_REF], but recombination occurring with hybridization can also break association between adaptive alleles [START_REF] Felsenstein | Skepticism Towards Santa Rosalia, or Why Are There so Few Kinds of Animals?[END_REF] and can accelerate divergence between diverging populations.

The geographical situation of populations also has a strong impact on the evolution of reproductive isolation. Populations in allopatry have no common distribution range and with time, they will eventually become reproductively isolated due to the action of genetic drift, the accumulation of mutations and divergent selection [START_REF] Turelli | Theory and speciation[END_REF][START_REF] Gavrilets | Perspective: Models of speciation: What have we learned in 40 years?[END_REF]. In parapatry, populations have a partly overlapping distribution range. Finally, in sympatry, the population have completely overlapping distribution ranges. Thus, the evolution of RI is conditioned by the level of gene flow, as the evolution of reproductive barriers will be challenged by gene flow. This classification has been criticized [START_REF] Butlin | Sympatric, parapatric or allopatric: the most important way to classify speciation?[END_REF][START_REF] Fitzpatrick | Climate change, plant migration, and range collapse in a global biodiversity hotspot: the Banksia (Proteaceae) of Western Australia[END_REF] because allopatry and sympatry do not really exist in nature as there is always some level of encounters in allopatry and distribution range can be slightly diverging in sympatry. As a result, most situations correspond to populations in parapatry with more or less gene flow. This classification remains important because it gives an idea of the geographical context but it does not take into account the extension and contractions of specie spatial distributions [START_REF] Willis | Genetic consequences of climatic oscillations in the Quaternary[END_REF]. For example, the glaciations during Pleistocene (two millions years ago), which finished with a global warming 10000 years ago [START_REF] Hewitt | Some genetic consequences of ice ages, and their role in divergence and speciation[END_REF], are thought to be the main historical event that has shaped the large-scale population structure of contemporary species [START_REF] Bernatchez | Comparative phylogeography of Nearctic and Palearctic fishes[END_REF][START_REF] Taberlet | Comparative phylogeography and postglacial colonization routes in Europe[END_REF]. After glaciations, the recolonization of northern areas are often linked to founder effects whereby populations have a reduced genetic diversity at the colonization front [START_REF] Hewitt | Some genetic consequences of ice ages, and their role in divergence and speciation[END_REF]. These large-scale movements can also lead to the formation of hybrid zones, in two ways. First, hybrid zones can result from divergent selection across an environmental gradient along the colonization way. Second, they can result from a secondary contact from species who have previously diverged in allopatry and are put into contact again following an environmental change [START_REF] Arntzen | Hybrid zone formation and contrasting outcomes of secondary contact over transects in common toads[END_REF]. During Pleistocene, there were episodes of species isolation in southern regions alternating with interactions with northern species / populations [START_REF] Hewitt | Post-glacial re-colonization of European biota[END_REF]. [START_REF] Arntzen | Hybrid zone formation and contrasting outcomes of secondary contact over transects in common toads[END_REF] argue that hybrid zones move until a genotype/environmental equilibrium is formed and that environmental conditions are stable. The hybrid zones are considered as "windows on evolution" [START_REF] Hewitt | Quaternary phylogeography: the roots of hybrid zones[END_REF] because they are like a snapshot of the hybridisation process at one time, thus giving great insights into the speciation process [START_REF] Barton | Analysis of hybrid zones[END_REF][START_REF] Abbott | Hybridization and speciation[END_REF]. In rare cases, hybridization can contribute to the formation of new species [START_REF] Desvignes | Intergeneric hybrids inform reproductive isolating barriers in the Antarctic icefish radiation[END_REF] when two distinct species hybridise, and a third one forms and ultimately becomes reproductively isolated from parental species (Figure 1; Uy et al., 2018).

When there is no spatial barrier, the interbreeding is only prevented by reproductive isolating mechanisms detailed in the next section.

REPRODUCTIVE ISOLATION

Reproductive isolation includes several "isolating mechanisms", without which evolution would not be possible [START_REF] Dobzhansky | Genetics and the origin of species[END_REF]. Following Coyne & Orr (2004) and [START_REF] Dobzhansky | Genetics and the origin of species[END_REF], reproductive barriers can be classified into premating isolation (PMI), postmating prezygotic isolation (PMPZ) and postzygotic isolation (Figure 2). Postzygotic barriers (local adaptation or intrinsic barriers) are expected to evolve first, followed by prezygotic barriers in the classical scenario of allopatric divergence followed by secondary contact [START_REF] Kulmuni | Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers[END_REF][START_REF] North | The role of copy-number variation in the reinforcement of sexual isolation between the two European subspecies of the house mouse[END_REF]. However, it is difficult to estimate which barriers evolve first and which ones complete the process of speciation.

a. Premating isolation

Premating isolating barriers prevent gamete transfer between species (Coyne & Orr, 2004). In the case where hybrids suffer reduced fitness compared to purebred individuals, they are the most important barriers to avoid reproduction costs. The process of developing divergent mate preferences as a response to selection against hybrids is referred to as reinforcement. The traits involved in mate choice, courtship and mating can evolve quickly [START_REF] Salzburger | Speciation[END_REF]. Thus, the sensory recognition systems (visual, chemosensory, tactile and acoustic) play a great role in PMI because diverse sensory receptors send signals that will be integrated and that can lead to an individual response that participates in the recognition and choice of potential mates (Smadja & Butlin, 2009).

A first barrier of PMI is called behavioural isolation. It includes all behavioural differences that conduct to a lack of interaction between members of different species, which prevent the initiation of mating behaviour thus cancelling any possibilities of copulation (Coyne & Orr, 2004). Coevolutionary changes in display traits promote assortative mating and thus lower the hybridisation rate. A good example of these is in birds, where the intensity of sexual selection act as a proxy for the speciation rate or the species richness (e.g. [START_REF] Barraclough | Sexual selection and taxonomic diversity in passerine birds[END_REF][START_REF] Mason | Song evolution, speciation, and vocal learning in passerine birds[END_REF]. Even slight changes in mating signals and preferences may cause behavioural isolation, especially at the beginning of population divergence (Uy et al., 2018). For instance, preference for plumage colour or song dialect, even if they are morphologically indistinguishable, evolve rapidly and promote assortative mating. Nesting behaviour can also influence the mate choice if the nest differ between species [START_REF] Bowen | The Diversity of Fishes: Biology, Evolution and Ecology[END_REF].

Chemosensory systems are also at play in mate recognition and have been extensively studied in Drosophila and moths species (Smadja & Butlin, 2009). In the later, mate finding generally involves long distance signalling by female pheromones. These pheromones have been considered to have a role in speciation because of the great structure diversity and the extensive radiation of moths. In most moth species, mate finding involves long-distance signalling by female-emitted sex pheromones. The great diversity of pheromone structures used by moth species, and the extensive radiation seen in moths, led to consideration of the role of these pheromones in the speciation process [START_REF] Linn Jr | Pheromone communication in the moths and its role in the speciation process[END_REF][START_REF] Greenfield | Signalers and Receivers: Mechanisms and Evolution of Arthropod Communication[END_REF] and in most cases, divergence in female pheromones correlates with a stronger attraction of conspecific males [START_REF] Monti | A genetic analysis of some components of reproductive isolation between two closely related species, Spodoptera latifascia (Walker) and S. descoinsi (Lalanne-Cassou and Silvain) (Lepidoptera: Noctuidae)[END_REF][START_REF] Groot | Experimental evidence for interspecific directional selection on moth pheromone communication[END_REF]. Behavioral isolation can also happen at the postmating prezygotic stage when behaviour does not allow a correct transfer of gametes that could initiate fertilization (Coyne & Orr, 2004) Mechanical isolation is defined as the incompatibility of reproductive structures. It can either be a structural isolation with a lack of mechanical fit between male and female genitalia, or a tactile isolation where the heterospecific genitalia cannot provide a necessary stimulation for mating (Coyne & Orr, 2004). In plants, this phenomenon is thought to be quite frequent (Grant, 1994). A study investigating the reproductive isolation mechanisms between Costus pulverulentus and C. scaber revealed that mechanical floral isolation acted as a complete barrier to block the interspecific pollen deposition on a C. pulverulentus maternal parent [START_REF] Kay | Reproductive isolation between two closely related hummingbird pollinated neotropical gingers[END_REF].

Mating system isolation is the evolution of partial/complete self-fertilization or asexual reproduction that impedes gene flow with members of the ancestral population (Coyne & Orr, 2004). For example (Martin & Willis, 2007), investigated the reproductive isolation between two closely related plant species: Mimulus guttatus and M. nasutus. They are morphologically distinct because of their divergent mating system (i.e. selfing rate) coming with divergence in floral traits [START_REF] Kiang | Reproductive isolation in the Mimulus guttatus M. nasutus complex[END_REF]. It was found that mating system isolation is a very strong barrier in this system, which can by itself almost completely block gene flow (Martin & Willis, 2007).

A fourth case of PMI is habitat isolation. It occurs when species display differential adaptation to different habitats even if they inhabit the same area. It limits or prevents completely the encounters of heterospecific individuals compared to conspecific individuals (Coyne & Orr, 2004). This type of isolation is based on genetic differences, different habitat choice and different survival ability in each habitat [START_REF] Rundle | Ecological speciation[END_REF]. Habitat isolation can happen for instance with host restricted insects that recognize their hosts through chemical cues (Smadja & Butlin, 2009). One famous example is the apple maggot fly, Rhagoletis pomonella that is a model of sympatric speciation by host shift. Originally infesting hawthorn, a shift appeared in the mid-1800s where a population derived and started infesting apple tree. This sympatric speciation is mainly due to host choice as the flies find their mate on their plant-host making an effective premating barrier to reproduction [START_REF] Feder | Host fidelity is an effective premating barrier between sympatric races of the apple maggot fly[END_REF]. The olfactory cues found by the fly in their host tree and a rapid adaptation to them might play a big role in the host fidelity in host race speciation [START_REF] Tait | Sensory specificity and speciation: a potential neuronal pathway for host fruit odour discrimination in Rhagoletis pomonella[END_REF]. Habitat isolation is considered as an ecological speciation mechanism. Ecological speciation a process by which populations become reproductively isolated due to diverging adaptations to the environment and / or to different ecological niches [START_REF] Rundle | Ecological speciation[END_REF][START_REF] Funk | Ecological divergence exhibits consistently positive associations with reproductive isolation across disparate taxa[END_REF].

Temporal isolation or "allochronic isolation" is another form of premating barrier when gene flow is impeded because two populations breed at different times of the day or the year (Coyne & Orr, 2004).

Heritable variation in reproductive times can lead to such genetic divergence between populations even in the absence of selection [START_REF] Hendry | Population structure attributable to reproductive time: isolation by time and adaptation by time[END_REF]. Seasonal allochrony is the most common mode of allochronic divergence. One of the best example is the pine processionary moth with two populations having no overlapping reproductive periods [START_REF] Santos | Genetic isolation through time: allochronic differentiation of a phenologically atypical population of the pine processionary moth[END_REF]. There is one summer and one winter population with highly heritable emergence time thus, representing "phenological races" [START_REF] Salzburger | Incipient allochronic speciation in the pine processionary moth (Thaumetopoea pityocampa, Lepidoptera, Notodontidae)[END_REF][START_REF] Branco | Experimental evidence for heritable reproductive time in 2 allochronic populations of pine processionary moth[END_REF]. A good example of true allochronic speciation is the one of the seven species of North American periodical cicadas (Magicicada spp.) which live underground as juveniles for either 13 or 17 years and then emerge to live for about 3 weeks at the adult stage. Thus, they are temporally divided into two groups: the 13 years species and the 17 years species (Marshall & Cooley, 2000). Every 13 th or 17 th year, there is a single mass emergence that will satiate predators and give the surviving adults a chance to find a mate and reproduce [START_REF] Karban | Increased reproductive success at high densities and predator satiation for periodical cicadas[END_REF][START_REF] Cooley | Allochronic speciation, secondary contact, and reproductive character displacement in periodical cicadas (Hemiptera: Magicicada spp.): genetic, morphological, and behavioural evidence[END_REF][START_REF] Kye | Sharp boundary formation and invasion between spatially adjacent periodical cicada broods[END_REF]. The hybridization can happen where their distribution ranges overlap and when the species of different life cycles co-emerge but that can happen only every 221 years [START_REF] Simon | Genetic evidence for assortative mating between 13-year cicadas and sympatric "17-year cicadas with 13-year life cycles" provides support for allochronic speciation[END_REF][START_REF] Sota | Independent divergence of 13-and 17-y life cycles among three periodical cicada lineages[END_REF]. Off-cycle emergence is also a possibility and no proof against or for it could be demonstrated [START_REF] Simon | Advances in the Evolution and Ecology of 13-and 17-Year Periodical Cicadas[END_REF]. Each species is more closely related to another species with an alternative life cycle, indicating that switches are involved in speciation and that allochrony is an important driving force in this case (Marshall & Cooley, 2000;[START_REF] Sota | Independent divergence of 13-and 17-y life cycles among three periodical cicada lineages[END_REF].

b. Postmating, prezygotic isolation

Postmating barriers act after pollen or sperm transfer but before fertilization (Coyne & Orr, 2004).

Gametic isolation includes all reproductive barriers ongoing between copulation and fertilization (Coyne & Orr, 2004). It is supposed to be one of the earliest for isolation to emerge [START_REF] Coyne | Patterns of Speciation in Drosophila[END_REF] and systems where female choice is not strong enough to generate assortative mating or where male tactics override female choice are likely to involve gametic isolation [START_REF] Ludlow | Gametic isolation in guppies (Poecilia reticulata)[END_REF].

Gametic isolation can be divided into two categories: non-competitive gametic isolation or competitive isolation.

Non-competitive gametic isolation occurs when heterospecific gametes are less successful at fertilizing the female gamete that the conspecific gametes (Coyne & Orr, 2004). The lower success can be explained by e.g. a lower sperm number transferred to the female in heterospecific crosses [START_REF] Price | Cryptic reproductive isolation in the Drosophila simulans species complex[END_REF]. The reproductive tract of females can be hostile to heterospecific sperm (Gregory & Howard, 1994) or seminal fluids proteins are different and cause lower fertilization [START_REF] Garlovsky | Seminal fluid protein divergence among populations exhibiting postmating prezygotic reproductive isolation[END_REF]. To be completed fertilization need the gamete attachment and fusion which is dependant of the proteins and enzymes at the cell surface [START_REF] Vieira | Gamete interaction: Is it species-specific?[END_REF]. Even if the heterospecific gametes meet, the fertilization may not take place [START_REF] Palumbi | Strong reproductive isolation between closely related tropical sea urchins (genus Echinometra)[END_REF]. Sperm morphology could also potentially contribute to postcopulatory prezygotic isolation like suggested for two closely passerine bird species (Luscinia megarhynchos and L. luscinia). In that case, the sperm morphology presents greater divergence in sympatric populations than in allopatric populations [START_REF] Albrecht | Sperm divergence in a passerine contact zone: Indication of reinforcement at the gametic level[END_REF] Competitive gametic isolation happens when heterospecific gametes is disadvantaged when put in competition with conspecific male gametes (Coyne & Orr, 2004). It is called conspecific sperm precedence (CSP) in animals and conspecific pollen precedence (CPP) in plants. For example, in guppies, it was shown that sperm from males of the same population as the female have precedence over sperm from divergent males [START_REF] Ludlow | Gametic isolation in guppies (Poecilia reticulata)[END_REF]. Divergence in sperm traits between species have a role in reproductive isolation as the heterospermic sperm can have a lower chance to fertilize the eggs compared to conspecific sperm in case of sperm competition. When the female mates with several males, mechanims that bias paternity toward one of the mates is a form of cryptic female choice. Many examples of reproductive isolation with cryptic female choice when the female bias the paternity toward a conspecific male have been documented in insects and birds like between collared and pied flycatcher (Ficedula albicollis and F. hypoleuca) [START_REF] Mendelson | Speciation by sexual selection: 20 years of progress[END_REF]. The female copulate with several males and inhibit heterospecific sperm performances [START_REF] Cramer | Females discriminate against heterospecific sperm in a natural hybrid zone[END_REF]. A famous example of cryptic female choice can found in fish. By bias of sperm competition trials (i.e. competition between sperm of two or more male for the same batch of eggs), Yeates et al., (2013) found that salmon and trout present a conspecific sperm precedence which is also enhanced by the ovarian fluid. However if the sperm of the salmon and the trout males are not in competition, a complete interfertility between both species is observed.

c. Postzygotic isolation

Postzygotic barriers can either be intrinsic or extrinsic (Coyne & Orr, 2004). Extrinsic postzygotic isolation depends on barriers linked to the environment, either biotic or abiotic and is characterized by decreased performances of hybrids in the environment of the parental species (Coyne & Orr, 2004).

Ecological inviability happens when viable hybrids have a lower fitness due to difficulties to fit an ecological niche (Coyne & Orr, 2004). Their behaviour and / or phenotype can be intermediate between parental species; hence, they are not fully adapted to either ecological niche of their parents.

For example, in the pea aphid (A. pisum) from North America, F1 hybrids between two host specific populations have a lower fitness on both host plants than their specialized parents (Via et al., 2000).

In contrast, behavioral sterility happens when hybrids have completely viable gametes but their fertility is lower than the parental species because they cannot find/attract mates. The signals used to attract mates are misplaced, like an intermediate phenotype, which causes reinforcement and selection against hybrids [START_REF] Dobzhansky | Genetics and the origin of species[END_REF]Coyne & Orr, 2004). Hybrids (F1, F2 and backcrosses) of the grasshopper species Chorthippus biguttulus and C. brunneus exhibit intermediate songs between those of the parental species in terms of phrase number and duration. The species-specific structure is lost and the song produced is irregular and imperfect so hybrid males are probably unattractive to females of either parental species [START_REF] Gottsberger | Behavioral sterility of hybrid males in acoustically communicating grasshoppers (Acrididae, Gomphocerinae)[END_REF].

Intrinsic barriers reflects a developmental problem that is independent of the environment (Coyne & Orr, 2004). Hybrid sterility and inviability are commonly known as hybrid incompatibilities [START_REF] Johnson | Hybrid incompatibility and speciation[END_REF]. Hybrid incompatibilities (HI) can take the form of sterile hybrids of inviable hybrids. [START_REF] Patten | Selfish X chromosomes and speciation[END_REF]. Hybrids can suffer sterility because of developmental problems in the reproductive system or in the gametogenesis. This is called physiological sterility (Coyne & Orr, 2004). In hybrids of Medaka fish (Oryzias latipes and O. curvinotus), the males from reciprocal crosses present a meiotic arrest and no production of sperm while the females exhibit a reduced fertility due to a disrupted phenotype [START_REF] Martinez-Bengochea | Sexual development dysgenesis in interspecific hybrids of Medaka fish[END_REF]. Hybrids can also suffer from behavioural sterility caused by neurological or physiological problems that make their courtship behaviour unsuccessful (Coyne & Orr, 2004). In the fall armyworm Spodoptera frugiperda (Lepidoptera), native to North and South America, two sympatric strains have been recognized: the rice and the corn strain [START_REF] Pashley | Host-associated genetic differentiation in fall armyworm (Lepidoptera: Noctuidae): a sibling species complex?[END_REF]. Female hybrids resulting from a cross between a female of the rice strain and a male from the corn strain exhibit a highly reduced fertility because they refrain from mating, which makes them behaviourally sterile. A Z-linked sterility locus likely to be incompatible with an autosomal or cytoplasmic factor is probably responsible for this sexual abstinence (Kost et al., 2016). Hybrid inviability reflects developmental problems leading to full or partial lethality in hybrids (Coyne & Orr, 2004). For example, the gene Hmr (Hybrid male rescue) causes lethality and female sterility in Drosophila melanogaster and other related species. The hybrid lethality is linked to disruption in gene regulation [START_REF] Barbash | A rapidly evolving MYB-related protein causes species isolation in Drosophila[END_REF]Cooper et al., 2019).

Taxa greatly vary in the relative importance of isolating barriers (Uy et al., 2018). For example, prezygotic barriers evolve before postzygotic barriers in Drosophila and several angiosperms [START_REF] Coyne | Patterns of Speciation in Drosophila" Revisited[END_REF][START_REF] Baack | The origins of reproductive isolation in plants[END_REF]. In contrast, in Tigriopus copepods intrinsic postzygotic barriers can appear with few or no prezygotic barriers [START_REF] Willett | The nature of interactions that contribute to postzygotic reproductive isolation in hybrid copepods[END_REF]. In general, prezygotic barriers in the form of gamete incompatibility evolve faster in annual than in perennial plants [START_REF] Baack | The origins of reproductive isolation in plants[END_REF], whereas intrinsic postzygotic barriers evolve faster in mammals than in birds and frogs [START_REF] Wilson | Two types of molecular evolution. evidence from studies of interspecific hybridization[END_REF][START_REF] Fitzpatrick | Rates of evolution of hybrid inviability in birds and mammals[END_REF]. Those differences are likely due to taxon specific aspects in their ecology, genetics and geography (Mayr, 1942).

Next, we will focus on the genetic and genomic basis of speciation.

SPECIATION GENETICS AND GENOMICS

The identification of genes involved in reproductive isolation is a central question in speciation research (Orr, 2005). Several studies suggested that such 'speciation genes' are involved but their number can greatly vary (Coyne & Orr, 2004;[START_REF] Orr | Speciation genes[END_REF]Rogers & Bernatchez, 2007;[START_REF] Bomblies | Arabidopsis and relatives as models for the study of genetic and genomic incompatibilities[END_REF][START_REF] Gagnaire | The genetic architecture of reproductive isolation during speciation-with-gene-flow in lake whitefish species pairs assessed by Rad Sequencing[END_REF]. The total number of genes involved in postzygotic isolation can be high (Coyne & Orr, 2004) but it is possible that the number of genes initiating RI is much lower [START_REF] Seehausen | Genomics and the origin of species[END_REF]. The size effect of each gene could thus be tightly linked to the number of genes involved [START_REF] Yeaman | Establishment and maintenance of adaptive genetic divergence under migration, selection, and drift[END_REF][START_REF] Yeaman | The genetic architecture of adaptation under migration-selection balance[END_REF]. These genes can be associated to any form of isolating barrier but the most studied are those linked to hybrid incompatibilities (Presgraves, 2010b).

a) The Bateson-Dobzhansky-Muller model and other genetic effects

Hybrid incompatibilities usually involve interactions among multiple genes. The evolution of such incompatibilities has been first modelled with the Dobzhansky-Muller (DM) model (also called Bateson-Dobzhansky-Muller model) from [START_REF] Bateson | Heredity and variation in modern lights[END_REF], [START_REF] Dobzhansky | Genetics and the origin of species[END_REF]Müller (1940). In its simplest case, the hybrid incompatibility involve only two loci (see Figure 3). Here we consider two loci A and B and assume that any combination of a allele at locus A and a allele at locus B would cause hybrid inviability. The ancestral genotype AABB could evolve into a aaBB population and a AAbb population. This would not generate any unfit individuals as a and b would not be in the same individual. However, any cross between the aaBB population and the AAbb population would generate inviable AaBb F1s [START_REF] Johnson | Hybrid incompatibility and speciation[END_REF]. This phenomenon is due potential deleterious interactions between genes, which will only occur when individuals from different populations mate together [START_REF] Orr | Speciation by postzygotic isolation: forces, genes and molecules[END_REF][START_REF] Maheshwari | The Genetics of hybrid incompatibilities[END_REF]. One famous example illustrating the DM model is found in Drosophila in which the interaction of functionally diverged genes cause hybrid lethality [START_REF] Brideau | Two Dobzhansky-Muller genes interact to cause hybrid lethality in Drosophila[END_REF]. In D. simulans, the Lethal hybrid rescue (Lhr) gene diverged and when hybridizing with D. melanogaster, it interacts with the also diverged Hybrid male rescue (Hmr) gene. This interaction cause lethality in F1 males. Some other models and extensions of the DM model have been proposed (see e.g. [START_REF] Nei | Roles of Mutation and Selection in Speciation: From Hugo de Vries to the Modern Genomic Era[END_REF]. The incompatibilities are expected to accumulate faster and faster with time, following the so-called "snowball effect" [START_REF] Orr | The evolution of postzygotic isolation: accumulating Dobzhansky-Muller Incompatibilities[END_REF][START_REF] Moyle | Hybrid incompatibility "snowballs" between Solanum species[END_REF], rather than linearly (Stelkens et al., 2015).

The incompatibilities do not always lead to the death of hybrids and can also just reduce the fertility or conduct hybrids to be sterile. The quantification of hybrid sterility is a common measure of reproductive isolation but its measure can be difficult in nature. In laboratory studies, the measure of sterility is very basic and often limited to the F1 generational though sterility can occur in later generations of hybrids (Figure 4; [START_REF] Wiley | Postzygotic isolation over multiple generations of hybrid descendents in a natural hybrid zone: How well do singlegeneration estimates reflect reproductive isolation?[END_REF]. are heterozygous for every homologous chromosomes (exception for sexual heterozygous chromosomes but we will not detail them here). The F2 are the produce of the cross with two F1. They still have heterozygous part in the genome but some of them are homologous like the parental species.

The first generation of backcrosses (BC 1) are the results of the cross between a F1 and one individual of the parental species. They are only homozygous for the backcross parent. Modified from [START_REF] Maheshwari | The Genetics of hybrid incompatibilities[END_REF].

The interaction between loci of different populations can result in hybrids and more commonly F1s to undergoing heterosis (hybrid vigor). This phenomenon makes the offspring exhibit superior traits compared to the average purebred parents (Wakchaure et al., 2015). For instance, the F1 hybrid males of the malaria Mosquito anopheles coluzzii present a higher fertility, fecundity and survival than individuals from pure strains [START_REF] Ekechukwu | Heterosis increases fertility, fecundity, and survival of laboratory-produced F1 hybrid males of the malaria mosquito Anopheles coluzzii[END_REF]. Two main models have been proposed to explain this phenomenon: the dominance model [START_REF] Jones | Dominance of linked factors as a means of accounting for heterosis[END_REF] and the overdominance model [START_REF] Shull | The composition of a field of maize[END_REF]. The dominance model proposes that the recessive deleterious alleles accumulating in in one parent by inbreeding will be silenced by the superior dominant alleles of the other parent in the F1.

The reciprocal complementation leads to the superiority over both parents [START_REF] Lippman | Heterosis: revisiting the magic[END_REF][START_REF] Li | Dominance, overdominance and epistasis condition the heterosis in two heterotic rice hybrids[END_REF]. The overdominance model proposes that the heterozygous advantage come from specific genes or alleles with specific phenotypic effects. These single loci with overdominance can remain in the population and enhance survival or reproductive fitness. With the dominance model, the heterosis may not persist due to recombination in latter generations and potential hybrid breakdown [START_REF] Lippman | Heterosis: revisiting the magic[END_REF]. Hybrid breakdown or outbreeding depression occurs when hybrids present reduced fitness compared to the purebred parents (Gharrett et al., 1999). The main breakdowns are expected to occur after the F1 generation, when the recessive DMI are more expressed (Templeton et al., 1986). For instance, the F2 hybrids of African haplochromine cichlid fish show lower viability than the F1 and the two parental species (Stelkens et al., 2015). In another study on genetic introgression between the non-native rainbow trout (Oncorhynchus mykiss) and the native westslope cutthroat trout (Oncorhynchus clarkii lewisi), first generation hybrids presented a high reproductive success while other hybrids had a reduced fitness highlighting hybrid breakdown [START_REF] Muhlfeld | Hybridization rapidly reduces fitness of a native trout in the wild[END_REF]. DMI can also involve sex-linked genes or sex-biased patterns of hybrid incompatibilities and the viability and fertility of individuals is often assymetrical (Johnson & Lachance, 2012). The Haldane's rule (Haldane, 1922) states that in general the heterogametic sex (XY or ZW) suffers more of hybrid incompatibilities than the homogametic sex (XX or ZZ). Most explanations for the Haldane's rule are direct consequences of the properties of sex chromosomes (Johnson & Lachance, 2012). However, when species lack differentiated sex chromosomes, the only known mechanism to maintain sexual conflict polymorphisms on autosomes is sex dependant dominance reversal. The level of dominance can spread only if heterozygotes are common at that locus [START_REF] Spencer | The evolution of sex-specific dominance in response to sexually antagonistic selection[END_REF].

In Oncorhynchus mykiss (rainbow trout), two different life history strategies coexist: an anadromous form and a freshwater resident one. Anadromy is more frequent in females because an anadromous female has a higher fecundity than a freshwater resident female. In contrast, males that mature in freshwater can avoid the mortality risks linked to the anadromous strategy. A supergene mediating sex specific migratory tendency was found in a large autosomal double inversion [START_REF] Pearse | Sexdependent dominance maintains migration supergene in rainbow trout[END_REF]. This inversion is linked to sex dependant dominance and provides conditions for a heterozygote advantage because the dominance can switch between sexes, thus avoiding sexual conflicts [START_REF] Pearse | Sexdependent dominance maintains migration supergene in rainbow trout[END_REF].

b) Chromosomal rearrangements

Most animals and plants have different karyotypes and/or different chromosomal rearrangements that have led researchers to look for a causative role of chromosomal structures in speciation [START_REF] Rieseberg | Chromosomal rearrangements and speciation[END_REF]. However, prezygotic barriers are thought to arise first and thus before any chromosomal changes [START_REF] Coyne | The evolutionary genetics of speciation[END_REF].

The first evidence that chromosomal rearrangements were involved in species distinctness was found by [START_REF] Dobzhansky | Inversions in the chromosomes of Drosophila pseudoobscura[END_REF] when studying Drosophila Pseudoobscura chromosomes of different localities. They found inversions of chromosome sections. More generally, chromosomal rearrangements include fusion and fission of chromosomes, insertions, deletions, inversions, translocation or duplication of chromosome parts. If an individual is heterozygous for a chromosomal rearrangement, it may lead to unbalanced gametes that will not be viable or that will consequently generate a non-viable zygote [START_REF] Rieseberg | Chromosomal rearrangements and speciation[END_REF]. Many sister species have different chromosomal rearrangements and produce hybrids with reduced fertility due to meiotic problems [START_REF] Wright | Modes of Speciation[END_REF]. However, within species, viable heterozygous individuals at an inversion have also been documented (e.g. Mérot et al. 2018). Recent advances in genome sequencing allow the identification of structural variants, especially inversions (Zhang et al., 2021).

Inversions have received a particular attention both in plants [START_REF] Strasburg | Genomic patterns of adaptive divergence between chromosomally differentiated sunflower species[END_REF][START_REF] Lowry | A widespread chromosomal inversion polymorphism contributes to a major life-history transition, local adaptation, and reproductive isolation[END_REF] and in animals (e.g. [START_REF] Noor | Chromosomal inversions and the reproductive isolation of species[END_REF]. Inversions suppress recombination when heterozygous, and can thus accumulate genetic differences contributing to pre-and postzygotic isolation [START_REF] Kirkpatrick | How and why chromosome inversions evolve[END_REF]. Inversions contribute to the maintenance of young species by creating large blocks that act as barriers to gene flow and that do not break up with hybridization [START_REF] Navarro | Accumulating postzygotic isolation genes in parapatry: a new twist on chromosomal speciation[END_REF][START_REF] Christmas | Chromosomal inversions associated with environmental adaptation in honeybees[END_REF]Mérot et al., 2023).

In the dune and non-dune ecotypes of Helianthus petiolaris, seven large islands of differentiation were detected and then identified as inversions. These regions present high heterozygosity between but not within ecotypes. The inversions seem to be associated with traits favouring local adaptation [START_REF] Huang | Multiple chromosomal inversions contribute to adaptive divergence of a dune sunflower ecotype[END_REF].

In the next part of this introduction, we will focus on the biological model of the thesis: the lampreys.

LAMPREYS a. General presentation

Lampreys (Petromyzontiforms), together with hagfishes, are considered as "living fossils", thus are invaluable to understand the evolutionary history of vertebrates (Docker, 2015). They have diverged from jawed vertebrates (Gnatostoma) more than 500 million years ago (Hedges et al., 2015). Lampreys are thus survivors of at least four of the five mass extinction documented since the Cambrian explosion (Docker, 2015). There is a large gap between the non-vertebrate chordates (Cephalochordates and Urochordates) and the vertebrates that is only filled by the study of jawless vertebrates [START_REF] Nicholls | Mouth to mouth: hagfish and lampreys are the only surviving fish without jaws. And they could solve an evolutionary mystery, finds Henry Nicholls[END_REF][START_REF] Shimeld | Evolutionary crossroads in developmental biology: cyclostomes (lamprey and hagfish)[END_REF]. The study of lampreys has significantly contributed to the understanding of vertebrates' evolution including the origins of jaws (Schilling et al., 2001), the development of the neural crest (Green & Bronner, 2014) and the evolution of the adaptive immune system (Boehm et al., 2012;Trancoso et al., 2020), neuroendocrine systems [START_REF] Sower | A lamprey view on the origins of neuroendocrine regulation of the thyroid axis[END_REF], anatomy, locomotion [START_REF] Bicanski | From lamprey to salamander: an exploratory modeling study on the architecture of the spinal locomotor networks in the salamander[END_REF][START_REF] Grillner | Evolution of the vertebrate motor system -from forebrain to spinal cord[END_REF] or eyes [START_REF] Lamb | Evolution of the vertebrate eye: opsins, photoreceptors, retina and eye cup[END_REF]. The sequencing of the first high quality lamprey genome by (Smith et al., 2013) provided valuable insight into the genetic basis of vertebrates' reproduction. Genomic studies in lampreys have also put into light key evolutionary mechanisms in the vertebrate lineage such as whole genome duplication (Mehta et al., 2013;[START_REF] Smith | The sea lamprey germline genome provides insights into programmed genome rearrangement and vertebrate evolution[END_REF].

Lampreys have also an important ecological significance. The larvae are a key component in the food chain and can represent a large portion of the biomass in the river they inhabit. For instance, the North American river lamprey, Lampetra ayresii, represents the majority of the biomass found in sediments of the Fraser River (British Columbia; [START_REF] Beamish | Life history and abundance of young adult Lampetra ayresi in the Fraser River and their possible impact on salmon and herring stocks in the strait of Georgia[END_REF]. Lamprey larvae also facilitate nutrient cycles in the substrate (Docker, 2015). At the adult stage, the dead lamprey spawners provide a great amount of nutrients to ecosystems, like pacific salmon [START_REF] Naiman | Pacific salmon, nutrients, and the dynamics of freshwater and riparian ecosystems[END_REF]. Lampreys are also ecosystem engineers: The larvae increase the substrate oxygen level with their burrowing and feeding activities [START_REF] Shirakawa | Lamprey larvae as ecosystem engineers: physical and geochemical impact on the streambed by their burrowing behavior[END_REF] and adults increase stream bed complexity when moving rocks to construct their nest, which benefits to other species [START_REF] Hogg | Anadromous sea lampreys (Petromyzon marinus) are ecosystem engineers in a spawning tributary[END_REF]Docker, 2015).

Forty-one species of lampreys are documented (Renaud & Cochran, 2019) and they are classified into three families: the Geotriidae (one species), the Mordaciidae (three species) and the Petromyzontidae (37 species). The Petromyzontidae are found in the Northern Hemisphere whereas the others are found in the Southern Hemisphere [START_REF] Potter | Petromyzontidae: Lampreys[END_REF]. Eighteen lamprey species are parasitic, either anadromous or freshwater resident. The remaining twenty-three lamprey species are non-parasitic (Docker, 2009;Renaud & Cochran, 2019).

b. Life cycle

All lampreys' species have the same larval phase spent in freshwater [START_REF] Hardisty | The behaviour, ecology and growth of larval lampreys[END_REF][START_REF] Potter | Ecology of larval and metamorphosing lampreys[END_REF]. The larvae of lampreys are called ammocoetes. They have a worm-like shape, with no tooth and no eyes. The ammocoetes live burrowed in the river or streambed substrate, capturing food (microorganisms or other source of organic matter like detritus) with a feeding mucus that entraps the food when filtering the water overlying their burrow [START_REF] Moore | Feeding of larval lamprey[END_REF]. The ammocoetes are relatively sedentary but they can passively move downstream, especially if a flood event occurs [START_REF] Maitland | Review of the ecology of lampreys in Northern Europe[END_REF][START_REF] Potter | Ecology of larval and metamorphosing lampreys[END_REF]. They are also capable of upstream movement against slow current [START_REF] Quintella | The use of PIT telemetry to study movements of ammocoetes and metamorphosing sea lampreys in river beds[END_REF]. After 3 to 7 years, the ammocoetes undergo a metamorphosis. In Western Europe species, it occurs from September to November. The metamorphosis involves the development of functional eyes, the growth of the dorsal fin and the development of the oral disc with a tongue likepiston to be able to suck and attach itself to different surfaces [START_REF] Hardisty | The behaviour, ecology and growth of larval lampreys[END_REF][START_REF] Maitland | Review of the ecology of lampreys in Northern Europe[END_REF][START_REF] Potter | Ecology of larval and metamorphosing lampreys[END_REF]. Sharpened teeth develop in parasitic species like the sea and river lampreys whereas in non-parasitic species like the brook lamprey, teeth are blunt [START_REF] Maitland | Review of the ecology of lampreys in Northern Europe[END_REF]Docker, 2009;[START_REF] Potter | The taxonomy, phylogeny, and distribution of lampreys[END_REF]. At metamorphosis, the parasitic lampreys will become sexually immature adults.

They will have to start a feeding phase that can occur at sea or in freshwater. The transformation of lampreys that migrate from freshwater to seawater is similar to the smoltification process of anadromous salmonids [START_REF] Sunga | Functional re-organization of the gills of metamorphosing sea lamprey (Petromyzon marinus): preparation for a blood diet and the freshwater to seawater transition[END_REF][START_REF] Ferreira-Martins | A review of osmoregulation in lamprey[END_REF]. The feeding phase varies from some month to 4 years (Renaud & Cochran, 2019). Four feeding mode have been reported: blood or flesh feeding, blood and flesh feeding, and carrion feeding. The blood feeders tend to attack larger host than the flesh feeders (Renaud & Cochran, 2019), thus lampreys have different host species [START_REF] Taverny | Les lamproies en Europe de l'Ouest : Écophases, espèces et habitats[END_REF] but when the main host population decreases it can directly affect the abundance of lampreys (COSEWIC, 2008). At the end of the parasitic phase, lampreys can reach a body length twice or more the maximum length of larvae [START_REF] Docker | Lampreys: biology, conservation and control[END_REF]. Anadromous lampreys make an upstream freshwater migration [START_REF] Docker | Life history evolution in lampreys: alternative migratory and feeding types[END_REF] following the pheromones from conspecifics [START_REF] Gaudron | First evidence of attraction of adult river lamprey in the migratory phase to larval odour[END_REF] but they do not express a homing behaviour like salmon [START_REF] Spice | Neither philopatric nor panmictic: microsatellite and mtDNA evidence suggests lack of natal homing but limits to dispersal in Pacific lamprey[END_REF]. This behaviour could result in high gene flow among rivers [START_REF] Bracken | Contrasting population genetic structure among freshwater-resident and anadromous lampreys: the role of demographic history, differential dispersal and anthropogenic barriers to movement[END_REF]. They become sexually mature and mate in freshwater before dying because of a semelparous life cycle [START_REF] Hardisty | The behaviour, ecology and growth of larval lampreys[END_REF][START_REF] Potter | Petromyzontidae: Lampreys[END_REF]. The life cycle of non-parasitic lampreys diverges at metamorphosis when the sexual maturation begins and adult do not feed and remain freshwater resident (Figure 5). The spawning phase happens in the following months and they die shortly after [START_REF] Huggins | Communal spawning of brook and river lampreys, Lampetra planeri Bloch and Lampetra fluviatilis L[END_REF]Docker, 2009). Although lampreys are very interesting for evolutionary, pest control and restoration studies, they do not constitute the easiest biological model because of their long and complicated life cycle [START_REF] Lampman | Lamprey aquaculture successes and failures: A path to production for control and conservation[END_REF]. The fertilization rates are often very high under controlled condition [START_REF] Moser | Lamprey Reproduction and Early Life History: Insights from Artificial Propagation[END_REF][START_REF] Decanter | Sperm competition experiments reveal low prezygotic postmating isolation between parasitic and nonparasitic lamprey ecotypes[END_REF] but some transitions between life stages can be tricky and high mortality rates are observed [START_REF] Hjort | Fluctuations in the great fisheries of northern Europe viewed in the light of biological research[END_REF][START_REF] Sifa | The critical period of high mortality of larvae fish -A discussion based on current research[END_REF]. For example, only 30% of survival were observed at first feeding [START_REF] Lampman | Chapter twenty two: Developing techniques for artificial propagation and early rearing of Pacific lamprey (Entosphenus tridentatus) for species recovery[END_REF]. Overall, the development of the rearing techniques are promising even if the production of metamorphosed juveniles is not yet routinely accomplished [START_REF] Lampman | Lamprey aquaculture successes and failures: A path to production for control and conservation[END_REF].

c. Case of paired species

The so-called "paired species" are pairs of closely related parasitic and non-parasitic lampreys, which can be considered as either ecotypes or distinct species (Docker, 2009). This debate has been ongoing for over a century, and it still bothering lamprey biologists [START_REF] Enequist | Das Bachneunauge als ökologische Modifikation des Flussneunauges[END_REF]Docker, 2015). 17 of the 23 non-parasitic lamprey species seem to have evolved from a parasitic species based on their morphology. The remaining non-parasitic lampreys are called the "southern relicts". They do not have an obvious parasitic ancestor still present in their environment [START_REF] Potter | The taxonomy, phylogeny, and distribution of lampreys[END_REF]. Most paired species are morphologically similar during the larval phase and can be distinguished only at the adult stage by the larger size of parasitic species. Some non-parasitic resident species (Lampetra planeri and L. richardsoni) still exhibit key elements for osmoregulation in marine water, like chloride cells that are only used by their parasitic counterparts. However, these chloride cells are only found absent in a very old species of non-parasitic lamprey so they seem to take a very long time be lost [START_REF] Bartels | Variations in the presence of chloride cells in the gills of lampreys (Petromyzontiformes) and their evolutionary implications[END_REF]. This result highlights the fact that morphological criteria are not enough to determine the evolutionary relationships of paired species. Two hypotheses have been put forward to explain how the nonparasitic lampreys have evolved. The first scenario is that because of a lack of preys or a barrier to migration resulting of the glaciation and deglaciation 10 000 15 000 years ago (for the European river lamprey and the brook lamprey), the parasitic species became non-parasitic. The second scenario proposes that the non-parasitic species appeared because some events were altering the balance of benefits and costs of migration. These scenarios are not exclusive but the first one proposed that nonparasitism was mandatory to survive bad conditions and the second one proposed that non-parasitism is sometimes the better choice [START_REF] Giuseppe Zanandrea | Speciation among Lampreys[END_REF][START_REF] Espanhol | Evolutionary history of lamprey paired species Lampetra fluviatilis (L.) and Lampetra planeri (Bloch) as inferred from mitochondrial DNA variation[END_REF]Docker, 2009). d. The case of L. fluviatilis and L. planeri In Western Europe, lampreys are represented by three species: the sea lamprey Petromyzon marinus, the largest of the three that is parasitic and anadromous, the river lamprey Lampetra fluviatilis (LF), also parasitic and anadromous and its sister species, the brook lamprey L. planeri (LP), which is nonparasitic and resident (Figure 6). The two Lampetra species splitted from the sea lamprey around 10-30 Mya ago [START_REF] Docker | Phylogeny of the lamprey genus Lampetra inferred from mitochondrial cytochrome b and ND3 gene sequences[END_REF][START_REF] Kuraku | Time scale for cyclostome evolution inferred with a phylogenetic diagnosis of hagfish and lamprey cDNA sequences[END_REF]. At the larval stage, the ammocoetes of the two species are indistinguishable but at the adult stage, their size make them easily distinguishable (Figure 8). Lampetra planeri is about 10 to 15cm whereas the body length of L. fluviatilis reaches 20 to 30 cm. That size difference is observed in a lot of paired species given the feeding phase of parasitic lampreys after metamorphosis (Figure 7; [START_REF] Potter | Ecology of larval and metamorphosing lampreys[END_REF]. Regarding their distribution (Figure 9), the two Lampetra species are sometimes found in sympatry where they reproduce in the same habitat at the same time [START_REF] Huggins | Communal spawning of brook and river lampreys, Lampetra planeri Bloch and Lampetra fluviatilis L[END_REF]Lasne et al., 2010). Rougemont et al. (2017) found high levels of gene flow between LF and LP in several French

Rivers and proposed to consider them as partially reproductively isolated ecotypes. While a partial reproductive isolation by body size has been observed in a similar case of paired species, the North American river and brook lampreys L. ayresii and L. richardsoni (Beamish & Neville, 1992), cases of interspecific communal spawning have been observed in LP and LF (Lasne et al., 2010). A "sneaker" strategy of L. planeri males has been observed in that situation and could potentially participate in the high gene flow observed between L. planeri and L. fluviatilis and in the observation of potential hybrids (Rougemont et al., 2017). However, the investigations of potential other reproductive barriers than assortative mating by size are often limited due to the rearing difficulties of lamprey larvae for more than a few months. Currently, the controlled crosses experiments have not revealed any barrier between LP and LF but studies stopped at the F1 generation where heterosis could occur (Rougemont et al., 2015). Moreover, the hybrids identified in Rougemont et al. (2017) seemed to be mostly F1s, as hybrids of second generation were extremely rare, hence the hypothesis of some degree of hybrid breakdown (Rougemont et al., 2017).

GOALS OF THE THESIS

The general goal of the thesis is to better understand the speciation between two closely related species of lampreys: Lampetra fluviatilis and L. planeri. Before the start of the thesis we already knew that there was some partial reproductive isolation by body size (Beamish & Neville, 1992), outliers SNPs can help discriminate LP from LF (Rougemont et al., 2017, Souissi et al., 2022) and F1 larvae are viable at least in the first weeks (Rougemont et al., 2015). We used a multidisciplinary approach to investigate the genome differences between LP and LF and we also experimentally tested their reproductive isolation.

In chapter 2, we investigated the genomic architecture of divergence between LP and LF. We built a high quality LP genome and a linkage map with RAD-Seq data. We also looked for chromosomal rearrangements and conducted a functional analysis of the differentiated loci between LP and LF.

In chapter 3, we investigated the potential existence of postmating prezygotic barriers between LP and LF. Given the low level of reproductive isolation, one of the first barrier to emerge could be a gametic barrier linked to cryptic female choice or sperm competition. We hypothesized a form of conspecific sperm precedence when the sperm of both male ecotypes are in competition for the eggs of one female ecotype. To test our hypothesis we performed an experiment of sperm competition with controlled crosses between LP and LF.

In chapter 4, we measured the frequency of hybrids in a natural hybrid zone (Figure 10). Our hypothesis was that F1s should be more frequent than second-generation hybrids due to hybrid breakdown. We identified discriminating SNPs between LP and LF and genotyped them in 1292 individuals sampled in the Oir River from 2010 to 2021. Controlled crosses between LP and LF and their hybrids were also conducted to experimentally test the existence of a hybrid breakdown in second-generation hybrids.

I finish this manuscript with a discussion summarizing the main results, and I propose new perspectives to address the unresolved questions.

CHAPTER 2

Genomic divergence between parasitic and non-parasitic lamprey ecotypes

Nolwenn Decanter 1 , Quentin Rougemont 2 , Arne Jacobs 3 , Gilles Lassalle 1 , Ahmed Souissi

Manuscript in preparation

ABSTRACT

Speciation is a key issue in evolutionary biology but the underlying mechanisms of genomic divergence remain largely unknown. Here we investigate the genomic architecture of divergence between two closely lamprey species, the freshwater resident brook lamprey (Lampetra planeri; LP) and the anadromous parasitic river lamprey (L. fluviatilis; LF). Despite their different life histories, the two species reproduce in the same habitats, can hybridize and are often considered as ecotypes. In previous studies, high levels of gene flow were found in sympatric populations and some outlier SNPs were documented between ecotypes. We assembled a high quality genome of LP and built a linkage map based on 32 681 SNPs to determine the divergent regions of the genome. We also looked for structural variants in those regions and annotated them. The most divergent regions were located in only two linkage groups out of 84. The most divergent linkage group included a putative large inversion. The annotation revealed genes involved in development, coagulation, immunity and inflammatory response thus potentially linked to the morphological and physiological differences between LP and LF. Our results highlight that genetic differences between ecotypes can be highly clustered within the genome and suggest the important role of a large inversion in genomic divergence.

INTRODUCTION

The study of speciation mechanisms is the key to understand how new species arise and how biodiversity is generated (Coyne & Orr, 2004). As populations start to diverge from one another, genetic barriers accumulate and eventually reduce the fitness of hybrids, which decreases gene flow and increases genomic divergence [START_REF] Roux | Shedding light on the grey zone of speciation along a continuum of genomic divergence[END_REF]. The genomic divergence includes nucleotide substitutions, changes in gene expression and chromosomal rearrangements. These changes can affect proteins coding genes, regulatory elements or non-coding RNAs and can ultimately lead to phenotypic changes between populations. Over the last decades, the technological progress made in sequencing boosted studies of the genomic architecture of divergence [START_REF] Ravinet | Interpreting the genomic landscape of speciation: a road map for finding barriers to gene flow[END_REF]. The barrier loci that contribute to the reduction of gene flow between populations can now be identified. These barrier loci will increase genetic differentiation typically measured with FST, the fixation index. Genome scans are used to analyse population differentiation at multiple loci across the genome to find outlier loci that fall outside the expected neutral distribution of differentiation [START_REF] Ravinet | Interpreting the genomic landscape of speciation: a road map for finding barriers to gene flow[END_REF]. The genomic differentiation is often heterogeneous during the speciation process and regions with outlier loci present a higher differentiation [START_REF] Wu | The genic view of the process of speciation[END_REF][START_REF] Nosil | Ecological Speciation[END_REF]. These regions are called "genomic islands" of speciation and are expected to contain genes responsible for reproductive isolation, which remain differentiated despite ongoing gene flow [START_REF] Turner | Genomic islands of speciation in Anopheles gambiae[END_REF]. For instance, 98 genomic islands were found between two cichlids ectomorphs Astatotilapia sp living in a crater lake in Tanzania. These genomic islands contain candidate genes linked with mate preference (sensory perception, hormone signalling and morphogenesis [START_REF] Malinsky | Genomic islands of speciation separate cichlid ecomorphs in an East African crater lake[END_REF]. In Drosophila yakuba subspecies, regions with high differentiation overlap with chromosomal inversions [START_REF] Ferreira | Genomic islands of divergence between Drosophila yakuba subspecies predominantly overlap with chromosomal inversions[END_REF]. Accordingly, chromosomal rearrangements, like inversions, could be implicated in reproductive isolation (Zhang et al., 2021).

Lampreys belong to the superclass Agnatha and are thus primitive jawless fish along with hagfish (Docker, 2015). The lamprey genome undergoes Programmed Genome Rearrangements (PGRs) that lead to the elimination of ~ 20% of the genome from somatic cells during early embryogenesis [START_REF] Smith | Genetic consequences of programmed genome rearrangement[END_REF][START_REF] Timoshevskiy | Cellular and molecular features of developmentally programmed genome rearrangement in a vertebrate (Sea Lamprey: Petromyzon marinus)[END_REF][START_REF] Timoshevskiy | Deep ancestry of programmed genome rearrangement in lampreys[END_REF][START_REF] Smith | The sea lamprey germline genome provides insights into programmed genome rearrangement and vertebrate evolution[END_REF]. Thus, the only way to sequence the whole genome is to use germline cells. Some pairs of lamprey species (parasitic and non-parasitic) are so alike morphologically and genetically that they are called "paired species". Generally, the nonparasitic species is thought to derive from the parasitic species (Docker, 2009(Docker, , 2015)). These "paired species" exhibit, at the adult stage, different morphology, behaviour and life histories and are considered as ecotypes or species depending on their degree of reproductive isolation (Docker, 2009(Docker, , 2015)). These closely related species with contrasted life histories make them an ideal biological model to study speciation. Here, we focus on the European paired species Lampetra fluviatilis (parasitic and anadromous; LF) and L. planeri (non-parasitic and freshwater resident; LP). Their populations can be found in sympatric areas that can be considered as hybrid zones. Despite a partial reproductive isolation by body length (Beamish & Neville, 1992) as LF is much larger than LP, matings between LP and LF have been observed and hybrids were identified in several hybrid zones (Lasne et al., 2010;Bracken et al., 2015b;Rougemont et al., 2017). As a result, they can be considered as partially reproductively isolated ecotypes (Rougemont et al. 2015(Rougemont et al. , 2017)). In this study, we aimed to describe and understand the genomic divergence between LP and LF using novel genomic resources. We assembled a high quality L. planeri genome, constructed a linkage map and generated long read data of LF to explore chromosomal rearrangements. We also explored the gene content of the most differentiated regions between LP and LF.

MATERIAL AND METHODS

Sampling, library construction and sequencing

A male brook lamprey (LP) was sampled in the Oir River (Normandy -France) in 2019 for genome sequencing. The testis cells and sperm were used for sequencing as the lampreys undergo PGRs (Smith et al., 2009a). High molecular weight (>50kb) genomic DNA extraction was performed according to [START_REF] Talsania | Genome assembly and annotation of the trichoplusia ni Tni-FNL insect cell line enabled by long-read technologies[END_REF] with minor modifications. Briefly, the sperm sample was embedded in a 2%

agarose plug, incubated with Proteinase K (50µg/mL) overnight at 50C, followed by RNaseA treatment (10µg/mL, NEB) at 37C for 1 hour and further washed in TE buffer. Plug degradation was performed by incubation of the sample at 55C for 10min and afterwards with beta-agarase (1 unit, NEB) at 43C for 1 hour. DNA purification was performed by drop dialysis; using a wide-bore micropipette tip for transfer onto a 0.1µm membrane placed on TE buffer and dialyzed for 30min. DNA concentration and quality control were assessed using Nanodrop, Qubit, Fragment Analyzer and Femto pulse methods. 15µg of genomic DNA was used for downstream procedures. The DNA was then sequenced with the HiFi PACBIO technology to obtain high-quality long reads. The Circular Consensus Sequencing (CSS) reads produced by the sequencing platform were used for subsequent analyses (see raw data report in Appendix A.1).

Genome assembly

The HiFi CSS reads were assembled with two different assemblers to compare their performances and get the best assembly: FALCON [START_REF] Chin | Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data[END_REF](Chin et al., , 2016) ) and HiFiasm [START_REF] Cheng | Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm[END_REF]. With FALCON, the CSS reads were passed through fc_run.py to realize the assembly and then to FALCON_UNZIP with fc_unzip.py that takes the contigs from FALCON to obtain partially phased contigs. The data file (.cfg) used as input for both falcon python scripts along with the complete assembly statistics can be found in Appendix A.2. The command lines used for the HiFiasm assembly and the complete assembly statistics can be found in Appendix A.3. The assembly statistics were computed with QUAST v5.0.2 [START_REF] Mikheenko | Versatile genome assembly evaluation with QUAST-LG[END_REF].

Linkage map

To construct our linkage map, we used the RAD sequencing data (609 individuals distributed in 17 populations) from (Rougemont et al., 2017). We first demutiplexed the raw data using the program process_radtags of the Stacks software v2.4 [START_REF] Catchen | Stacks: an analysis tool set for population genomics[END_REF][START_REF] Rochette | Stacks 2: Analytical methods for paired-end sequencing improve RADseq-based population genomics[END_REF] ; example of command line in Appendix A.4). process_radtags was used to check reads overall quality, trimmed read to 85bp and removed reads containing Illumina adapters. The reads of each individual were aligned to the HiFi LP genome using BWA-mem [START_REF] Li | Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM[END_REF]. The output was then transformed from a

.sam file to a sorted .bam file with SAMtools [START_REF] Danecek | Twelve years of SAMtools and BCFtools[END_REF].

We then took advantage of our LP genome to run gstacks (Stacks v2.4) in reference-based mode. As advised by Catchen (personal communication), we removed the PCR duplicates via gstacks. The unpaired reads were removed; a minimum PHRED-scaled mapping quality of 25 was set to consider a read. We also applied a maximum soft clipping level of 5% of the read length to reduce sequencing errors [START_REF] Li | Fast and accurate short read alignment with Burrows-Wheeler transform[END_REF].

The populations program (Stacks) was used to export the results in a .vcf file (Variant Call Format) for further filtering and only keep the useful individuals for the linkage map: the 2 parents (female LF and male LP from Oir population) and their 90 offspring. The SNPs at positions 60 were removed due to a sequencing problem occurring at this position (Rougemont et al. 2017). We filtered SNPs using VCFtools [START_REF] Danecek | The variant call format and VCFtools[END_REF] to remove low quality genotypes and SNPs. We kept genotypes with a coverage ranging from 5x to 100x and a mean coverage ranging from 5x to 100x. We also only included bi-allelic sites with less than 60% missing data, and a minor allele count of one. Then we filtered out two individuals with a mean depth inferior to 10 and more than 25% missing data. We checked for mendelian inheritance errors with BCFtools [START_REF] Danecek | Twelve years of SAMtools and BCFtools[END_REF]. We removed one individual with many mendelian errors and then made a second round of SNPs filtering, the same as before but we only kept SNPs with less than 30% missing data. We also sought singletons with the option --singletons of VCFtools and removed them (see filtering command line Appendix A.5).

We used LepMap3 (Rastas, 2017) to construct the linkage map. Two different pipelines were tested to construct the map: with or without Filtering2. When using filtering, Filtering2 was run with the following parameters: removeNonInformative=1 and dataTolerance=0.0001. Small dataTolerance like this is advised in single-family crosses. Then, for the SeparateChromosomes2 module we computed different linkage maps testing different lodLimit ranging from 6 to 12 and removed the LG computed with less than 20 SNPs with sizeLimit=20. When not using Filtering2, we ran SeparateChromosomes2 the same way as before but with the parameter distorsionLod=1 as advised for single family data (see example of command lines in Appendix A.6). We expected approximatively 82 chromosomes in L.

planeri and L. fluviatilis [START_REF] Robinson | The nuclear DNA content of lampreys[END_REF]. We chose to continue the analysis with the pipeline with filtering because without, the first LGs contained too many markers and very few were left in the others. The selected map was passed through the module JoinSingles2All with parameters lodLimit=6 and lodDifference=2.

To compute FST between LP and LF for the mapped SNPs, we used the data of (Rougemont et al., 2017) from 121 individuals (66 LF and 55 LP) coming from 4 sympatric populations (Oir, Risle, Aa, Bethune).

Twelve individuals were removed from the dataset because they had more than 40% missing data. The FST was computed in these 109 individuals and also only in a dataset made of pure LF and LP identified in the Oir population in Chapter 4 using the diffCalc function of the package diversity [START_REF] Keenan | diveRsity: An R package for the estimation and exploration of population genetics parameters and their associated errors[END_REF]; See command line in Appendix A.7). Two Manhattan plots of FST along the linkage groups (one with the 109 individuals (57 LF and 52 LP) and one with only the pure individuals of the Oir River ( 17LF and 22 LP) were computed using ggplot2 v 3.4.0 [START_REF] Wickham | ggplot2[END_REF]. The eigenvalues and eigenvectors were computed for the SNPs found in LG6 (n = 804) and LG26 (n = 457) with PLINK (v1.9; www.coggenomics.org/plink/1.9/ ; [START_REF] Chang | Second-generation PLINK: rising to the challenge of larger and richer datasets[END_REF]See command line in Appendix A.8). A PCA was then realized using ggplot2 v 3.4.0. The heterozygosity was calculated with the option --het of VCFtools [START_REF] Danecek | The variant call format and VCFtools[END_REF] and boxplots were realized using ggplot2 v 3.4.0.

Structural variants

We made a Continuous Long Reads (CLR) PacBio sequencing of a LF individual of the Oir population (see the raw data report in Appendix A.9). These long reads were aligned against the previously assembled LP HiFi genome with PBMM2 (a SMRT C++ wrapper for minimap2 and part of the tools of PacBio Secondary Analysis Tools on Bioconda; https://github.com/PacificBiosciences/pbbioconda) and with minimap2 [START_REF] Li | Minimap2: pairwise alignment for nucleotide sequences[END_REF]. Then the structural variants where searched for with PBSV (PacBio structural variant (SV) calling and analysis tools; https://github.com/PacificBiosciences/pbbioconda).

Structural and functional annotation

For the genome annotation, we chose to use RNA-Seq data combined with our reference LP genome. Four LP adults (two males and two females), LF adults (two males and two females), and four larvae that were caught by electrofishing in the Oir River in April 2018, anesthetized, and then killed with a lethal dosage of benzocaine. We collected their liver, brain, gonad and gill (total = 32 samples) for the adults and the entire larvae, which were immediately frozen in liquid nitrogen.

Total RNA was extracted from brains and gonads with the NucleoSpin® RNA Plus XS extraction kit following manufacturer's instructions (Macherey-Nagel); other organs were processed with TRIzol® Reagent. Paired-end RNA sequencing was performed with a HiSeq3000 Illumina sequencer. Individual libraries were sequenced in 5 lanes, with an average of 3x10 7 reads per sample. Raw sequences were trimmed from Illumina adaptors, and resulting sequences shorter than 75bp were removed. The average quality per base was high (quality score above 28). The RNA-Seq data were aligned against the genome using STAR aligner, a universal RNA-Seq aligner [START_REF] Dobin | STAR: ultrafast universal RNA-seq aligner[END_REF].Then Braker2 was used with RNA-Seq evidence to make predictions of gene models [START_REF] Brůna | BRAKER2: automatic eukaryotic genome annotation with GeneMark-EP+ and AUGUSTUS supported by a protein database[END_REF]. An annotation of transcripts using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) was realized via OmicsBox (https://www.biobam.com/omicsbox) with

EggNOGmapper v2 [START_REF] Cantalapiedra | eggNOG-mapper v2: Functional annotation, orthology assignments, and domain prediction at the metagenomic scale[END_REF] and the EggNOG 5.0 database [START_REF] Huerta-Cepas | eggNOG 5.0: a hierarchical, functionally and phylogenetically annotated orthology resource based on 5090 organisms and 2502 viruses[END_REF]. We then extracted the annotation of GO and KEGG terms for the two mostly differentiated linkage groups between LF and LP. The GO terms were first passed through the CateGOrizer free web tool [START_REF] Hu | CateGOrizer: A web-based program to batch analyze gene ontology classification categories[END_REF] to helps hierarchize the terms and create relationships between the terms in a biological context. The GO_slim GO classification method was used. The output of CateGorizer then served as an input to REVIGO [START_REF] Supek | REVIGO summarizes and visualizes long lists of gene ontology terms[END_REF] to summarize and visualize the list of GO terms according to the Uniprot annotation. The KEGG annotations [START_REF] Kanehisa | KEGG as a reference resource for gene and protein annotation[END_REF] were given as input to KEGG Mapper tool Reconstruct [START_REF] Kanehisa | KEGG Mapper for inferring cellular functions from protein sequences[END_REF] to recover the distribution of our KEGG terms in KEGG pathways.

RESULTS

L. planeri reference genome

The PACBIO HiFi sequencing produced 715 Gb of raw data (See Appendix A.1) that were converted into 19.3 Gb of CCS reads by the sequencing platform. The expected genome size of L. planeri is between 1.31 and 1.39Gbp [START_REF] Gregory | Animal Genome Size Database[END_REF] thus we chose to keep the HiFiasm assembly for further analyses given the 1.27 Gb length of this assembly (Table 1).

Linkage map

A dataset of 89 individuals (87 offspring and 2 parents) and 39 896 SNPs was used as input in Lepmap3.

The ParentCall2 module informed us that 35 295 markers were informative and after using Filtering2, 32 875 SNPs were kept for the pipeline using it. Finally, we chose the linkage map with the best LOD score and the lowest number of single markers (Table 2). After passing thought JoinSingle2All, the final dataset held 32 681 SNPs distributed in 85 linkage groups The PCAs of LG 6 and LG 26 are completely different. In LG 6 the differentiation between LP and LF is important but the two groups are overlapping (Figure 2a). In contrast, in LG 26 the two ecotypes are distributed into three groups with no overlapping and a vertical distribution (Figure 2b). The heterozygosity in the second group (mean ± SD; 0.539 ± 0.119) of the PCA from LG 26 is much higher than in the first (0.19 ± 0.032) and third groups (0.083 ± 0.021; Figure 3). The full PBSV protocol revealed a very high number of chromosomal rearrangements in LG 6 and LG 26 (Table 3). Regarding the inversions in LG 26, overall they are quite small (range of length of the structural variants: from 144bp to 2 077bp). In contrast, with the alignment from minimap2, only 12 structural variants were detected and no inversion was identified.

Search of structural variants

Structural and functional annotation

In total, 41 201 potential transcripts/genes sequences were available for annotation and half of the sequences (n = 21 891) were fully annotated. These fully annotated genes are strong candidates for being actual complete genes in the genome. A complete report of the EggNOG annotation for the whole genome can be found in Appendix A.10. An exploration of the GO and KEGG annotations were performed on the two linkage groups showing the highest differentiation: LG 6 and LG 26. Out of 876 transcripts on LG 6, 698 were assigned to at least one GO term and in LG 26, out of 180 transcripts, at least 152 were assigned to at least one GO term. In the two LGs, we observed similar proportions of the main GO terms: Biological Process (BP), Cellular Component (CC) and Molecular Function (MF). For

LG 6, we found 71.6 % of BP GO terms, 20% of CC terms, and 8.4% MF terms. For LG 26, we found 72.1 % of BP GO terms, 20.3% of CC terms, and 7.6% MF terms. The transcripts were mainly categorized into 51 functional groups in LG6 (Figure 4a) and into 52 functional groups in LG 26 (Figure 4b). For the BP category, the three principal groups in LG 6 and LG 26 were the "multicellular organism development", "metabolic process" and "cellular component organization". For the CC category, in both LG 6 and LG 26, the transcripts involved in binding and catalytic process were the largest groups.

For MF in LG 6 and LG 26, the main two groups were the intracellular anatomical structure and the cytoplasm. The third group was the plasma membrane for LG 6 and the nucleus for LG 26.

To further investigate the biological function present in those LGs we then used the KEGG annotations.

For LG 6, out of 876 transcripts, 637 were assigned to at least one KEGG pathway and for LG 26, out of 180 transcripts, 149 were assigned to a KEGG pathway. In both LGs, the most represent KEGG pathways are the signal transduction, the immune system and the endocrine system (Figure 5 a-b).

Furthermore, we identified 103 different genes in the LG 26, (see list in Appendix A.11). Twenty-seven genes, containing the 119 SNPs with the highest FST (all with FST = 1) were more specifically analysed (see Appendix A.12). On of 119 SNPs, only 75 fall into detected transcripts and 43 are in annotated transcripts corresponding to 27 different genes. Out of the 27, eight are genes involved in development, one in coagulation, and three in immunity and inflammatory process.

DISCUSSION

The HiFi LP genome assembled here is currently the first available for this taxon but it could be further improved at the chromosome level like the sea lamprey (Smith et al., 2013) or reissner lamprey genomes [START_REF] Zhu | Chromosome-level genome assembly of Lethenteron reissneri provides insights into lamprey evolution[END_REF]. To better understand the genome structure with this assembly we made a linkage map that is also the first for the Lampetra genus. Most of the divergence between LP and LF seems to occur in two linkage groups: LG6 and LG 26. These peaks of differentiation in the genome are in accordance with the genic view of speciation in which, when populations are in contact, gene flow is first restricted to barrier loci [START_REF] Wu | The genic view of the process of speciation[END_REF]. If speciation progresses, the differentiation will spread due to linkage disequilibrium [START_REF] Ravinet | Interpreting the genomic landscape of speciation: a road map for finding barriers to gene flow[END_REF]. Compared to e.g. the 98 peaks of differentiation found by [START_REF] Malinsky | Genomic islands of speciation separate cichlid ecomorphs in an East African crater lake[END_REF] between cichlid ecotypes, we detected a low number of genomic islands of differentiation. This situation is thus congruent with a scenario of genomic islands of speciation involving only few genes [START_REF] Nosil | How many genetic changes create new species?[END_REF]. A similar example is the one of the large genomic regions containing 81 out of 82 fixed differences between all-black carrion crowns and gray-coated hooded crowns. These regions include genes involved in prezygotic isolation (genes coding for pigmentation and visual perception). It proves that even localized genomic divergence could still maintain phenotypic divergence [START_REF] Poelstra | The genomic landscape underlying phenotypic integrity in the face of gene flow in crows[END_REF]. The two bird species Sporophila iberaensis and S.

hypoxantha, also display very low genome wide differentiation with only three small regions on three different chromosomes presenting a high differentiation. The genes contained in those peaks code for plumage coloration and thus, promote premating isolation by assortative mating based on plumage coloration and song [START_REF] Turbek | Rapid speciation via the evolution of pre-mating isolation in the Iberá Seedeater[END_REF]. Sex chromosomes also display higher divergence levels than autosomes between closely related species [START_REF] Irwin | Sex chromosomes and speciation in birds and other ZW systems[END_REF][START_REF] Presgraves | Evaluating genomic signatures of "the large X-effect" during complex speciation[END_REF]. However, we could not investigate this hypothesis because the sex determination of lamprey is poorly known and up to date, no sex chromosome have been identified and environmental factors are probably involved in sex determination [START_REF] Smith | Programmed loss of millions of base pairs from a vertebrate genome[END_REF][START_REF] Smith | Genetic consequences of programmed genome rearrangement[END_REF]. However, a recent study on germlines specific genes (GSGs)

of the sea lamprey suggest that these genes have a major role in testicular differentiation and potentially in sex determination. These GSGs are mostly expressed in males undergoing spermatogonial differentiation while most of them are lowly expressed in females [START_REF] Yasmin | Pervasive male-biased expression throughout the germline-specific regions of the sea lamprey genome supports key roles in sex differentiation and spermatogenesis[END_REF].

Our search for putative chromosomal rearrangements was mostly not conclusive. We tried two different pipelines to detect the SVs and one detected a lot of such SVs but not the other. In the future, it will be necessary to sequence more individuals with long reads to align them against our reference genome.

Interestingly, the high differentiation between LP and LF found in LG 26 highlighted the low recombination in this LG. Accordingly, in this LG the PCA revealed three discrete clusters with the middle one having a higher heterozygosity, which suggests the presence of an inversion in this region [START_REF] Huang | Multiple chromosomal inversions contribute to adaptive divergence of a dune sunflower ecotype[END_REF]Mérot, 2020). In contrast, it is predicted that regions under selection present continuous patterns of population structure in PCA like we found for LG 6 [START_REF] Huang | Multiple chromosomal inversions contribute to adaptive divergence of a dune sunflower ecotype[END_REF]. Multiple chromosomal inversions have been found to be involved in adaptive divergence as for instance in plants like Helianthus petiolaris dune and non-dune ecotypes [START_REF] Huang | Multiple chromosomal inversions contribute to adaptive divergence of a dune sunflower ecotype[END_REF] or in seaweed flies [START_REF] Mérot | Balancing selection via life-history trade-offs maintains an inversion polymorphism in a seaweed fly[END_REF]). Chromosomal rearrangements have long been known to promote genomic differentiation [START_REF] Rieseberg | Chromosomal rearrangements and speciation[END_REF]. Among SVs, the inversions directly supress recombination and have been suggested to allow differentiation by protecting genome regions from gene flow and thus possibly protect beneficial allelic combinations [START_REF] Rieseberg | Chromosomal rearrangements and speciation[END_REF][START_REF] Kirkpatrick | Chromosome inversions, local adaptation and speciation[END_REF][START_REF] Christmas | Chromosomal inversions associated with environmental adaptation in honeybees[END_REF].

In addition, we wanted to evaluate which genes were present in LG 6 and LG26. The most abundant transcripts concerned development and metabolic processes that are potentially involved in phenotypic differences of LP and LF and in their different metabolism at the adult stage. Lampetra fluviatilis develops a piston-like tongue and razor-like teeth to be able to pierce the skin of the fish it will feed on (Borowiec et al., 2021). Buccal secretions with anti-coagulant properties aid this mechanical action. The lampreys produce lamphredin that is a mix of anticoagulants, immune suppressors and ion channels blockers to facilitate the feeding on living hosts [START_REF] Xiao | The buccal gland of Lampetra japonica is a source of diverse bioactive proteins[END_REF]. In our annotation we found a gene (F10) which is involved in the coagulation system and thus, might potentially be involved in lamprey parasitic life [START_REF] Hu | Genome editing of factor X in zebrafish reveals unexpected tolerance of severe defects in the common pathway[END_REF]. In the same way, genes involved in the immunity system like NOD2 could be linked to immune suppressors production or be immune receptors and could thus potentially explain the high level of immunity system genes found in the KEGG pathways [START_REF] Klinker | Multiple mechanisms of immune suppression by B lymphocytes[END_REF][START_REF] Drake | Modulation of the NOD-like receptors NOD1 and NOD2: A chemist's perspective[END_REF].

The most represented KEGG pathway was signal transduction. Signal transduction cascades are important for lampreys that use many chemicals cues in their communication. For reproduction, sex pheromones are released to attract individuals of the opposite sex. The signal transduction from the recognition of the pheromone to the guiding behaviour to potential mates is important [START_REF] Li | Sex pheromone communication in the sea lamprey: Implications for integrated management[END_REF][START_REF] Buchinger | Chemical cues and pheromones in the sea lamprey (Petromyzon marinus)[END_REF]. During migration, adult lampreys recognize chemical cues of larvae bile acids to enter rivers [START_REF] Li | The olfactory system of migratory adult sea lamprey (Petromyzon marinus) is specifically and acutely sensitive to unique bile acids released by conspecific larvae[END_REF]. Moreover, the endocrine system is also highly represented and is important for the biosynthesis and the release of pheromones [START_REF] Li | Sex pheromone communication in the sea lamprey: Implications for integrated management[END_REF]. In addition, we found 10 SNPs locating in an ortholog of DAND5, among them 9 are in exons and 1 locate in a CDS (coding DNA sequence). DAN gene family plays a fundamental role in morphogenesis, left-right axis specification, kidney formation and limb bud formation [START_REF] Opazo | Evolution of the DAN gene family in vertebrates[END_REF]. Thus, it is a good candidate for phenotypic difference between L. planeri and L. fluviatilis. Furthermore, FNDC3A

(Fibronectin) have 11 SNPs in intron regions. In lampreys, fibronectins plays a major role in cell matrix adhesion and tissue organisation and is found in all its connective tissues and in plasma [START_REF] Wright | Immunocytochemical study of fibronectin in the sea lamprey, Petromyzon marinus, and the Atlantic hagfish, Myxine glutinosa[END_REF].

However, further analyses are needed to validate the candidate genes detected in this study.

CONCLUSION

This study showed that the genomic divergence between LP and LF is concentrated into two genomic regions. In one of these regions, a large inversion is probably responsible for the high differentiation observed. Sequencing more individuals from both ecotypes (in CLR, HiFi or Hi-C) could improve the genome assembly and better characterize the putative large inversion in LG 26. In addition, a less fragmented assembly could allow the detection of inversion breakpoints. Besides, the results of the GO and KEGG annotation are promising because the transcripts found are potentially involved in key differences between LP and LF. A more precise gene annotation and differential gene expression analyses in LG 6 and LG 26 could further help to reveal the genetic determinants of the divergence between LP and LF.

CHAPTER 3

Sperm competition experiments reveal low prezygotic postmating isolation between parasitic and nonparasitic lamprey ecotypes

Nolwenn Decanter 1 , Romane Normand

ABSTRACT

Postzygotic isolation is a key mechanism in the speciation process. Postzygotic barriers can be identified with direct experimental approaches or with population genetics tools to infer their effect on gene flow. Here used both direct and indirect approaches to infer postzygotic isolation between two species of lampreys of the genus Lampetra. L. fluviatilis (LF) is parasitic of other fish species and anadromous, whereas L. planeri (LP) is non-parasitic and resident in freshwater. Both species reproduce in the same habitats, can hybridize, and are considered as ecotypes of the same species. However, the viability and fecundity of hybrids in the wild are not well known. Previous population genetics studies have revealed the occurrence of F1 hybrids but F2 (i.e. F1 x F1) or backcrosses (i.e. F1 x F0) seem rare, hence the hypothesis that those types of hybrids could experience hybrid breakdown. To investigate this hypothesis we developed a panel of discriminating SNPs between LP and LF to identify different types of hybrids in 1292 individuals sampled between 2010 and 2021 in a hybrid zone. In parallel, individual-based simulations of various scenarios of hybrid viability and partial premating barriers were performed to explain the observed frequency of hybrids in the wild. We also made experimental crosses between LP, LF and their hybrids to study potential sterility or inviability in hybrids of second generation. Our results revealed a high proportion of hybrids in the wild, which could not be mirrored by any of our simulated scenarios. In addition, no postzygotic barrier was detected in experimental crosses. Interestingly, this high proportion of hybrids might be linked to a heterozygous advantage for an inversion.

INTRODUCTION

The speciation is a gradual process of evolution of reproductive isolation by which an ancestral species populations could result in two or more species [START_REF] Rabosky | Reproductive isolation and the causes of speciation rate variation in nature[END_REF][START_REF] Ravinet | Interpreting the genomic landscape of speciation: a road map for finding barriers to gene flow[END_REF]Stankowski & Ravinet, 2021). The mechanisms of reproductive isolation are numerous and consists of the establishment of incompatibilities that reduce or prevent the reproduction between populations.

These barriers to reproduction can be endogenous or exogenous and can happen at the premating, postmating prezygotic or the postzygotic stage (Coyne & Orr, 2004). The areas where populations of different species are in contact and can reproduce are called hybrid zones. Hybrid zones are veritable snapshots of the speciation process and thus are the best spot to study speciation [START_REF] Seehausen | Hybridization and adaptive radiation[END_REF][START_REF] Hewitt | Quaternary phylogeography: the roots of hybrid zones[END_REF]. In hybrid zones, barriers to reproduction can limit the fertility and/or viability of hybrids (Coyne & Orr, 2004). For instance, in hybrid zones between Mus musculus and M. domesticus, a reduced male hybrid fertility is commonly found. This sterility varies in severity depending on the hybrid zones but helps to maintain the separation between the two subspecies [START_REF] Albrechtová | Sperm-related phenotypes implicated in both maintenance and breakdown of a natural species barrier in the house mouse[END_REF][START_REF] Turner | Genome-wide mapping in a house mouse hybrid zone reveals hybrid sterility loci and Dobzhansky-Muller interactions[END_REF]. The incompatibilities found in hybrid zones are often associated with maladaptative genotypes, which results in a partial sterility and more rarely in full sterility and inviability [START_REF] Hagberg | Extensive introgression at late stages of species formation: Insights from grasshopper hybrid zones[END_REF]. In Drosophila and in the melanogaster species complex, the hybrids are often inviable [START_REF] Matute | Comparative studies on speciation: 30 years since Coyne and Orr[END_REF][START_REF] Hagberg | Extensive introgression at late stages of species formation: Insights from grasshopper hybrid zones[END_REF].

Lampreys are jawless fish that are with the hagfish, the oldest group of vertebrates (Docker, 2015). In lampreys, 17 of the 23 non-parasitic species seem to have evolved from parasitic species, thus forming what is called "paired species". These paired species are closely related and have very similar life histories and morphology until metamorphosis. The parasitic ones feed on bony fish for several month or years before reproduction whereas the non-parasitic species reproduce directly after metamorphosis. It is still debate whether these paired species are distinct species or just ecotypes or forms of the same species [START_REF] Enequist | Das Bachneunauge als ökologische Modifikation des Flussneunauges[END_REF]Docker, 2009Docker, , 2015)). Several cases of hybrid zones were described between two European lamprey species: Lampetra fluviatilis (LF) and L. planeri (LP) (Rougemont et al., 2017). LF is anadromous and parasitic of other fish whereas LP is freshwater resident and non-parasitic. Despite these different life histories, populations of both species can be found in the same reproduction area, thus forming hybrid zones. Both species have been spotted reproducing together (Lasne et al., 2010) and some individuals were identified as hybrids in the Oir River by Rougemont et al. (2017) using a RAD-Seq approach. This previous study has shown the presence of a significant number of individuals that could be F1 (20 out of 316 sampled individuals) but found very few F2 (i.e. F1 x F1; one of 316 sampled individuals) or backcrosses (BC, i.e. F1 x F0; one of 316 samples individuals). As a result, those hybrids of second generation may have a lower fitness due to hybrid breakdown caused by reproductive barriers between species. Hybrid breakdown is observed after the first hybrid generation due to the expression of recessive Dobzhansky-Müller incompatibilities that were silenced in heterozygous F1 (Dobzansky, 1946;Templeton et al., 1986). For example, in cichlid fish F2s present a significant reduction of fitness compared to the F1 and non-hybrid crosses. To explore our hypothesis of hybrid breakdown in F2 and BC between LP and LF, we developed a panel of 189 SNPs and genotyped 1292 individuals sampled over 10 years in a hybrid zone to measure the frequency of hybrids. We expected higher hybrid proportions in juveniles than in adults due to hybrid breakdown, and also potential temporal variations in admixture due to demographic variations of parental species. To investigate which factors may explain observed hybrid frequencies, we developed a simulation approach mimicking the life cycle of our biological model. In particular, we tested the effects of assortative mating, dispersal rate, fecundity and hybrid fitness on hybrid frequencies. Finally, analyses of sperm quality and experimental crosses between pure and hybrid individuals were performed to experimentally test the existence of intrinsic barriers between LF and LP.

MATERIAL AND METHODS

Markers detection

To develop a set of about 200 SNPs allowing the distinction of LP, LF and their hybrids, we used the RAD sequencing data (609 individuals distributed in 17 populations) from Rougemont et al. (2017). We first demutiplexed the raw data using the program process_radtags of the Stacks software (v2.4; [START_REF] Catchen | Stacks: an analysis tool set for population genomics[END_REF][START_REF] Rochette | Stacks 2: Analytical methods for paired-end sequencing improve RADseq-based population genomics[END_REF]. process_radtags was used to check reads overall quality, trimmed read to 85bp and removed reads containing Illumina adapters. The reads of each individual were aligned to the HiFi LP genome using BWA mem [START_REF] Li | Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM[END_REF]. The output was then transformed from a .sam file to a sorted .bam file SAMtools [START_REF] Danecek | Twelve years of SAMtools and BCFtools[END_REF].

We then took advantage of our LP genome to run gstacks (Stacks v2.4) in reference-based mode. As advised by Catchen (personal communication), we removed the PCR duplicates via gstacks. The unpaired reads were removed; a minimum PHRED-scaled mapping quality of 25 was set to consider a read. We also applied a maximum soft clipping level of 5% of the read length to reduce sequencing errors [START_REF] Li | Fast and accurate short read alignment with Burrows-Wheeler transform[END_REF].

The populations program (Stacks) was used to export the results in a VCF file for further filtering (see command line Appendix B.1). The SNPs at positions 60 were removed due to a sequencing problem occurring at this position (Rougemont et al. 2017).

Detection of the outlier SNPs

First, we wanted to identify a set of SNPs potentially non-neutral but well differentiated between LP and LF in order to discriminate both ecotypes and their hybrids. Only individuals coming from populations with a genome wide fst < 0.1 were retained for downstream analysis (Rougemont et al., 2017; Table 1; n = 121). These populations experiencing high gene flow were more appropriate to investigate reproductive isolation than allopatric populations with low levels of gene flow since only regions involved in reproductive isolation should resist the homogenising effect of gene flow. In addition, highly differentiated markers in allopatric populations could be population-specific due to random genetic drift in isolated populations. After data visualisation, we kept genotypes with a mean coverage ranging from 5x to 100x and a minimum minor allele count of 5. We only included biallelic sites with a maximum of 50 % missing genotypes. Then we removed 8 individuals with more than 40% missing data. We excluded sites with more than 20% missing data in LF or in LP individuals. Lastly, we kept biallelic sites with a minimum minor allele count of 5, and that were at least genotyped in 85% of individuals (see command lines used for filtering in Appendix B.2). The filtered .vcf file was then transformed into a genepop datafile with PGDspider v2.1.1.5 [START_REF] Lischer | PGDSpider: an automated data conversion tool for connecting population genetics and genomics programs[END_REF].

The same filtering steps were used with just the LP and LF individuals of our focal populations: the Oir River. The resulting .vcf was also transformed into a genepop file using PGDspider v2.1.1.5 [START_REF] Lischer | PGDSpider: an automated data conversion tool for connecting population genetics and genomics programs[END_REF].

The FST was computed for the two genepop files using the function diffCalc of the package diveRsity [START_REF] Keenan | diveRsity: An R package for the estimation and exploration of population genetics parameters and their associated errors[END_REF] ; see R script in Appendix B.3). Finally, a custom script (python script in Appendix 11) was used to retrieve the common loci with a FST > 0.3 in both result files (i.e. one with the 4 sympatric populations and one with only the Oir population). We obtained a total of 190 outlier loci.

Population

Phenotype N

Oir

DNA extraction, preparation and sequencing

We used the kit NucleoSpin® 96 Tissue (Macherey-Nagel, Düren, Germany) to extract DNA of 1292 individuals (LP = 536; LF = 480, larvae and non-identified juveniles = 275, and one P. marinus; Figure 1) from fin clips according to the manufacturer's instructions. All individuals (n=1292) were genotyped using a modified version of the Hi-Plex protocol (Hammet et al., 2019;[START_REF] Meek | The future is now: Amplicon sequencing and sequence capture usher in the conservation genomics era[END_REF]Larson, 2019, Besnard et al. 2023, in prep.). The Hi-Plex protocol is an amplicon sequencing technique [START_REF] Meek | The future is now: Amplicon sequencing and sequence capture usher in the conservation genomics era[END_REF]) that allows the co-amplification of all SNPs in a multiplex reaction before a Ion-Torrent sequencing or an Illumina sequencing (here we used Illumina sequencing). A dual indexing of each sample is realized for demultiplexing. The reads are aligned to the reference sequences with BWA-MEM [START_REF] Li | Fast and accurate short read alignment with Burrows-Wheeler transform[END_REF][START_REF] Li | Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM[END_REF] and alleles are counted with SAMtools [START_REF] Danecek | Twelve years of SAMtools and BCFtools[END_REF]. In each individual and for each single SNP genotype, the posterior probabilities were computed using the likelihood of all possible genotypes considering a multinominal distribution of the number of times each nucleobase was genotyped at one SNP locus within one individual. In order to obtain a robust dataset, a genotype was only assigned when its posterior probability of allele dosage was superior to 70%; otherwise, it was considered as missing.

We then divided our dataset into two: one with outlier SNPs only and one with all SNPs (outliers and neutral). For both, we removed SNPs that presented missing genotypes in more than half of the individuals and then we further removed individuals with more than half of missing genotypes.

Regarding the larvae samples, we had to sort out potential sea lamprey (Petromyzon marinus) LGs. Eigenvalues and eigenvectors were computed with PLINK v1.9 [START_REF] Chang | Second-generation PLINK: rising to the challenge of larger and richer datasets[END_REF] for the 21 outliers SNPs found in LG 26. We then realized a PCA and calculate the heterozygosity of the observed groups on the PCA (group 1 < -0.03, group 2 in the range [-0.03 : 0.03] and group 3 > 0.03). All the figures were plotted using ggplot2 v3.4.0 [START_REF] Wickham | ggplot2[END_REF].

Hybrid assignment

We aimed at discriminating parental or 'pure' LP and LF (F0), first generation hybrids (F1), secondgeneration hybrids (F2) and backcrosses (LP x F1 or LF x F1). First, we needed to identify reference 'pure' LP and LF. We used individuals from the Oir population sampled by Rougemont et al. (2017) and which were also used here in the selection of markers. Structure software (Pritchard et al., 2000a) was run with K=2 and a burn-in of 250 000 and 500 000 iterations to remove any admixed individual using either the outlier SNPs dataset (Panel 1) or the entire dataset (Panel 2). In both cases, the same individual was identified as admixed (q-value < 0.90 for a category) and thus removed (Appendix B.14) [START_REF] Van Wyk | Quantitative evaluation of hybridization and the impact on biodiversity conservation[END_REF]. A total of 39 individuals (17 LF and 22 LP) were selected as 'pure' references samples. Then we simulated F1, F2, LF backcrosses and LP backcrosses genotypes (n= 100 for each group) with a hybrid simulation tool called recom-sim (https://github.com/salanova-elliott/recom-sim)

based on the program Hybridlab [START_REF] Nielsen | hybridlab (version 1.0): a program for generating simulated hybrids from population samples[END_REF]Appendix B.15). These simulated reference genotypes were included in our empirical datasets for subsequent analyses with Structure (Pritchard et al., 2000) and Newhybrids [START_REF] Anderson | A model-based method for identifying species hybrids using multilocus genetic data[END_REF]Anderson, 2008).

For Structure analyses, we ran five independent replicates (Appendix B.16) on the entire dataset with a burn-in of 50 000, 100 000 iterations and a K=2. Individuals were considered pure if their q-value was greater than 0.9, thus individuals were considered admixed if 0.1 < q-value <0.9 [START_REF] Van Wyk | Quantitative evaluation of hybridization and the impact on biodiversity conservation[END_REF][START_REF] Knipler | Genome-wide SNPs detect hybridisation of marsupial gliders (Petaurus breviceps breviceps × Petaurus norfolcensis) in the wild[END_REF].

We then used Newhybrids to make a finer analysis of all hybrid categories. For both datasets (panels 1 and 2), Newhybrids was run using a burn-in period of 100 000 MCMC followed by 250 000 sweeps using Jeffreys-like priors for the distribution of allelic frequency and mixing proportions (See command lines Appendix B.17). We used simulated genotypes to assess the power, accuracy and type I error of Newhybrids runs. The definition by [START_REF] Vähä | Efficiency of model-based Bayesian methods for detecting hybrid individuals under different hybridization scenarios and with different numbers of loci[END_REF] of accuracy, power and type I error was used: accuracy = (number of correctly identified individuals in a category)/(total number of individuals assigned to this category), power = (number of correctly identified individuals in a category)/(true number of individuals of that category) and the type I error = (number of mis-assigned assigned hybrids) / (total number of pure individuals). All individuals that fell into a category with a posterior probability higher than 0.8 were assigned to that category. For the comparison of Newhybrids and

Structure results, we grouped all hybrids together into an admixed category, as Structure does not allow a clear differentiation between F1 and F2 and Newhybrids does not allow a clear differentiation between F2 and backcrosses.

Hybrid simulation

We aimed at comparing the observed proportions of each hybrid class with simulated proportions computed according to various scenarios of hybrid breakdown. We used the individual-based simulation software NEMO v.2.3.55 (Guillaume & Rougemont, 2006) that makes forward-in-time evolutionary genetics simulations. We aimed at simulating a life cycle as close as possible as the one of lampreys. First, a model of assortative mating was implemented to allow for different levels of assortative mating (i.e. within ecotype). We tested either 10%, 30% or 50% of random matings (i.e.

within or between ecotypes). Next, a model of hybrid incompatibility was implemented with the survival of hybrid juveniles being dependent upon their pedigree. This model is deterministic in the sense that survival is not the result of incompatibilities at the genetic level. Instead, we tested different values of fitness for F1 (parents are LF and LP), F2 (two parents are F1 hybrids), and backcrosses (one parent is a F1 or F2, the other is a pure LF or LP). In addition, different fecundity levels were implemented between LP and LF populations: LF fecundity was multiplied by either 1, 3 or 5 after each breeding event. The phenotype of the offspring (LP or LF) was inherited from the mother or by ancestry backcrosses as these two categories could be hardly distinguished in our analyses. Thus, any cross containing an admixed individual was classified as an Admixed cross (Table 3). 21 individuals were not clearly classified with Newhybrids and thus, they were removed from further analyses along with any cross they were part of. We measured the body length of progenitors, fertilization rate of eggs, embryo survival, length of larvae and number of larvae with developmental abnormalities. All statistical analyses were performed using R software v. 4.0.3. A one-way ANOVA was used to compare the body length and sperm quality of LP, LF and hybrids. Post hoc comparisons were computed using tukey_hsd(). The fertilization rate and embryo viability, number of disabled individuals were analysed using GLMMs with a binomial distribution and a logit-link function [START_REF] Bates | Fitting linear mixed-effects models using lme4[END_REF]. The larval length was analysed with Linear Mixed Models (LMM). The models included the cross type (LF, BC, F1, F2, Admixed) as a fixed effect and the male ID and female ID as neutral effects.

Hybrid assignment

First, we compared the Hybrid assignment results with Structure and Newhybrids (Table 4 for panel 1

and Table 5 for panel 2). Here the power of panel 2 to detect pure LF is lower than panel 1 but panel 2 has a better power to identify F2 individuals. However, since the power was low for both panels to detect F2s, we preferred to continue the investigations with the Newhybrids results and the Panel 1 that performs well to detect pure LP and LF, F1s and backcrosses. For further analyses we created the category "admixed" that includes all second generations (or further) hybrids (i.e. In adult individuals, both ecotypes present a high proportion of hybrid individuals: 35% and 41% of F1s

for LF and LP, respectively. These F1 proportions seem huge given the low proportion of pure individuals: 27% and 17% for LF and LP, respectively. The proportion of admixed individuals is also high:

30% and 29 % for LF and LP, respectively (Figures 3 and4). In larvae and Lampetra sp. juveniles, the proportion of hybrids are also high (44% and 34% of F1s and admixed individuals, respectively) with no pure LF found (Figure 6). When analysed for each sex separately, the proportions of hybrids are also high and similar between sexes (Figure 7). The PCA on adult genotypes of both phenotypes at SNPs of LG 26 revealed three groups corresponding to both ecotypes and a third groups of intermediates genotypes including both phenotypes (figure 8).

The heterozygosity of the second group (777 ± 0.139) was higher than the one of the first (267 ± 0.108) and third group (0.165 ± 0.162; Figure 9).

Hybrid simulation

All the simulated scenarios were significantly different from the observed data. In particular, no scenario presented such a high proportion of F1s like the one observed in both ecotypes (Figure 10, full simulation results in Appendix B.21). The individuals have either all the same fitness or hybrids present a lower fitness than pure individuals.

The fecundity factor is of the factor by which the LF fecundity was multiplied compared to the LP fecundity. The 10% and 50% correspond to the percentage of random mating between ecotypes.

Sperm quality, body size and controlled crosses

We found a significant length difference (F = 358.57, p < 0.001) among hybrid classes but this difference was found only between individuals with distinct phenotypes (LF phenotype (n = 54): 229 ± 14.1 mm (mean ± sd) ; LP phenotype (n = 79): 131 ± 10.1 mm; Figure 11). Thus, hybrids had a similar size than pure individuals with a similar phenotype (Figure 11).

Similarly, sperm quality analyses revealed significant differences between phenotypes for both sperm concentration and sperm velocity (Figure 12a and12b), but no difference between hybrids and individuals of the same phenotype.

Discussion

The study revealed high hybrid proportions in the Oir population based on both Newhybrids and Structure results. With Structure, we could not distinguish F1 from F2 as these hybrids have on average 50 % of their genome coming from each species (Anderson, 2008). With Newhybrids, we detected large proportions of F1 and other hybrids (backcrosses and F2). The fact that we could not discriminate backcrosses and F2s was certainly due to a lack of power with our low number of SNPs. For example, [START_REF] Knipler | Genome-wide SNPs detect hybridisation of marsupial gliders (Petaurus breviceps breviceps × Petaurus norfolcensis) in the wild[END_REF] used more than 10 000 SNPs and Newhybrids is usually used with about 200 SNPs (Anderson, 2008). Nevertheless, the results of Structure and Newhybrids were consistent for the panel 1 of SNPs.

The hybrid proportions were also comparable between adults and larvae or transformers. These proportions combined to the results of experimental crosses that do not reveal any sign of hybrid breakdown, tend to prove that the level of reproductive isolation between the LP and LF is very low.

In addition, if we combine the F1 and admixed individuals, the hybrid proportions reached 65 and 70%

in LF and LP respectively, which is extremely high and suggests some degree of hybrid vigour.

The explanation for this high rate of hybrids could lie in the LG 26 identified in chapter 2. Indeed, we found a potential inversion reducing the gene flow in that part of the genome. The 21 SNPs coming from LG 26 used in this study gave a similar PCA than the one in Chapter 2. Three distinct groups could be observed with the intermediate one expressing a high heterozygosity. Inversions can arise to preserve an association of loci conferring a high fitness genotype. When recombination is suppressed in an organism that has two different versions of an inverted segment (known as an inversion heterozygote), it can result in a type of advantage for the heterozygote [START_REF] Charlesworth | On the fixation or nonfixation of inversions under epistatic selection[END_REF].

The maintenance of this inversion polymorphism could be maintained with epistatic selection [START_REF] Kimura | A model of a genetic system which leads to closer linkage by natural selection[END_REF][START_REF] Charlesworth | Inversion polymorphism in a two-locus genetic system[END_REF]. It is possible that the inversion cause segregation disorder, but further study are needed [START_REF] Charlesworth | On the fixation or nonfixation of inversions under epistatic selection[END_REF]. Considering this, it is possible that inversion heterozygotes are positively selected, explaining their high frequency in the population. Such high proportions of heterozygotes for an inversion have been observed in the seaweed fly Coelopa frigida (Mérot et al. 2018[START_REF] Mérot | Balancing selection via life-history trade-offs maintains an inversion polymorphism in a seaweed fly[END_REF]. Furthermore, the PCA also showed that the sex of LP and LF individuals was not linked to a potential sex-dependant dominance pattern like in the inversion polymorphism discovered in rainbow trout [START_REF] Pearse | Sexdependent dominance maintains migration supergene in rainbow trout[END_REF].

The simulation analyses did not provide any scenario with a high proportion of F1s like the one we found in the Oir population. Different modelling approaches like heterosis or simulating the inversion genotype with segregation distortion or overdominance may help to explain the pattern we observed (Mérot et al. 2023).

Our empirical data on body size showed that there was no intermediate hybrid phenotype: hybrids are either with a LF phenotype or with a LP phenotype. In contrast, intermediate phenotypes were described in stickelbacks [START_REF] Schluter | Adaptive radiation in sticklebacks: size, shape, and habitat use efficiency[END_REF] and more recently in Asiatic black bears [START_REF] Zou | .recode.vcf -T pedigree_bcftool.txt -c > output_mendelian row['loc6']==0 and row['loc7']==0 and row['loc8']==0 and rowloc9']==0 and row['loc10']==0 and row['loc11']==0 and loc12']==0 and row[[END_REF].

We did not find any sign of hybrid transgression either (i.e. extreme phenotypes compared to the parental lines; [START_REF] Rieseberg | Transgressive segregation, adaptation and speciation[END_REF]. We can propose that there is a stabilizing selection in parental species and the optima of hybrids match those of parental species [START_REF] Moran | The genomic consequences of hybridization[END_REF]. We did not identify the mechanisms that allow the development of either phenotype in hybrids. In the literature, the only reproductive barrier described between LP and LF is a partial reproductive isolation by body size (Beamish & Neville, 1992). However, the identified hybrids do not differ in size compared to the parental species thus are not disfavoured by this size assortative mating. We also showed that the sperm quality of males just changes according to the phenotype and not the hybrid classes they belong to [START_REF] Decanter | Sperm competition experiments reveal low prezygotic postmating isolation between parasitic and nonparasitic lamprey ecotypes[END_REF].

Furthermore, the experimental crosses did not highlight any post-zygotic barrier during fertilization or in larvae development. The fertilization rate of the pure LF was significantly lower than F2 and BC but the low sample size for pure individuals did not allow us to conclude about a superiority of hybrids for that trait.

To conclude, we discovered that the hybridization between LP and LF might be more frequent than expected and possibly linked to hybrid vigour or overdominance as a direct consequence of a chromosomal inversion.

This thesis dealt with three questions related to the speciation between L. fluviatilis and L. planeri. We studied how the divergence between L. planeri and L. fluviatilis is structured in the genome, we experimentally investigated potential postmating prezygotic and we explored a potential hybrid breakdown in second-generation hybrids with multiples approaches (experimental crosses, and comparison of simulation and empirical population genetic data). We used L. planeri as a genomic reference and found two keys regions of divergence between L. planeri and L. fluviatilis with one including a potential inversion of several Mb. We highlighted that the partial reproductive isolation by body size might be the only prezygotic barrier ongoing between the two species (Beamish & Neville, 1992). Finally, we discovered a high frequency of adult hybrids of first and second generation in the wild, thus refuting our hybrid breakdown hypothesis.

Genomic basis of divergence

Little was known about the genomic differences between L. planeri and L. fluviatilis apart from several SNPs that could differentiate them to a certain extent (Rougemont et al., 2017;Souissi et al., 2022).

Nevertheless, as we previously lacked genomic data, we did not know how the genomic divergence was structured within the genomes of LP and LF. LP and LF provide an excellent situation to study speciation genomics as they are undergoing the homogenizing effect of gene flow, thus leading to the right conditions to highlight the genomic regions truly involved in reproductive isolation and speciation (Rougemont et al., 2017).

To discover the architecture of genomic divergence, a new high quality lamprey genome was assembled and a linkage map was constructed to see where the diagnostics SNPs locate in the genome.

In addition, we investigated potential chromosomal rearrangements by direct or indirect approaches.

Regions of high differentiation are thought to arise due to reproductive barriers. Genome scans are now powerful enough to reveal this genomic heterogeneity [START_REF] Nosil | Divergent selection and heterogeneous genomic divergence[END_REF]. However, there are some difficulties to detect barriers loci of local adaptation [START_REF] Ravinet | Interpreting the genomic landscape of speciation: a road map for finding barriers to gene flow[END_REF] because without gene flow, high peaks of genomic differentiation arise but they are caused by selection and selective sweeps after isolation [START_REF] Noor | Islands of speciation or mirages in the desert? Examining the role of restricted recombination in maintaining species[END_REF][START_REF] Cruickshank | Reanalysis suggests that genomic islands of speciation are due to reduced diversity, not reduced gene flow[END_REF]. Barriers loci can have different forms, from a single nucleotide change to chromosomal rearrangements [START_REF] Ravinet | Interpreting the genomic landscape of speciation: a road map for finding barriers to gene flow[END_REF]. The barriers can evolve in relation to the environment or not [START_REF] Servedio | The role of reinforcement in speciation: theory and data[END_REF][START_REF] Presgraves | Speciation genetics: epistasis, conflict and the origin of species[END_REF][START_REF] Nosil | Ecological Speciation[END_REF]. It is important to note that FST at loci close to the barrier loci will also be high due to linkage disequilibrium [START_REF] Ravinet | Interpreting the genomic landscape of speciation: a road map for finding barriers to gene flow[END_REF]. If there is only a few barrier loci, their effect can be small as the majority of the genome still recombines [START_REF] Ravinet | Interpreting the genomic landscape of speciation: a road map for finding barriers to gene flow[END_REF].

Here our results demonstrate that the genetic differences between LP and LF co-localize in the genome in two major zones that can be called genomic islands of divergence [START_REF] Turner | Genomic islands of speciation in Anopheles gambiae[END_REF]. These islands of divergence are expected, with ongoing gene flow, the only regions that will present a low recombination rate will be linked to barrier loci [START_REF] Wu | The genic view of the process of speciation[END_REF][START_REF] Nosil | How many genetic changes create new species?[END_REF]. The two diverging regions we found, which are called here LG 6 and LG 26, present very different patterns of divergence.

LG6

presents a continuous pattern of differentiation like "normal" regions under selection [START_REF] Huang | Multiple chromosomal inversions contribute to adaptive divergence of a dune sunflower ecotype[END_REF]. However, LG 26 presents three discrete clusters with one having a high heterozygosity rate. This pattern suggests an inversion of several Mb [START_REF] Huang | Multiple chromosomal inversions contribute to adaptive divergence of a dune sunflower ecotype[END_REF]Mérot, 2020). Chromosomal rearrangements such as inversions could be involved in speciation as they drastically reduce gene flow [START_REF] Kirkpatrick | Chromosome inversions, local adaptation and speciation[END_REF][START_REF] Wellenreuther | Eco-evolutionary genomics of chromosomal inversions[END_REF]. For example, inversions have been involved in several cases of speciation in plants [START_REF] Lowry | A widespread chromosomal inversion polymorphism contributes to a major life-history transition, local adaptation, and reproductive isolation[END_REF][START_REF] Huang | Multiple chromosomal inversions contribute to adaptive divergence of a dune sunflower ecotype[END_REF] or animals [START_REF] Noor | Chromosomal inversions and the reproductive isolation of species[END_REF][START_REF] Satou | Chromosomal inversion polymorphisms in two sympatric ascidian lineages[END_REF].

To improve these results, novel long read sequencing and Hi-C sequencing could allow a better genome assembly to reach the chromosome level [START_REF] Belton | Hi-C: A comprehensive technique to capture the conformation of genomes[END_REF]. Furthermore, new long reads could allow a better depth to search for structural variants. The breakpoints of the potential inversion could be found or at least these new data could enhance our genetic map and clarify the length of the inversion.

We decided to explore the functional annotation of the divergent regions and it revealed genes function associated with development, metabolic processes, immunity and signal transduction. All these genes are potentially involved in phenotypic and physiological difference between LP and LF at the adult stage. For example, there are genes involved in coagulation and immunity that could be linked to the buccal secretions of parasitic lamprey, which contains anticoagulant and immune suppressors that facilitate the life on living host [START_REF] Klinker | Multiple mechanisms of immune suppression by B lymphocytes[END_REF][START_REF] Xiao | The buccal gland of Lampetra japonica is a source of diverse bioactive proteins[END_REF]. The abundance of signal transduction transcripts found could be linked to lamprey communication and recognition. Indeed, it can be linked to either the recognition of sex pheromone for reproduction or either to recognize the chemicals clues of larvae bile acids that are used for the recognition of potential river to go and reproduce while at sea [START_REF] Li | The olfactory system of migratory adult sea lamprey (Petromyzon marinus) is specifically and acutely sensitive to unique bile acids released by conspecific larvae[END_REF][START_REF] Li | Sex pheromone communication in the sea lamprey: Implications for integrated management[END_REF][START_REF] Buchinger | Chemical cues and pheromones in the sea lamprey (Petromyzon marinus)[END_REF][START_REF] Buchinger | Intra-and interspecific variation in production of bile acids that act as sex pheromones in lampreys[END_REF]. It is possible that the genes in this region are involved in the phenotype and the migratory behaviours like it is the case for respectively, Drosophila [START_REF] Hoffmann | Revisiting the impact of inversions in evolution: From population genetic markers to drivers of adaptive shifts and speciation? Annual Review of Ecology[END_REF] and Atlantic cod [START_REF] Berg | Trans-oceanic genomic divergence of Atlantic cod ecotypes is associated with large inversions[END_REF].

More work on the annotation is needed to validate our genes annotations and we could extend gene annotations to the whole LG 26 and not just SNPs with a FST = 1. It will be interesting to see if we find similarities with annotations of highly differentiated SNPs described in previous studies [START_REF] Mateus | Strong genome-wide divergence between sympatric European river and brook lampreys[END_REF]. To better understand the function of genes present in LG 26, we plan to make differential expression analyses between L. planeri and L. fluviatilis. To go further in the understanding on those genes and their function, one promising future is a CRISPR/Cas9 approach [START_REF] Bono | Connecting genotypes, phenotypes and fitness: harnessing the power of CRISPR/Cas9 genome editing[END_REF], which could give insights on the role of barrier in divergence [START_REF] Seehausen | Genomics and the origin of species[END_REF].

Reproductive isolation

The speciation process is characterized by a gradual accumulation of reproductive barriers, and consequently genetic incompatibilities. Premating, prezygotic or postzygotic barriers can isolate species. The hybrid zones provide insight on ongoing diverging species, subspecies or populations [START_REF] Barton | Analysis of hybrid zones[END_REF]. Usually, only partial sterility is observed in hybrid zones instead of full sterility and inviability [START_REF] Nachman | Recombination rate variation and speciation: theoretical predictions and empirical results from rabbits and mice[END_REF]. Relatively little was known about the reproductive barriers between LF and LP. (Beamish & Neville, 1992) identified a partial reproductive isolation by body size in two Lampetra paired species and [START_REF] Hume | Post-zygotic hybrid viability in sympatric species pairs: a case study from European lampreys[END_REF]Rougemont et al., 2015) showed that fertilization rate and hatching rate in artificial crosses between LP and LF were close to 100%. However, good cross-fertilization does not mean the absence of prezygotic barriers. (Yeates et al., 2013) showed perfect cross-fertilization between salmon and trout in controlled conditions. However, when sperm of one salmon and one trout compete for a batch of eggs, the conspecific male has a significant advantage and sires more eggs than the heterospecific male. (i.e. conspecific sperm precedence;

Howard, 1999).

a. Prezygotic barriers

Lampreys are ideal models to study postmating prezygotic barriers as they are external fertilizers and thus in vitro fertilization is easy to perform.

We analysed the sperm traits of both ecotypes and we realized in vitro fertilization and sperm competition trials between ecotypes. Our results showed that LF sperm have a higher velocity and a lower concentration than LP. A higher fertilization rate of LP was shown when the sperm velocity of the LF male was low. Both ecotypes had great fertilization rates with either ecotype in control crosses.

For the sperm competition trials, no cryptic female choice or conspecific sperm precedence was highlighted. We did not find any prezygotic barrier but we highlighted that sperm traits could potentially have an impact on fertilization success, thus potentially have a role in the speciation process between L. planeri and L. fluviatilis.

It could be interesting to measure these traits in allopatric population to see if the higher concentration found in LP phenotype is an adaptation to "sneak" fertilization to LF in sympatric areas. Another point is that we also have sperm longevity data that were not analysed in this thesis due to time constraints.

An improvement to this study could be to redo sperm competition trials but with the addition of the ovarian fluids that can have an impact on sperm. For example, in controlled crosses between salmon and trout, sperm of the conspecific male doubles motile life by straightening the swimming trajectory when in contact with conspecific ovarian fluid (Yeates et al., 2013).

b. Post zygotic barriers

The genetic incompatibilities can be explained by the Bateson-Dobzhansky-Müller model [START_REF] Bateson | Heredity and variation in modern lights[END_REF][START_REF] Dobzhansky | Genetics and the origin of species[END_REF][START_REF] Muller | Bearing of the Drosophila work on systematics[END_REF]. Adaptative or neutral mutations in a species can become deleterious in a foreign genomic background (Presgraves, 2010a). Hybrid sterility and hybrid inviability are characteristics of advanced speciation, as they are thought to be irreversible [START_REF] Sobel | The biology of speciation[END_REF].

The understanding of their evolution in differentiating species is an important topic in evolutionary biology [START_REF] Presgraves | Evaluating genomic signatures of "the large X-effect" during complex speciation[END_REF][START_REF] Coughlan | The importance of intrinsic postzygotic barriers throughout the speciation process[END_REF]. Often the incompatibilities are recessive [START_REF] Masly | High-resolution genome-wide dissection of the two rules of speciation in Drosophila[END_REF] thus, their deleterious effects are silenced in autosomes and in the homogametic sex XX females or ZZ males. However, the deleterious effects can affect the heterogametic sex (ZW females and XY or XO males) following what is called the Haldane's rule (Haldane, 1922).

Previous study on the lamprey paired species LP and LF have found that the F1s, until early larval stage are viable [START_REF] Hume | Post-zygotic hybrid viability in sympatric species pairs: a case study from European lampreys[END_REF]Rougemont et al., 2015) and that potential hybrids are found in the wild (Rougemont et al., 2017). However, it is not uncommon that F1s undergo heterosis whereas the later hybrid generation undergo hybrid breakdown due to the breakup of co-adapted gene and the expression of recessive deleterious alleles [START_REF] Edmands | Heterosis and outbreedind depression in interpopulation crosses spanning a wide range of divergence[END_REF]Stelkens et al., 2015) In this thesis, we developed a panel of diagnostic SNPs associated with neutral SNPs randomly distributed in the genome. We then used a modified HiPlex protocol [START_REF] Besnard | Workflow for SNP genotyping using the Hi-Plex method[END_REF] allowing us to pool and genotypes 1192 individuals coming from 10 years of sampling in the same hybrid zone (Oir River -France). Then the categorization of the individuals into pure or hybrids category was realized with two approaches: Structure (Pritchard et al., 2000b) and Newhybrids (Anderson, 2008b). Both software revealed a high proportion of hybrids. Such a High proportion of juveniles and adults hybrids in the wild tend to reject the hypothesis of extrinsic postzygotic barriers (ecological maladaptation or hybrid inferiority; Coyne & Orr, 2004). Surprisingly, we found a large proportion of individuals with a high heterozygosity in LG 26, hence the presence of a potential large inversion.

An inversion could be responsible for the maintenance of vital polymorphisms and alternatively, it could come from secondary contact to protect from endogenous incompatibilities [START_REF] Berg | Trans-oceanic genomic divergence of Atlantic cod ecotypes is associated with large inversions[END_REF].

A good knowledge on the ecological, geographical and demographic context of the model species is mandatory [START_REF] Ravinet | Interpreting the genomic landscape of speciation: a road map for finding barriers to gene flow[END_REF] and we know that the sympatry between LP and LF results from a secondary contact (Rougemont et al. 2017). However, the hypothesis that the inversion is a protection against incompatibilities might not be the good one as we observed a high frequency of heterozygous individuals at the inversion and a large proportion of F1s [START_REF] Berg | Trans-oceanic genomic divergence of Atlantic cod ecotypes is associated with large inversions[END_REF]. This, combined with no evidence of endogenous incompatibilities in the controlled crosses tends to prove that the explanation lies somewhere else. Another hypothesis be that broad selective agents alter several biological functions [START_REF] Berg | Trans-oceanic genomic divergence of Atlantic cod ecotypes is associated with large inversions[END_REF].

LG 26 is likely to be a target of selection and involved in the maintenance of divergence between LP and LF. A different hypothesis could lie in the study of [START_REF] Mérot | Balancing selection via life-history trade-offs maintains an inversion polymorphism in a seaweed fly[END_REF].

Indeed, they found that large inversion blocking the recombination behave as a single locus, which is comparable to our LG 26. They observe an overdominance effect linked to antagonistic pleiotropy, which can generate excess of heterozygous, which can conduct to the limitation of the genetic load, thus reinforcing overdominance.

Considering the demographic history of L. planeri and L. fluviatilis, which is thought to be a case of secondary contact, endogenous barriers (DMIs) are supposed to accumulate across the genome and not exogenous barriers due to ecological selection [START_REF] Salewski | Satellite species in lampreys: a worldwide trend for ecological speciation in sympatry?[END_REF][START_REF] Rundle | Ecological speciation[END_REF]. In addition, it is possible that potential positively inversion heterozygous selection discussed in chapter 2, somehow blocks recessive deleterious alleles. Accordingly, it could be interesting to see if outlier loci present segregation distortion patterns with the linkage map.

It could be also interesting to see if the observed high number of heterozygous for that region/inversion is the same in other hybrid zones of LP and LF. Similar patterns in other zone of hybridization could strengthen our results (Mérot et al., 2023) and demonstrate that the identified regions and loci are indeed involved in reproductive isolation like it was shown in Cichlids of the Victoria Lake [START_REF] Meier | Genomics of parallel ecological speciation in Lake Victoria Cichlids[END_REF].

A simulation approach was used to confront these empirical results with simulated hybrid frequencies.

However, none of the simulated scenarios gave comparable results as those from empirical data. We plan to model an inversion with an overdominance effect. We also propose to make parameter inferences using Approximate Bayesian Computation (ABC). This method would allow us to perform simulations using parameter values drawn from prior distributions and compare how similar are the result from observed data [START_REF] Csilléry | Approximate bayesian computation (ABC) in practice[END_REF]. This model is under construction with the help of Frédéric Guillaume.

The experimental work consisted in measurements of individuals and their sperm traits when they were males, and in the realization of in vitro fertilization between ecotypes. Our results did not highlight any heterosis or hybrid breakdown but these results could be pushed further. We need more crosses between pure individuals to be able to make statistics that are more reliable. In addition, another improvement would be to be able to keep the larvae longer to see if a length or survival difference appears. Lampreys are extremely difficult to rear due to their long life cycle [START_REF] Lampman | Chapter twenty two: Developing techniques for artificial propagation and early rearing of Pacific lamprey (Entosphenus tridentatus) for species recovery[END_REF][START_REF] Lampman | Lamprey aquaculture successes and failures: A path to production for control and conservation[END_REF][START_REF] Moser | Lamprey Reproduction and Early Life History: Insights from Artificial Propagation[END_REF]. During my thesis, we tried different rearing methods but we noticed high mortality during the transfer from petri dishes to tanks and / or at the first feeding (data not shown). However, the current trend with trials in the understanding of the correct way to rear lampreys is promising [START_REF] Lampman | Lamprey aquaculture successes and failures: A path to production for control and conservation[END_REF].

We found that hybrids either have a LF or a LP phenotype (i.e. not an intermediate phenotype) and thus do not suffer from assortative mating (Beamish & Neville, 1992). As no problem of fertility and viability have been highlighted, this similar size should favour the introgression of the heterozygous inversion in the population. Furthermore, overdominance could be present even if we did not detect the viability advantage for the heterozygous yet. Indeed, depending on the environmental and spatiotemporal condition, advantage of heterozygotes might not be strong [START_REF] Mérot | Balancing selection via life-history trade-offs maintains an inversion polymorphism in a seaweed fly[END_REF]. This phenotypic similarity prevents the identification of hybrids in the field and genotyping is thus necessary. We recommend to genotype with SNPs from both LG 6 and LG 26, because among individuals genotyped with only the diagnostic SNP diagLpf, some individuals thought to be pure were in fact backcrosses (Souissi et al., 2022). Another perspective could be to precisely quantify the partial reproductive isolation by body size in our system and try to quantify the potential overdominance linked to the inversion potentially explaining the high F1 proportion observed.

General conclusion

In this thesis, we investigated the ongoing speciation process between L. fluviatilis and L. planeri using different and complementary approaches. We provided an overview of the genomic differences and observable reproductive barriers.

The successful hybridization observed during experimental crosses and in the field challenges the classification of paired species [START_REF] Enequist | Das Bachneunauge als ökologische Modifikation des Flussneunauges[END_REF]. Our results confirm that L. fluviatilis and L. planeri can be considered as partially reproductively isolated ecotypes, which could impact their conservation (Docker, 2009). Appendix A.11. List of the gene found in LG 26

ABCC4 DROSHA MAP4K4 RNASEH1 ACSL4 ERBB2IP MB21D2 RNF13 AGFG1 ERCC5 MBNL3 RNF169 AKAP11 F10 MCF2L2 Rnf213 APOBEC3H FAM195A MED11 RSF1 ARHGEF4 FARP2 MFF RUNDC1 ARHGEF7 FGFBP3 MINK1 RYK ARRB1 FIS1 MRE11A SAP130 ATG16L1 FNDC3A MYO16 sina ATP11B FXR1 MYO7A SLC15A1 BOK fxr2 NLGN1 SLC19A3 CAPN5 GPR83 NLGN3 SLC25A30 cldn1 GRK7 NLGN4X SLC5A7 CLYBL GTF2F2 NOD2 SLITRK3 COA4 HEPHL1 P2RY1 SOX3 COG6 HMGXB4 pak2 SUCLA2 COL4A5 HTR2C PAK3 TAF6 COL4A6 IL13RA2 PANX3 TFDP1 CPT2 INPPL1 PCOLCE2 TMEM255B CUL4B IRS1 PDK1 TMTC4 dally JRKL PFN1 Trpc2 DAND5 KRT8 PLS3 TSC22D3 DCDC2 LAMP1 PRKX UBE2G2 dlg1 LCP1 PSMD1 YAP1 DLG2 LIMS1 RAB6A ZNF500 DOCK9 LIMS2 Rbmxl2
Appendix A.12. Annotation of the SNPs with a FST = 1 (n=119) of the linkage group 26 -> allele 0 codes for small size, allele 1 codes for large size. The alleles are set with the "quanti_allele" parameters. Initialization is done with the "quanti_init" LCE, which set the allele frequencies in each population at the start with the "quanti_init_freq" parameter. For this test, there two populations, one with "big" individuals (patch 1) and the other with "small" individuals (patch 2). Allele 0 has frequency 0 in patch 1 and frequency 1 in patch 2. # -> phenotype inheritance is not based on the genotype but on the phenotype expressed in the mother! It is thus an "epistatic" inheritance. This is hard coded in the "breed_assortative" LCE. However, the phenotypes are initialized from the allele frequency at generation 0. This is why we start "pure" big and pure small patches.

####################################################### ## VCF
############################################################ #Check if
#################################################################### # End of parameters tests #################################################################### VCF_CsvReader=csv.
####################################################### #Control
######################################################## #Check if
########################################################## dico = {}
# The mating is assortative based on the phenotype, thus not directly linked to the genotype. A female accepts to mate with a male if his size is <= to her size. # The assortative criteria is set with the "breed_assortative" parameters.

# Migration is unidirectional, from patch 1 to patch 2 only (first row of the dispersal matrix) with 0.1% migration: {0.999, 0.001}. ## NEMO SUB sub_program sbatch sub_script_directive SBATCH sub_parameters (--account=project_2005585 --partition=small --time=00:10:00 --ntasks=1 --mem-per-cpu=1000) sub_script_args nemo2. ## DISPERSAL ## dispersal_matrix {{0.999,0,0.001}{0,0.999,0.001}{0,0,1}} {{0.995,0,0.005}{0,0.995,0.005}{0,0,1}} {{0.99,0,0.01}{0,0.99,0.01}{0,0,1}} {{0.95,0,0.05}{0,0.95,0.05}{0,0,1}} {{0.9,0,0.1}{0,0.9,0.1}{0,0,1}} {{0.7,0,0.3}{0,0.7,0.3}{0,0,1}} ## STATS ## newOutfile.write("%s\n" % (str(results.filename)+"\t"+str(rep)+"\t"+str(compteurLF)+"\t"+str(c ompteurLP)+"\t"+str(compteurF1_LF)+"\t"+str(compteurF1_LP)+"\t"+str( compteurF2_LF)+"\t"+str( compteurF2_LP 2 same 3 50 0,00033 0,001 0,1 0,4 0,5 0,7 0,0 0,0 0,0 0,0 999,3 0,9 0,1 0,3 EQUAL 2 same 3 50 0,00167 0,005 0,8 0,8 1,5 1,4 0,0 0,0 0,0 0,0 997,4 1,7 0,3 0,7 13,9 0,5 1,1 0,1 0,5 835,7 15,3 7,5 4,5 EQUAL 5 same 3 30 0,00033 0,001 0,0 0,2 0,4 0,6 0,0 0,0 0,0 0,0 999,5 0,9 0,1 0,7 EQUAL 5 same 3 30 0,00167 0,005 0,4 0,5 1,8 1,1 0,0 0,0 0,0 0,0 997,7 1,3 0,1 0,5 EQUAL 5 same 3 30 0,00333 0,01 0,4 0,7 3,8 2,1 0,0 0,0 0,0 0,0 995,0 2,4 0,7 Such hybrids may have a lower fitness, which could explain the limited gene flow and maintenance of the two ecotypes. However, an analysis of hybridization in 1000 individuals from a single population revealed that most individuals were hybrids including about 40% of F1s. At the genome scale, two regions of the genome seem to concentrate the majority of differences between LP and LF. Structural differences as well as the genes present in those regions were identified. Sperm competition experiments revealed no prezygotic post-mating barrier even though sperm quality differs between LP and LF males. To conclude, it seems that the intensity of reproductive barriers between LF and LP is low, in relation with a genetic divergence concentrated in two regions of the genome.

EQUAL 2 same 3 50 0,00333 0,01 2,1 1,6 4,1 2,1 0,0 0,0 0,0 0,0 993,0 3,1 0,

  afin d'approcher un assemblage au niveau chromosomique. Une recherche des réarrangements chromosomiques a aussi été réalisée avec l'utilisation de génomes nouvellement assemblés de L. planeri et de long reads Pacbio de L. fluviatilis. L'assemblage de haute qualité combiné à la carte de liaison nous ont permis d'identifier deux groupes de liaison présentant une forte différenciation entre L. planeri et L. fluviatilis par rapport à ce qui est observé sur le reste du génome. Le plus divergent des groupes de liaisons est potentiellement inclus dans une inversion chromosomique. Son annotation fonctionnelle a permis l'identification de 103 gènes, parmi lesquels 27 contiennent des SNPs avec un FST élevé entre L. planeri et L. fluviatilis. Ces 27 gènes sont notamment liés au développement ou à des processus indispensables à la vie parasitaire adoptée par la lamproie, tels que la coagulation, l'immunité ou l'inflammation. Ces résultats mettent en évidence une divergence entre écotypes très localisée dans le génome et suggère qu'une inversion joue un rôle important dans la divergence entre L. planeri et L. fluviatilis. Chapitre 3 : Les expériences de compétition spermatique révèlent une faible isolation prézygotique postcopulatoire entre les écotypes de lamproies parasite et non-parasite Dans le Chapitre 3, nous avons cherché à identifier l'existence de potentielles barrières prézygotiques. Nous avons tout d'abord étudié les traits spermatiques (vitesse et concentration) chez les mâles des deux espèces. Nous avons trouvé qu'il existait une plus forte concentration en spermatozoïdes dans l'éjaculat des mâles L. planeri comparés aux mâles L. fluviatilis mais que les spermatozoïdes des mâles L. fluviatilis étaient plus rapides que ceux des mâles L. planeri. L'indice gonadosomatique (GSI) a été mesuré chez les mâles et nous avons trouvé une quantité semblable de spermatozoïdes totaux chez les deux espèces. Néanmoins le GSI des lamproies de Planer est plus fort, montrant un investissement plus fort dans les gamètes ce qui pourrait aller dans le sens d'une tactique de « sneaker » des L. planeri quand ils essayent de se reproduire avec des femelles L. fluviatilis. Nous avons ensuite réalisé des expériences de compétition spermatique entre écotypes à concentration égale de spermatozoïdes ou à volume égal de sperme qui n'ont ni révélé de choix cryptique de la femelle, ni de barrière à l'hybridation. Ces résultats soutiennent l'hypothèse que L. planeri et L. fluviatilis sont deux écotypes dont la principale barrière à la reproduction est une barrière précopulatoire dû au différentiel de tailles entre eux. Chapitre 4 : Un niveau élevé d'hybridation suggère une vigueur hybride entre les écotypes de lamproies parasite et non-parasite Dans le chapitre 4, nous avons tenté de déterminer de potentielles barrières postzygotiques via une approche multidisciplinaire. Dans un premier temps nous avons utilisé une approche empirique de génétique des populations pour répondre à notre question. Il nous fallait être capable de clairement distinguer à quelle classe hybride appartenait les 1292 individus échantillonnés entre 2010 et 2021 dans la rivière de l'Oir et un seul marqueur n'était pas suffisant. Nous avons donc d'abord recherché des marqueurs discriminants L. planeri et L. fluviatilis, puis nous avons vérifié s'ils étaient amplifiables.

  of paired species…………………………………………………………………………………….17

  d. The case of L. fluviatilis and L. planeri………………………………………………………………18 5. Goals of the thesis………………………………………………………………………………………………………21 CHAPTER 2: Genomic divergence between parasitic and non-parasitic lamprey ecotypes…………………………………………………………………………………………………………24 CHAPTER 3: Sperm competition experiments reveal low prezygotic postmating isolation between parasitic and non-parasitic lamprey ecotypes………………………44 CHAPTER 4: High level of admixture suggest hybrid vigour between parasitic and non-

Figure 1 :

 1 Figure 1: Example of the speciation continuum with two species (A and B) originating from a common ancestral species. The speciation is completed when the reproductive isolation between the two is complete. From Salzburger (2020).

Figure 2 :

 2 Figure 2: Main mechanisms of reproductive isolation classified by their order of appearance in the life cycle and their endogenous/exogenous components. Modified fromCoyne & Orr (2004)

Figure 3 :

 3 Figure 3: Dobzhansky-Muller model. An ancestral population with AABB genotypes will split into two populations that evolve independently. The A evolves into a in the first population while in the second B evolves into b. a an b are mutually incompatible but as they are not present in the ancestral species, the evolution of incompatibility is possible. The red arrow indicates the incompatibility. Modified from Wu & Ting, 2004.

Figure 4 :

 4 Figure 4: Genetic background of hybrids. Chromosome of species A are shown in orange and chromosomes of species B are shown in green. The F1 received on allele from each parent and thus

Figure 5 :

 5 Figure 5: Life cycle of an anadromous parasitic lamprey and a freshwater resident non-parasitic lamprey.

Figure 6 :

 6 Figure 6: Picture presenting recently metamorphosed lampreys (in order from top to bottom): Petromyzon marinus, two Lampetra fluviatilis and one Lampetra planeri. Taken at the Cerisel station (Normandy) the 18/09/2019.

Figure 7 :

 7 Figure 7: Picture presenting (from top to bottom) an adult L. fluviatilis and an adult L. planeri. Taken at the Cerisel station (Normandy -France) the 22/04/2021.

Figure 8 :

 8 Figure 8: Picture of lampreys, from top to bottom: ammocoetes of Lampetra sp., metamorphosing Lampetra sp. with apparent eyes (also called 'transformer') and juvenile Lampetra fluviatilis (Picture by Nolwenn Decanter).

Figure 9 :

 9 Figure 9: Distribution of a) L. fluviatilis and b) L. planeri from observations between 2000 and 2022 in GBIF Secretariat (2021). (GBIF Backbone Taxonomy. Checklist dataset https://doi.org/10.15468/39omei accessed via GBIF.org on 2022-11-02.)

Figure 10 :

 10 Figure 10: a) Position of the natural hybrid zone (Oir River -France) and b) example of electrofishing in the Oir River

Figure 1 :Figure 2 :

 12 Figure 1: Manhattan plot of FST computed (a) between 57 LF and 52 LP from four sympatric populations sampled by (Rougemont et al., 2017) and (b) between 17 pure LF and 22 pure LP from the Oir River (Normandy -France) . The 85 linkage groups are displayed and red dots indicate FST values higher than 0.25.

Figure 3 :

 3 Figure 3: Observed heterozygosity in the three groups detected in the PCA of LG 26. The group 1 (n = 48) is mainly made of LF and group 3 (n = 43) mainly of LP. The second group (n = 18) is composed of intermediate individuals of the LG 26 PCA, from both ecotypes.

Figure 4 :

 4 Figure 4: GO classifications for LG 6 (a) and LG 26 (b). The classification colors correspond to the main categories and each bar indicates the number of times a given GO term was highlighted.

Figure 5 :

 5 Figure 5: Percentage of each KEGG pathways found in LG6 (a) and LG 26 (b).

Figure 1 :

 1 Figure 1: Cumulative barplot of the 1292 genotyped individuals of the Oir population by year and phenotype.

  percentage and generations were non-overlapping. Ten Dobzhansky Müller Incompatibilities (DMI) loci were modelled to allow the simulation of fitness differences among individuals and simulate hybrid breakdown. The passage from the juvenile stage to the adult stage was dependent on individual fitness that was dependant of the individual genotype. We simulated three populations: one sink population mimicking a hybrid zone receiving a unidirectional migration from two other populations containing either LP or LF individuals (Figure2). At the first generation, the sink population contained only LP individuals. We recorded the individual genotypes at the DMI loci and phenotypes in the sink population after 200 generations for further analyses. The populations' carrying capacities were set at 1000. We performed 50 replicates of each combination of parameters (dispersal, fecundity, assortative mating, hybrid fitness, and transmission of the phenotype, see table2). We performed a total 43 200 runs (bash script and Nemo .ini file example in Appendix B.18). To keep the same dispersal between LP and LF, LF dispersal was divided by the fecundity factor. The classification of hybrids with the Nemo output was performed with a custom python script (see Appendix B.19). Given the combination of different parameters values, we simulated a total of 865 scenarios.

Figure 2 :

 2 Figure 2: Scheme of the LF and LP populations simulated with the NEMO software.

Figure 3 :

 3 Figure 3: Plot of q-values from the Structure software of a) all individuals sampled in the hybrid zone (n = 1099). The Red color is for the LF population (n = 457) and the green color is for the LP population (n = 502). Population 1 represents individuals with a LF phenotype, population 2 the individuals with a LP phenotype and population 3 represents the larvae. q-values of the reference (i.e. baseline) and simulated individuals (b). Populations 1 to 6 in (b) represent the reference LF (n = 17), reference LP (n = 22), F1 (n = 100), F2 (n = 100), BC with LF (n = 100) and BC with LP (n = 100), respectively.

Figure 4 :

 4 Figure 4: Pie charts of the percentage of adults assigned to each hybrid class (a) LF phenotype (n=457) and b) LP phenotype (n=502)).

Figure 5 :

 5 Figure 5: Cumulative barplots per year of the percentage of individuals with a LF phenotype (a) assigned to each hybrid class (2010 (n = 33), 2011 (n = 46), 2014 (n = 38), 2015 (n = 35), 2017 (n = 46), 2018 (n = 47), 2019 (n = 138) and 2021 (n = 45)). Only years with more than 30 samples are displayed. Barplots for individuals with a LP phenotype (b) (2012 (n = 36), 2015 (n = 52), 2017 (n = 65), 2018 (n = 167), 2019 (n = 76) and 2021 (n = 68) ).

Figure 6 :

 6 Figure 6: Pie-charts of the percentage of larvae (n=91) and b) transformers (n = 49) assigned to each hybrid class with the diagnostic markers.

Figure 7 :

 7 Figure 7: Pie charts of the percentage of LP and LF males and females assigned to each hybrid class (males with a LF phenotype (n = 89), females with a LF phenotype (n=50), males with a LP phenotype (n=126) and females with a LP phenotype (n=80).

Figure 8 :

 8 Figure 8: PCA computed with the adult individuals sampled in the Oir River between 2010 and 2021 and genotyped with 21 SNPs from LG26. (a) LF phenotypes (n = 451), are in blue and LP phenotypes (n = 468), are in green; (b) the dots represent the individuals with a LF phenotype (n = 451),the triangles are LP phenotype (n = 468), the red color represent females (n = 126), the blue color represents males (n = 209) and the grey color individuals whose sex could not be determined (n = 584).

Figure 9 :

 9 Figure 9: Observed heterozygosity in the three groups detected in the PCA of LG 26. The group 1 (n = 207) is mainly made of LF and group 3 (n = 177) mainly of LP. The second group (n = 535) is composed of intermediate individuals of the LG 26 PCA, from both ecotypes.

Figure 10 :

 10 Figure 10: Examples of the simulated hybrid proportions for a limited range of the parameters tested.

Figure 11 :Figure 12 :

 1112 Figure 11: Comparison of the body length of adult lampreys according to their hybrid status: Pure LF (n = 7), pure LP (n = 17), F1 with LF phenotype (n = 16), F1 with LP phenotype (n = 16) and Admixed individuals with LF (n = 21) and LP phenotype (n = 35).

  submit = /bin/bash -c "${JOB_SCRIPT}" > "${JOB_STDOUT}" 2> "statistics of the FALCON assembly All statistics are based on contigs of size >= 3000 bp, unless otherwise noted (e.g., "# contigs (>= 0 bp)" and "Total length (>= 0 bp)" include all contigs).

  (0,len(liste_snps)): pos_5 = int(liste_snps[i][2])+35 #position 5' pos_3 = int(liste_snps[i][4])-35 #position 3' pos_inferieure = int(liste_snps[i][2]) pos_superieur=int(liste_snps[i][4]) #print("position inf : "+ str(liste_snps[i][2])+" position moins 40 : "+str(pos_moins40)+" position snp : "+str(liste_snps[i][3])+ " position plus 40 : "+str(pos_plus40)+ " position sup : "+str(liste_snps[i][4])) #print(range(int(liste_snps[i][2]),pos_moins40)) #print(range(pos_plus40,int(liste_snps[i][4])+1)) #print(len(range(int(liste_snps[i][2]),pos_moins40))) #if (position_snp in (range(pos_inferieure,pos_5+1)) or (position_snp in range(pos_3,pos_superieur+1))):

Appendix B. 18

 18 . a) Bach script to launch Nemo with the ini files and an b) example of ini file a) Bash script #$1 = init file, contains the whole path to the file (e.g. jobs/init/filename.ini) #$2 = nemo exec module load gsl filename=`basename $1` simname=`awk '/^filename/{if($2 != "")print $2}' $1` root_dir=`awk '/^root_dir/{print $2}' $1` ##output directory of nemo result files echo "starting sim in: " $1 command="$HOME/bin/$2 $1" echo "executing $command... " files #tar cfz ${simname}.tgz $root_dir/$simname* --remove-files tar cfz ${simname}.tgz $root_dir/{./,quanti/,dmi/}$simname* -remove-files b) .ini file example # N E M 0 # Lamprey assortative mating # # This test simulation shows how to model assortative mating on a trait value coded by a quantitative trait. The trait here is very simple. It has one locus with two alleles:

  Figure: Pie chart of the percentage of individuals assigned to Newhybrids categories for the individuals with a a) LF (n=466) and b) LP phenotype (n=510) with the diagnostics markers

Figure :

 : Figure: Cumulative barplots of the percentage of individuals assigned to Newhybrids categories for the individuals with a LP phenotype in 2012 (n = 37), 2015 (n = 52), 2017 (n = 66), 2018 (n = 168), 2019 (n = 78) and 2021 (n = 70) with panel 2.

Figure :

 : Figure: Pie chart of the percentage of individuals assigned to Newhybrids categories for the individuals of the sedentary larvae (n = 91) and transformers (n = 50) witht the panel 2.

Figure :

 : Figure: Pie chart of the percentage of individuals assigned to Newhybrids categories for the individuals of male (n = 89) and female (n = 52) with a LF and male (n = 129) and female (n = 80) with a LP phenotype with the diagnostics markers

Titre:

  Divergence génomique et spéciation entre la lamproie de rivière -parasite (Lampetra fluviatilis) et la lamproie de Planer -non-parasite (L. planeri) Mots clés : Barrières reproductives -Lamproie -Hybridation -Sympatrie Résumé : L'isolement reproducteur est une question clé dans l'étude de la spéciation. Nous avons étudié les mécanismes de l'isolement reproducteur entre deux espèces proches de lamproies. La lamproie de rivière Lampetra fluviatilis (LF) est anadrome et parasite alors que la lamproie de Planer L. planeri (LP) est résidente en eau douce et non parasite. Malgré des histoires de vie différentes, les deux espèces utilisent les mêmes habitats de reproduction, peuvent s'hybrider et sont considérées comme des écotypes avec un isolement reproducteur partiel. Grâce à de précédents travaux, nous savons que la première génération d'hybrides est viable et des marqueurs génétiques permettent de différentier les deux écotypes. Jusqu'ici, peu d'hybrides de seconde génération ou de rétrocroisements ont été identifiés. Ces hybrides pourraient avoir une valeur sélective moins importante, ce qui expliquerait le flux de gènes limité et le maintien des deux écotypes. Néanmoins, une analyse de l'hybridation sur plus de 1000 individus d'une même population a montré que la majorité des individus étaient hybrides avec environ 40% de F1s. A l'échelle du génome, deux régions semblent concentrer la majorité des différences entre LP et LF. Les différences structurales ainsi que les gènes présents dans ces régions ont été identifiés. En outre, des expériences de compétition spermatique n'ont révélé aucune barrière prézygotique malgré des différences de qualité spermatique entre les mâles LP et LF. En conclusion, il semble que l'intensité des barrières à la reproduction entre LF et LP soit faible, en lien avec une divergence génétique concentrée dans deux régions du génome.Title: Genomic divergence and speciation between the parasitic river lamprey (Lampetra fluviatilis) and non-parasitic brook lamprey (L. planeri) Keywords: Reproductive barriers -Lamprey -Sympatry -Hybridization Abstract: Reproductive isolation is a key issue in the study of speciation. Here, we investigated the mechanisms of reproductive isolation between two closely related lamprey species. The river lamprey Lampetra fluviatilis (LF) is anadromous and parasitic of other fish species whereas the brook lamprey L. planeri (LP) is freshwater resident and non-parasitic. Despite different life histories, the two species use the same breeding habitats, can hybridize and are considered as partially reproductively isolated ecotypes. From previous studies, we know that first-generation hybrids are viable and molecular markers allow the distinction of both ecotypes. So far, few second-generation hybrids or backcrosses have been identified.

Table 1 :

 1 Genome statistics of the FALCON and HiFiasm assemblies of the LP male with PACBIO HiFi sequencing. The statistics were obtained with QUAST v5.0.2[START_REF] Mikheenko | Versatile genome assembly evaluation with QUAST-LG[END_REF].

	Assembler	FALCON	HiFiasm
	Number of contigs (>= 0bp)	5187	6078
	Longest contig (bp)	13 346 483	13 587 778
	Total length of the assembly (<= 0bp)	1 044 614 692	1 267 568 030
	GC content (%)	49.03	49.36
	N50 (bp)	1 452 700	1 181 465
	N75 (bp)	329 179	268 516
	L50	154	211
	L75	546	792

Table 2 :

 2 Statistics

	Filtering	LOD	Number of linkage groups	Number of markers in a linkage group	Number of single markers
		6	2	32740	135
		7	9	32696	179
		8	39	32670	205
	With	9	72	32611	264
		10	83	32565	310
		11	85	32463	412
		12	90	32297	578
		6	1	34534	5362
		7	12	34426	5470
		8	52	34351	5545
	Without	9	74	34259	5637
		10	84	34155	5741
		11	86	34024	5872
		12	89	33830	6066

of the linkage maps built with or without using Filtering2. LOD score stand for "logarithm of the odds" and is a statistical estimate of the relative probability that two loci are located near each other on a chromosome.

Table 3 :

 3 Type and number of structural variants in LG6 (~37Mbp) and LG26 (~8 Mbp) discovered with PBSV program with the two alignments made with pbsv or minimap2.

	Aligners	PBSV			Minimap2
	SVs type	LG 6	LG 26	LG 6	LG 26
	Insertion	1 898	1 330	3	0
	Deletion	1 845	1 344	2	0
	Inversion	13	5	0	0
	Duplication	51	27	0	0
	Translocation	273	40	6	1
	Copy number variation	23	2	0	0
	Total	4 103	2 748	11	1

  1 , Ahmed Souissi 1 , Catherine Labbé 2 , Eric Edeline 1 ,

	CHAPTER 4
	High levels of admixture
	suggest hybrid vigour
	between parasitic and non-
	parasitic lamprey ecotypes
	Nolwenn Decanter 1 , Frédéric Guillaume 2 , Romane Normand 1 , Anne-Laure Besnard 1 , Gilles
	Lassalle 1 , Solenn Stoeckel 3 , Eric Petit 1 , Guillaume Evanno 1
	Guillaume Evanno 1
	1: DECOD (Ecosystem Dynamics and Sustainability), INRAE, Institut Agro, IFREMER Rennes, France
	1: DECOD (Ecosystem Dynamics and Sustainability), INRAE, Institut Agro, IFREMER Rennes, France 2: Organismal and Evolutionary Biology Research Programme, University of Helsinki, Biocenter 3,
	2: UMR 1037 LPGP (Fish Physiology and Genomics), Campus de Beaulieu, 35000, France 00014 Helsinki, Finland
	3: IGEPP (Institute for Genetics, Environment and Plant Protection), INRAE, Institut Agro, Université de
	Rennes, Le Rheu, France
	Published
	Manuscript in preparation

Table 1 :

 1 Number of individuals kept per location and per phenotype.

  individuals among the larvae (i.e. ammocoetes) sampled at a small size (i.e. < 5 cm) when they cannot be discriminated from Lampetra larvae. As a result, we performed Principal Component Analyses (PCA) for each of the two datasets to test whether the single P. marinus sample could be easily discriminated from Lampetra samples. Unfortunately, the P. marinus sample could not be distinguished from other samples (see Appendix B.13) hence we chose to remove all larvae with a length lower than 8 cm to ensure not to include P. marinus larvae in our analyses. Overall, we got a data set of 32 outlier SNPs genotyped in 1099 individuals and a second dataset of 148 SNPs genotyped in 1117 individuals. The outlier SNPs were mostly located on the two highly differentiated linkage groups (LG) between LP and LF (see Chapter 2). There were 9 SNPs on LG6, 21 on LG26, and two on other

	300
	250		L. planeri Larvae		L. fluviatilis P. marinus						1
	200								51
	150									146	275
	100					35		47	174			47
	0 50	33	5 46	42 15	16 38	52	18 22	73		81		75
		2010	2011	2012	2014	2015	2016	2017	2018	2019	2020	2021

Table 3 :

 3 Number of crosses per type of cross

	Cross type	Number of crosses
	LF	3
	LP	0
	BC	24
	F1	3
	F2	20
	Admixed	87
	LF
	RESULTS

Table 4 :

 4 Results of hybrid assignment with Panel 1 in Structure and Newhybrids. ST stand for Structure and NH stand for Newhybrids.

		All		Adults with LF phenotype	Adults with LP phenotype	Larvae	Female		Male
	Software	ST NH	ST	NH	ST	NH	ST NH	ST	NH	ST	NH
	Pure LF	169 157	145	137	23	20	1	0	14	12	25	25
	Pure LP	118 95	5	5	93	84	10	6	18	18	39	36
	Admixed 812 705	307	279	386	355	80 71	98	91 151	136
	NAs	0	93	0	36	0	43	0	14	0	9	0	18

Table 5 :

 5 Results of hybrid assignment with Panel 2 in Structure and Newhybrids. ST stand for Structure and NH stand for Newhybrids.Overall, the results with panel 1 were very similar between Structure and Newhybrids. In contrast, for the panel 2 results between Newhybrids and Structure were different because Newhybrids assigned much fewer pure LF than with panel 1 whereas results were similar with Structure for both panels. As

		All		Adults with LF phenotypes	Adults with LP phenotypes	Larvae	Female	Male
	Software	ST NH	ST	NH	ST	NH	ST	NH	ST	NH	ST	NH
	Pure LF	170 36	147	30	22	6	1	0	15	4	26	9
	Pure LP	119 79	7	3	92	72	10	4	18	18	38	31
	Admixed 828 848	313	375	395	392	80	81	99	102 154 157
	NAs	0	86	0	46	0	40	0	6	0	8	0	21

a result, it seems that the panel 1 is more appropriate with both methods. Then we further compared the performances of Newhybrids between both panels (table

6

).

Table 6 :

 6 Comparison of Newhybrids performances on reference and simulated data for the panels 1 and 2.

	Categories	n		Accuracy	Power	Type I error
	Panel	Panel 1 Panel 2	Panel 1	Panel 2	Panel 1	Panel 2	Panel 1	Panel 2
	Pure LF	17	17	1	1	1	0.76	-	-
	Pure LP	22	22	1	1	1	0.91	-	-
	F1	100	100	0.99	0.99	1	1	-	-
	F2	100	100	1	1	0.14	0.41	-	-
	Backcrosses
	LF	100	100	0.85	0.88	1	1	-	-
	Backcrosses
	LP	100	100	0.9	0.99	0.99	0.99	-	-
		-	-	-	-	-	-	2.2	1.5

  the output file can be created in the given path

	if results.OutFile:
	try:
	newOutfile=open(results.OutFile,"w")
	except:
	print("Impossible to create output file. Verify rights
	problem")
	sys.exit()
	else:
	print("-o missing. Program end")
	sys.exit()

Detection of neutrals SNPs

We decided to add putative neutral SNPs randomly distributed in the genome (hereafter called "neutral SNPs") to have a better genome representativeness as outliers SNPs were mostly located on only 2 linkage groups described in Chapter 2. For neutrals SNPs, we ran the populations program with individuals of the Oir River. A minimum of 2% of individuals must present the locus and finally a minimum minor allele count of 3 was required to process the loci. Next, we splitted the dataset in two, one per phenotype, and we kept genotypes with a mean coverage ranging from 5x to 100x and a minimum minor allele count of 5. We only included biallelic sites with a maximum of 50 % missing genotypes. Individuals with more than 60% missing data were removed. With VCFtools we removed loci with more than 15% missing data, loci that were not in Hardy-Weinberg Equilibrium and loci with a minor allele frequency inferior to 0.1. Then, with the populations program we filtered out loci with a heterozygosity inferior to 0.5. We kept markers present in both LP and LF individuals with a custom python script (Appendix B.4). In total, 13 236 loci were retrieved.

Sequences and primers design

We used the .vcf information on the loci positions in the LP genome contigs to extract the sequences around our candidate SNPs. Using a custom script (python script in Appendix B.5) we located each locus, cut a 145 bp sequence and stored the results in a fasta file. These 145 bp sequences were blasted against our reference genome with Blastn [START_REF] Camacho | BLAST+: architecture and applications[END_REF] and only blast match with a e-value superior to 1E-20 were considered (python script in Appendix B.6). A custom script (python script in Appendix B.7) was designed to parse the blastn output and we only kept the sequences with a unique blast match. The original fasta file was filtered with a custom python script (Appendix B.8) and we obtained 138 fasta sequences for outlier SNPs and 9105 for neutrals SNPs.

Then, we checked for the presence of repeats in our sequences with the Rad4Snps programme Rad4SnpsRepbaseFilter.py (command line in Appendix B.9). A total of 9 sequences were removed in outliers SNPs sequences and 1037 sequences were removed in neutrals SNPs. A custom script was used to keep only SNPs whose sequences do not overlap (python script in Appendix B.10) and then another custom script was used to filter out non-sequencable sequences (python script in Supplementary material Appendix B.11). At that stage, we had obtained 63 candidate sequences for outlier SNPs and 5120 for neutral SNPs. The 5120 candidate sequences were randomly chosen using a custom python script (Appendix B.12) to only keep one sequence per contig and we ended up with 585 sequences.

The resulting 648 sequences were sent for the design of primers to an Australian team who developed the Hi-Plex protocol to genotype SNPs (Hammet et al., 2019). The primers were then blasted against our LP genome to remove repeated loci and aspecific amplifications.

Fertilization rates of pure LF (0.763 ± 0.285) were lower than those of BC (0.84 ± 0.241) and F2 (0.851 ± 0.192; 𝜒2 = 11.099; p < 0.05; Figure 13). FST_boot_sympatry<-diffCalc("genepop_for_sympatric_ind_map_DP5_ind.txt", outfile = "results_genepop_for_fst_carto_IND", fst = TRUE, pairwise = FALSE,bs_locus = TRUE, bs_pairwise = FALSE, boots = 1000, ci_type = "individuals", alpha = 0.05, para = TRUE) Appendix A.8. PLINK command line plink --vcf LG26_for_PCA.recode.vcf --double-id --allow-extra-chr -set-missing-var-ids @:# --make-bed --pca --

VCF_CsvReader=csv.reader(One_File,delimiter="\t") Nombre_Seq = 0 #compteur pour savoir combien de SNP on a dans le fichier à la fin CsvFile1=open(file1Match,"w") print"creation du fichier CSV1 ok!" except:

print("Unexpected error:", sys.exc_info()[0]) sys.exit() try:

CsvFileMulti=open(fileMulti,"w") print"creation du fichier CSVmulti ok!" except:

print("Unexpected error:", sys.exc_info()[0]) sys.exit() #si le snp intru est dans l intervalle (pos inf -pos 5') ou (l intervalle pos 3' -pos sup) #print("position 5' :"+str(pos_5)+" position du SNP :"+ str(position_snp)+" position 3' :"+str(pos_3)) #print(liste_snps

for seq_id in liste_mauvaise_seq: newOutfile.write("%s\n" % (seq_id))

Appendix B.11. python script to retrieve the good fasta sequences using sequence name (Extract_good_fasta.py)

parser.add_argument('-f', action='store', dest='InFile', help='Input Fasta File') parser.add_argument('-o', action='store', dest='OutFile', help='Outdir Fasta File') parser.add_argument('-r', action='store', dest='RefFile', help='Reference File.txt') parser.add_argument ('--version', action='version', version='%(prog) ('-f', action='store', dest='InFile', help='Input Fasta File') parser.add_argument('-o', action='store', dest='OutFile', help='Outdir Fasta File : IDSNPcomplet CHROM, POS,length scaffold, ref alt') parser.add_argument('--version', action='version', version='%(prog)

#Control path and good opening of files replicates = ["01","02","03","04","05","06","07","08","09","10","11","12","13","1 4","15","16","17","18","19","20","21","22","23","24","25","26","27", "28","29","30","31","32","33","34","35","36","37","38","39","40","41 ","42","43","44","45","46","47","48","49","