# Génétique des populations et phylogéographie du nématode à kyste de la pomme de terre (Globodera pallida) au Pérou

Damien Picard

► To cite this version:

Damien Picard. Génétique des populations et phylogéographie du nématode à kyste de la pomme de terre (Globodera pallida) au Pérou. Sciences du Vivant [q-bio]. Ecole Nationale Supérieure Agronomique de Rennes, 2005. Français. NNT: 2005NSARC076. tel-04225873

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# THÈSE

Présentée devant

AGROCAMPUS RENNES

Pour Obtenir le grade de:
DOCTEUR DE L'AGROCAMPUS RENNES.

Mention: Biologie

Par

DAMIEN PICARD

## GÉNÉTIQUE DES POPULATIONS ET PHYLOGÉOGRAPHIE DU NÉMATODE À KYSTE DE LA POMME DE TERRE (Globodera pallida) AU PÉROU

Unité mixte de Recherche I.N.R.A/Agrocampus Rennes, Biologie des Organismes et des Populations appliquée à la Protection des Plantes, le Rheu Laboratoire de Nématologie

École Doctorale Université de Rennes 1/Agrocampus Rennes: Vie-Agro-Sante

Soutenue le 22 Mars 2005 devant la commission d'examen

J. MALLARD

Professeur

Président

M. PHILLIPS

Professeur

Rapporteur

J. Y. RASPLUS

Directeur de Recherche

Rapporteur

D. ESMENJAUD

Directeur de Recherche

Membre

E. PETIT

Maître de Conférence

Membre

D. MUGNIÉRY

Directeur de Recherche

Directeur de Thèse

O. PLANTARD

Chargé de Recherche

Directeur de Thèse associé
REMERCIEMENTS

Je voudrais tout d'abord remercier Olivier Plantard pour s'être tant investi au cours de ma
thèse, ainsi que dans ma formation scientifique. Olivier est l'une des rares personnes qui ont la
générosité de donner pour les autres sans rien attendre en retour. Et pour moi, il n'a jamais compté
le temps passé pour m'apporter son savoir. Pour ta participation capitale à l'écriture du manuscrit,
pour les nombreux allers-retours des articles entre nos deux bureaux, pour ton investissement
altruiste, pour m'avoir apporté une large part de mes connaissances scientifiques actuelles, pour ta
patience, ta bonne humeur, tes rires allant jusqu'aux larmes et pour tous les bons moments passés
ensemble, je tiens à exprimer ma profonde reconnaissance et toute ma satisfaction de t'avoir eu
comme mentor.

Ce travail de thèse a été réalisé à l'UMR BiO3P, au centre INRA du Rheu. Je remercie très vivement les directeurs, Messieurs Charles-Antoine Dedryver, Bernard Tivoli et Didier Andrivon de m'avoir accueilli et permis de conduire mon travail dans les meilleures conditions.

J'adresse mes remerciements à Messieurs Mark Phillips, Jean-Yves Rasplus, Jacques Mallard, Daniel Esmenjaud et Eric Petit pour le jugement qu'ils ont porté sur ce travail.

Les échanges scientifiques au cours des comités de pilotage de cette thèse m'ont été d'un
apport certain dans la conduite des expérimentations et dans l'analyse des résultats. Je remercie
donc Messieurs François Bonhomme, Jean-Pierre Hugot, Philipe Castagnone-Sereno, Jacques
Mallard et Madame Claire Neema.

Je tiens à remercier tout particulièrement Didier Mugniéry, Eric Grenier et Sylvain Fournet pour tous nos échanges scientifiques, votre aide à l'écriture du manuscrit, aux présentations orales et pour votre grande disponibilité.

Sylvie Valette et Lucie Mieuzet, je vous remercie pour votre travail m'ayant permis d'acquérir une partie des données de ma thèse.

Je suis aussi particulièrement reconnaissant à toutes les personnes qui travaillent
quotidiennement au bon fonctionnement du laboratoire de biologie moléculaire.

Manuel Plantegenest, merci pour ton coup de pouce.

Malgré les fortes tensions entre le gouvernement ivoirien et le gouvernement français ayant
eu cours pendant ma thèse, les relations diplomatiques entre Abou et moi n'ont jamais connu
d'ombrage. Comment décrire cette parfaite cohabitation dans ce bureau du portakabin, peut-être
en relatant qu'elle a conduit à une amitié tellement balèze que même la distance qui va bientôt nous
séparer aura bien du mal à estomper. Ce bureau a vu passer une jeune fille à la grâce ensorcelante et
au parfum salvateur. Abou et moi avons vécu ce moment avec le plus grand des plaisirs, en tout bien tout honneur. Laure nous a charmés par sa rhétorique portant au plus haut l'art oratoire. La bonne ambiance régnant au portakabin ne s'est pas limitée à notre bureau. Il m'est encore très agréable de songer à mes deux égéries du portakabin : j'avoue que la belle Flora au sourire envoûtant et ma Nanabelle au regard troublant (hein ! Abou) peuplent encore mes rêves les plus doux. Un joyeux bordel ambiant a été mené de main de maître par Sabine, Fabian, Christophe et Sébastien et leur départ a été regretté par tous et a laissé un vide. Je tiens aussi à saluer toutes les autres personnes que j'ai côtoyées avec plaisir dans ce portakabin, comme Florian, Morvan, Delphine, Josselin, Valérie, Marie...

Pendant ces trois ans et demi, j'ai vécu une belle ambiance de sympathie et de convivialité avec l'ensemble de l'équipe de nématologie. J'ai vécu avec beaucoup de gaieté les amicales conversations avec vous, Géraldine et Alexandra. Je vous souhaite beaucoup de bonheur pour la suite. Ces phrases me rappellent ce voyage à Rome qui est classé numéro 1 dans le top 10 des séminaires. J'ai passé un chouette moment avec vous en Italie, bien qu'aillant failli dormir dehors car confronté à un italien revêche qui refusait avec véhémence d'ouvrir la porte de l'appartement, qui n'était pourtant habité que par d'éminents chercheurs de l'INRA. Sylvain, Môssieu MUGNIÉRY, Eric, Olivier, Laurent, Didier, Marie-Christine, Catherine, Lionel, Claudia, Sylvie, Magali, merci pour votre accueil chaleureux.

A toi Adrien, j'ouvre mon cœur pour te déclarer ma flamme. Tu es mon dieu grec, avec toi, j'en perds mon latin, poil aux mains. Sublimant l'humour, la gaîté et la franche rigolade, je pense qu'ensemble, nous en avons atteint le paroxysme. De dieu de dieu et tu vas être papa, ça me fait chaud au cœur. A la tienne Adrien, vis heureux avec ta petite famille et surtout ne perds jamais ton humour et ta grandiloquence (je te fais plein de bisous...). Aude, bon ça ne va pas être facile après cette déclaration d'amour. Ne pleure pas, non ne pleure pas tu as, tu as toujours de beaux yeux, ne pleure pas, non ne pleure pas tu m'as souvent rendu heureux, dis-moi Audinette ma thèse vient de s'achever, Adrien va s'en aller et toi tu vas rester, alors ne perds pas courage et prends un petit poisson, glisse-le entre tes jambes, il n'y a pas de raison, pour se tirer la langue, ne me regarde pas comme ça tout de travers, qui fait le premier pas, pour s'aimer à l'envers. Maga, tu es rayonnante, une vraie perle. El gringo Laurent de los Estados Unidos, I'm very proud to meet you Lorenzo. Frédéric-le-normal, avec toi c'est le pied, avec toi j'ai passé des instants dignes d'une randonnée dans le Cantal. Je décerne le ballon d'or à : GAËL (oooooh ! bravo, si si, toutes mes félicitations, j'y tiens j'y tiens, quel homme...). Virgil, fieffé coquin, c'est toi qui l'as eue, hein ! Je m'incline et te souhaite un joyeux mariage plein de grâce, pour votre progéniture je ne me fais pas de soucis elle sera obligatoirement une réussite esthétique. Je me demande seulement qui de vous ou d'Adrien-
Maga fera la plus jolie des jolies jeunes filles. Fabien Halkett, je t'ai toujours estimé comme le meilleur des thésards de BiO3P, alors félicitations pour être arrivé N°1 dans mon classement et bonne chance pour la suite mi amigo. Eduardo, Beatriz, Alexandra : viva España! Viva Italia!

Los tres meses en las Andes del Peru ont été un épisode fantastique de la thèse, une expérience inoubliable pour Pauline et moi. L'ensemble de l'échantillonnage réalisé dans ces montagnes grandioses a été un réel succès grâce à de nombreuses personnes qui nous ont aidés tout au long de notre périple. Monsieur MUGNIÉRY nous a accompagnés pendant les 15 premiers jours. Ses connaissances du pays et de la langue nous ont permis de nous adapter en douceur. Maria Scurrah nous a accueillis avec beaucoup de chaleur et de bienveillance. Maria a été la personne sans qui rien n'aurait été possible. Elle a notamment obtenu l'autorisation du gouvernement péruvien d'échantillonner et d'exporter les nématodes, ce qui n'est pas une mince affaire. Susana Chumbiauca nous a accompagnés dans le nord du pays. Son dévouement à notre cause et ses compétences ont fortement contribué à notre réussite. Par la suite, elle a suivi de Lima notre expédition avec attention et a préparé notre arrivée dans toutes les villes où nous nous sommes rendus. Mario 1 et Mario 2, nos épisodiques chauffeurs de choc nous ont permis de nous déplacer sans encombre et de façon efficace sur des « routes » chaotiques et parfois dangereuses. La SENASA est un organisme gouvernemental péruvien qui possède un réseau d'ingénieurs agronomes répartis dans tout le pays. Ces gens nous ont accompagnés dans tous les champs échantillonnés. Je tiens donc ici à les remercier toutes pour leurs compétences, leur dévouement et leur accueil amical. Pauline m'a bien plus qu'accompagné au Pérou. Elle a participé avec beaucoup de punch à ce travail parfois épuisant et m'a suppléé avec efficacité quand les aléas du voyage me clouaient au lit.

Maman, papa, belle-maman, beau-papa, Audrey, Rahim, Ulrich, Cynthia, Juliette, Romain et Marie, Sophie et Nico, votre amour a été un soutien permanent pour moi.

Pauline est la compagne de ma vie, le pilier de mon équilibre, mon petit poussin, mon capuchon. Ton aide efficace, ton indéfectible soutien et ton amour m'auront permis de mener à bien cette thèse.
## Préambule : agronomie et évolution

Les nouvelles méthodes de lutte contre les bioagresseurs issues des biotechnologies (plantes génétiquement modifiées, sélection assistée par marqueur pour l'obtention de nouvelles variétés résistantes) ainsi que le contrôle biologique du parasite à l'aide d'organismes génétiquement modifiés ou non enrichissent l'arsenal disponible contre les bioagresseurs. Néanmoins, de par la capacité à évoluer des bioagresseurs, l'efficacité de ces méthodes est souvent de courte durée. Dès lors la biologie évolutive, en s'intéressant à l'impact de la libération dans l'environnement de tels organismes tant sur les nuisibles des cultures que sur le milieu naturel, constitue une approche indispensable (Roderick et Navajas 2003). En outre ce type de libération a souvent montré la nature imprévisible de tels organismes introduits. En Amérique du nord, 16% des espèces introduites (insecte phytophage) contre une plante adventice ont aussi attaqué des plantes sauvages endémiques (Hawkins et Marino 1997). Le potentiel évolutif des espèces introduites, des nuisibles et des autres espèces en interactions dans cet environnement cultural, déterminant le succès du contrôle du nuisible, peuvent être appréhendés par des outils génétiques à travers une approche populationnelle ou phylogéographique. Les résultats de ces études aideront les chercheurs à évaluer les risques associés à l'utilisation à grande échelle d'organismes issus des biotechnologies ou à des nouvelles pratiques agricoles, en particulier les changements évolutifs à long terme. De plus, l'introduction d'organismes pour limiter les intrants chimiques peut être considérée comme un champ d'expérimentation semi-naturel qui permettrait d'élargir nos connaissances fondamentales sur l'évolution des êtres vivants.
# SOMMAIRE

## Introduction générale.................................................................................................................. 1

## Chapitre I

### Introduction : cadre évolutif du parasite, contexte biologique et agronomique................................ 5

1.1. De l'apparition des nématodes parasites des plantes à l'endoparasitisme : l'importance des données moléculaires dans la reconstruction de leur histoire.................................................................................... 6

1.2. Principales caractéristiques biologiques pouvant influencer le patron génétique................................ 11

1.2.1. Interactions fines entre le parasite et l'hôte, reproduction amphimictique et forme de conservation.................................................................................................................... 11

1.2.2. Capacité de dispersion du nématode à kyste.................................................................................................................... 13

1.3. La plante hôte et l'homme................................................................................................................................. 16

1.3.1. Le contexte évolutif : la plante hôte (Solanum sect. petota)................................................................. 16

1.3.2. Le contexte agronomique et culturel.................................................................................................................... 18

## Chapitre II

### Description du fonctionnement génétique des populations de Globodera pallida dans son berceau d'origine (le Pérou) à partir d'un échantillonnage hiérarchisé.................................................. 21

Article 1: Inbreeding and population structure of the potato cyst nematode (Globodera pallida) its native area (Peru).................................................................................................................... 23

## Chapitre III

### Patron d'« isolement par la distance » le long d'un transect de 1.500 km dans la cordillère des Andes et structuration génétique chez un organisme du sol de 500 micromètres................................................ 33

Article 2: Isolation by distance only above 50 km for a 500 µm long soil nematode................................................ 35

3.1. Abstract.................................................................................................................................................................................... 35

3.2. Introduction.................................................................................................................................................................................... 36

3.3. Materials and Methods.................................................................................................................................................................................... 37

3.3.1. Sampling design, field collection of nematodes and DNA sample preparation.................................................... 37

3.3.2. Population differentiation and IBD.................................................................................................................................................... 39

3.3.3. Genetic model-based clustering.................................................................................................................................................... 40

3.4. Results.................................................................................................................................................................................................................................................... 41

3.4.1. Population differentiation and IBD.................................................................................................................................................... 41

3.4.2. Genetic model-based clustering and hierarchical population structure................................................................. 41

3.5. Discussion.................................................................................................................................................................................................................................................... 46

3.5.1. No evidence of genetic differentiation for fields less than 50 km apart................................................................. 46

3.5.2. Increasing genetic differentiation among fields with geographic distance above 50 km.................................................... 47

3.5.3. What is a population?.................................................................................................................................................................................... 47

References.................................................................................................................................................................................................................................................... 49

## Chapitre IV

### Une colonisation du sud vers le nord de la Cordillère Andine par le nématode à kyste de la pomme de terre au cours des temps géologiques suggère de multiples changements d'hôtes des espèces sauvages aux espèces cultivées................................................................................ 53

Article 3: A South-to-North colonisation of the Andean Cordillera by the potato cyst nematode during geological times suggests multiple host shifts from wild to cultivated potatoes.................................................................................... 57

4.1. Introduction.................................................................................................................................................................................... 58

4.2. Materials and Methods.................................................................................................................................................................................... 59

4.3. Results.................................................................................................................................................................................................................................................... 63

4.3.1. Phylogeographical pattern based on the cytochrome B sequences................................................................. 63

4.3.2. Phylogeographical pattern based on the microsatellite loci.................................................................................................................... 65

4.3.3. Comparison of the trees based on the mitochondrial and the nuclear markers.................................................... 65

4.4. Discussion.................................................................................................................................................................................................................................................... 66

References.................................................................................................................................................................................................................................................... 71
# ANNEXE

<table><tr><td>Annexes 1 : Le Pérou, berceau d'origine du nématode de la pomme de terre</td><td>131</td></tr><tr><td>Annexes 2 : Génétique des populations de Globodera pallida dans son berceau d'origine (le Pérou) : consanguinité et fort flux de gènes</td><td>139</td></tr><tr><td>Annexes 3 : Orientation et datation de l'orogenèse des Andes induits par la phylogéographie de Globodera pallida</td><td>141</td></tr><tr><td>Annexes 4 : Listes des espèces du genre Globodera, du genre Punctodera et autre espèces citées dans la thèse</td><td>155</td></tr><tr><td>Annexes 5 : Tailles (axe Y) et fréquences (diamètre des cercles) des allèles microsatellites représentées à l'aide de graphiques bulles</td><td>159</td></tr><tr><td>Annexes 6 : Gène cytochrome b (Test de la saturation dans le jeu de donnée, Divergence nucléotidique et Séquences)</td><td>171</td></tr><tr><td>Annexes 7 : Abréviation</td><td>183</td></tr></table>
# INTRODUCTION GÉNÉRALE
Contexte de la thèse

Le nématode *Globodera pallida* est un endoparasite des racines de la pomme de terre. Originaire des hauts plateaux Andins, comme les espèces de *Solanum* sauvages auxquelles il est inféodé depuis des millions d'années, on le trouve actuellement associé aux champs à diverses espèces de *Solanum* domestiquées au Pérou depuis 5.000 ans. Plus récemment, il a été introduit sur le vieux continent suite à l'importation de *Solanum tuberosum* sous-espèce *tuberosum*. Il peut y occasionner des pertes de rendement allant jusqu'à 50% et est classé comme parasite de quarantaine dans la plupart des pays du monde. L'utilisation de nématicides est progressivement abandonnée en Europe en raison de la toxicité de ces produits pour l'homme et l'environnement. La lutte variétale, reposant sur la recherche de gènes de résistance isolés à partir de *Solanum* sauvages péruviens est actuellement privilégiée. Néanmoins, ces gènes de résistance ont une efficacité variable suivant l'origine géographique des nématodes, attestant de la diversité de ces derniers. L'étroite association entre le nématode et sa plante hôte (manipulation de l'expression des cellules végétales par le nématode pour son développement), ainsi que l'existence de nombreux gènes de résistance dans diverses espèces de *Solanum* sauvages des hauts plateaux andins, suggèrent une longue évolution commune entre ces deux protagonistes dans leur berceau d'origine. Le Pérou constitue donc une zone d'étude cruciale pour appréhender le patron génétique du parasite. De par la longue histoire de l'association entre l'hôte et le parasite, la diversité génétique des populations du nématode y est en effet maximale.

Notre étude se place en amont de l'introduction en Europe de nouvelles variétés de pommes de terre résistant aux nématodes à kyste *G. pallida*. Elle vise à appréhender la diversité génétique, le flux génique, l'histoire et le potentiel évolutifs d'un nématode du sol dans son bassin d'origine et secondairement dans ses zones d'introduction. Par la suite, cette étude permettra de suivre les modifications de la composition génétique des populations et d'estimer leur potentiel évolutif lors de l'introduction d'un gène de résistance qui constitue une pression de sélection. Ainsi, les informations obtenues permettraient d'adapter des stratégies d'utilisation des gènes de résistance pour limiter leur contournement, mais aussi d'enrichir un modèle épidémiologique pour combattre le parasite.
# Objectif de la thèse

Dans cet étude, nous avons eu recours à une approche évolutive (génétique des populations et phylogéographie). Jusqu'alors, tant en génétique des populations qu'en phylogéographie, peu d'études ont été réalisées chez les nématodes phytoparasites et plus généralement chez l'ensemble des nématodes. L'objectif de ce travail a été d'approfondir les connaissances sur la biologie des nématodes à kyste de la pomme de terre (mode de reproduction, capacité de dispersion) mais aussi les connaissances sur son histoire comme la localisation de son origine géographique et sa dispersion dans la Cordillère des Andes. Les résultats obtenus ont été aussi comparés au patron génétique de *G. pallida* dans une zone d'introduction (l'Europe) afin de déterminer l'origine géographique des populations introduites.

Après un premier chapitre introductif, œtte thèse est articulée autour de quatre chapitres complémentaires. Dans un premier temps (chapitre II), nous nous sommes intéressés aux questions suivantes :

* ✓ À quelle échelle spatiale observe-t-on une unité panmictique ?

* ✓ Comment se distribue la variabilité génétique à différentes échelles spatiales ?

* ✓ Quel est le niveau de la structuration génétique des populations et quelle est l'importance des flux géniques ?

Pour répondre à ces questions, nous avons réalisé un échantillonnage hiérarchisé au Pérou à différentes échelles spatiales (parcelle, région et pays). Huit loci microsatellites (marqueurs neutres et codominants) ont été utilisés pour génotyper les individus.

Dans un second temps (Chapitre III), nous nous sommes intéressés aux limites géographiques d'une population. Nous avons abordé ce sujet central en génétique des populations, en se posant les questions suivantes :

* ✓ Les tailles et les fréquences des allèles sont-elles géographiquement distribuées aux hasards ou s'associent-elles (ou se corrèlent-elles) en fonction de leur distribution géographique (l'association la plus commune est une augmentation de la différentiation génétique en fonction de la distance géographique ce qui traduit un isolement par la distance) ?

* ✓ Quel est le niveau de structure génétique observé à l'aide d'un algorithme bayésien de regroupement entre individu ou entre groupe d'individus ?

Grâce à un échantillonnage de 360 parcelles le long d'un transect nord - sud dans le massif andin (cf. Annexe 1), nous avons pu choisir les parcelles à analyser pour obtenir un jeu de données adapté pour répondre à nos questions et ainsi complémenter les données déjà analysées dans le chapitre II.
Dans un troisième temps (chapitre IV), nous avons cherché à retracer l'histoire de la dispersion des populations (phylogéographie) dans le bassin d'origine de *G. pallida*. En plus des données analysées dans les deux chapitres précédents, la séquence du gène cytochrome b (ADN mitochondriale) a été utilisée pour génotyper des individus choisis parmi les 360 échantillons du transect.

Dans un dernier temps (chapitre V), nous avons confronté les données obtenues en génétique des populations et en phylogéographie dans le berceau d'origine de *G. pallida* avec de nouvelles données obtenues par ailleurs au sein de l'équipe après analyse des populations d'une zone d'introduction (six pays européens). La comparaison des données obtenues en Europe et au Pérou, nous permettra d'estimer les modifications de la composition génétique des populations consécutive à une ou plusieurs introductions, d'estimer la diversité génétique introduite en Europe et de déterminer l'origine des populations introduites en Europe.

**Glossaire :**

**Allèle** : une des formes d'un gène dont la séquence d'ADN diffère dans la position d'au moins une paire de base.

**Flux génique** : le mouvement des gènes entre les populations. Cela nécessite la migration des individus d'une population à une autre et la réussite subséquente de la reproduction.

**Hétérozygotie** : un individu diploïde est considéré hétérozygote si les deux copies du même gène (allèles) sont différents.

**Homozygotie** : un individu diploïde est considéré homozygote si les deux copies d'un même gène sont identiques.

**Phylogenèse** : l'histoire évolutionniste d'un groupe d'organismes typiquement représentée par un arbre à bifurcations.

**Phylogénie** : l'étude des affinités évolutives de groupes d'organismes.

**Phylogéographie** : l'étude de la répartition géographique de la variation génétique soulignant les relations évolutives (phylogénétiques) entre les populations.
CHAPITRE I

Introduction : cadre évolutif du parasite,
contexte biologique et agronomique
Une bibliographie sur différentes thématiques en lien avec la thèse est présentée dans ce chapitre, afin d'établir un état des lieux des connaissances antérieures à la thèse et de développer des sujets discutés dans les chapitres qui suivront. Les points abordés sont le cycle parasitaire, la capacité de dispersion du parasite, la systématique des nématodes, la systématique de l'hôte et l'histoire de la culture de la pomme de terre dans les Andes, éléments essentiels et indispensables pour une approche de génétique des populations et de phylogéographie.

# 1.1. De l'apparition des nématodes parasites des plantes à l'endoparasitisme : l'importance des données moléculaires dans la reconstruction de leur histoire

La phylogéographie de *Globodera pallida* s'insère dans l'histoire du phylum Nematoda (figure 1.1) et particulièrement dans celles des nématodes endoparasites de plantes. Nous allons donc retracer l'histoire du parasitisme des plantes de ce phylum en mettant en lumière l'importance des données moléculaires (utilisées de façon privilégiée dans la thèse) dans sa reconstruction. La difficulté à établir une horloge moléculaire fiable dans le phylum et par conséquent dans le genre *Globodera* sera abordée.

La classification actuellement acceptée pour le phylum Nematoda (figure 1.1) est établie à partir de la phylogénie moléculaire basée sur le séquençage de la totalité du gène 18S de l'ADN ribosomique (Blaxter *et al.* 1998 ; Dorris *et al.* 1999 ; De Ley et Blaxter 2002). La précédente classification, basée exclusivement sur des caractères morphologiques, en a été profondément remaniée et ce, bien que les phylogénies moléculaires possèdent évidemment leurs propres limites (l'attraction des longues branches, la saturation due à des substitutions multiples au même site et les problèmes lié à l'alignement des séquences). Au sein du phylum Nematoda, la grande homogénéité morphologique des taxa rend difficile une résolution robuste de la phylogénie des nématodes basée sur ces seuls caractères (Dorris *et al.* 1999 ; Baldwin *et al.* 2004). À la lumière des données moléculaires, certains caractères morphologiques des nématodes se sont avérés similaires par convergence due à des pressions de sélection fonctionnelle dans des lignées différentes, ce qui peut avoir entraîné la formation de groupe paraphylétique ou polyphylétique (Baldwin *et al.* 2004). Par exemple, deux ordres composés de parasites de plantes (Dorylaimida et Triplonchida) qui étaient considérés comme formant un groupe monophylétique sur la base des données morphologiques se sont finalement avérés polyphylétique sur la base des données moléculaires (figure 1.1). À un rang taxonomique inférieur, l'analyse morphologique ne permet pas de discriminer certaines espèces entre elles. En effet, *Caenorhabditis elegans* et *C. briggsae* sont deux espèces morphologiquement semblables qui pourtant divergent depuis approximativement de 23 à 120 millions d'années (Prasad
et Baillie 1989 ; Heschl et Baillie 1990 ; Lee *et al.* 1992 ; Kennedy *et al.* 1993 ; Thacker *et al.* 1999 ; Coghlan et Wolfe 2002). De même au sein du genre *Globodera*, il est difficile de discriminer les espèces entre elles sans l'aide d'un outil moléculaire.

<figure><img src="image_2.png" /></figure>

**Figure 1.1** : Arbre phylogénétique du phylum Nematoda obtenu avec le gène 18S (d'après Dorris *et al.* 1999). Les nombres au dessus des branches indiquent la valeur de « bootstrap ». Cinq clades majeurs sont identifiés et numérotés de I à V. Les Chromadorida sont paraphylétiques et leur relation avec les Secernentea (clade III, IV et V) reste incertaine.
**Glossaire :**

**Apomorphe** : Qualifie, dans une séquence de transformations évolutives d'un caractère, l'état dérivé d'un état ancestral. On dit aussi état apomorphe, ou, par extension caractère apomorphe ou encore caractère dérivé.

**Clade** : Vient du grec clados qui signifie branche. Taxon* strictement monophylétique*, c'est-à-dire contenant un ancêtre et tous ses descendants.

**Convergence** : Ressemblance apparue indépendamment dans différents taxons*, par conséquent non héritée de l'espèce ancestrale à ces taxons. Une convergence apparue chez des taxons proches parents est appelée parallélisme*. La convergence est un cas particulier de l'homoplasie*.

**Homoplasie** : Similarité chez une ou plusieurs espèces, d'organes, de parties d'organes, ou de séquences d'ADN ou de protéines, lorsque l'on peut présumer que cette correspondance ne provient pas de l'héritage d'un ancêtre commun. On distingue plusieurs cas d'homoplasies : l'analogie, la convergence*, le parallélisme*, la réversion. Remarque : Dans le cas des caractères moléculaires, l'homoplasie n'est souvent pas détectable a priori et elle est révélée par l'arbre le plus parcimonieux. Dans le cas des caractères morphologiques, l'analyse fine des caractères et de leur homologie primaire permet plus souvent de détecter des homoplasies. Comme dans le cas précédent, celles qui n'auront pas été décelées seront révélées par l'arbre le plus parcimonieux.

**Horloge moléculaire (hypothèse de l')** : Hypothèse selon laquelle les molécules d'une même classe fonctionnelle évoluent régulièrement dans le temps et à un rythme égal dans différentes Ignées. Ainsi la quantité des différences moléculaires constatées de nos jours dans des séquences homologues d'espèces distinctes peut être utilisée pour estimer le temps écoulé depuis le dernier ancêtre commun à ces deux espèces (ou temps de divergence).

**Monophylétique (groupe)** : En Systématique phylogénétique, groupe qui comprend une espèce ancestrale et tous ses descendants. Un groupe monophylétique est défini par au moins une synapomorphie*. On dit aussi d'un groupe monophylétique qu'il est un clade*.

**Parallélisme** : Ressemblance apparue indépendamment dans différents taxons* proches parents. Le parallélisme est un cas particulier de la convergence*.

**Paraphylétique (groupe)** : En Systématique phylogénétique, groupe qui comprend une espèce ancestrale, et une partie seulement de ses descendants. Un groupe paraphylétique est défini par au moins une symplésiomorphie* ou une absence de caractère.

**Plésiomorphe** : Se dit de l'état ancestral d'un caractère. Les adjectifs ancestral ou primitif sont synonymes.

**Polyphylétique (groupe)** : En Systématique phylogénétique, groupe qui contient un certain nombre d'espèces ou de taxons*, mais ne contient pas l'ancêtre commun à tous. En d'autres termes, un groupe polyphylétique dérive de deux ou plusieurs espèces ancestrales. Un groupe polyphylétique est défini par au moins une homoplasie*.

**Taxon** : Ensemble des organismes reconnus et définis dans chacune des catégories de la classification biologique hiérarchisée. En d'autres termes : contenu concret d'une catégorie. Exemple : Canis lupus, le Loup, est un taxon de rang spécifique (catégorie : espèce) ; les canidés (Chien, Loup, Renard .) constituent un taxon de rang familial (catégorie : famille).

**Symplésiomorphe** (caractère) : Caractère plésiomorphe* présent chez deux ou plusieurs taxons.

**Synapomorphe** (caractère) : Caractère apomorphe* partagé par deux ou plusieurs taxons*.

L'homogénéité morphologique observée chez les nématodes est étonnante de par leur longue évolution et leur importante diversité (Dorris *et al.* 1999). En effet, le phylum Nematoda serait probablement apparu au début du Cambrien vers moins 550 millions d'années (Ayala *et al.* 1998 ; Rodriguez-Trelles *et al.* 2002). Les données moléculaires et paléontologiques suggèrent que les plantes terrestres sont apparues depuis 425 à 490 millions d'années (Sanderson 2003). Les nématodes sont probablement à l'origine des organismes libres, leur coexistence dans le sol avec les plantes pendant une très longue période leur a permis d'exploiter en tant que parasite cette nouvelle ressource. Ce parasitisme des plantes semble être apparu de façon indépendante à au moins trois reprises au cours de l'évolution (l'ordre des Dorylaimida, des Triplonchida et des Tylenchida ; figure 1.1 ; Blaxter *et al.* 1998). L'endoparasitisme sédentaire des plantes est apparu chez les Tylenchida, indépendamment dans la sous-famille des Meloidogyninae avec les nématodes à galles
et dans la sous-famille des Heteroderinae avec les nématodes à kystes (Baldwin *et al.* 2004). La distribution mondiale des espèces de la sous-famille des Heteroderinae suggère que les ancêtres de ce groupe étaient présents à l'époque de la Pangée (Ferris 1979), donc il y a au moins 185 millions d'années (Veevers 2004). Cependant, l'absence de fossile limite la fiabilité des estimations temporelles et du calibrage d'une horloge moléculaire dans le phylum [Il faut noter l'existence de quelques nématodes fossiles (entomopathogène) conservés dans l'ambre : le plus ancien provient du Crétacé (Poinar et Poinar 2003)].

Chez les Heteroderinae, la phylogénie obtenue avec les séquences d'ITS « internal transcribed spacers » (Subbotin *et al.* 2001) ne permet pas de résoudre les relations phylogénétiques entre les espèces du genre *Globodera* et celles du genre le plus proche de *Globodera: Punctodera* (figure 1.2 ; Subbotin *et al.* 2001). En effet, on observe une trifurcation entre trois clades : les *Globodera* parasitant les Solanacées (Amérique), les *Globodera* parasitant les Astéracées (Eurasie) et les *Punctodera* parasitant les Poacées. Cependant, les espèces du genre *Globodera* (cf. Annexe 4) sont morphologiquement bien différenciées des espèces du genre *Punctodera* (Luc *et al.* 1988 ; forme du cône vulvaire, forme du kyste...). La systématique de ces deux genres est en cours de révision. Jusqu'alors, l'homogénéité morphologique au sein du genre *Globodera* a rendu difficile la détermination des relations phylogénétiques de ces espèces. De plus, les efforts de description et d'identification des espèces du genre *Globodera* se sont portés quasi-exclusivement sur des espèces d'intérêt agronomique. La répartition géographique de nombreuses espèces n'est connue qu'à travers une prospection dans des parcelles cultivées. En dehors des parasites de plantes cultivées dans le genre *Globodera* et plus largement dans la famille des Heteroderinae, beaucoup d'espèces ont été décrites à partir de peu d'individus et n'ont parfois aucun hôte connu ; quant à leur répartition géographique, elle se limite souvent à une unique localité (*G. leptonepia*, *G. zelandica*...). Une prospection des espèces du genre *Globodera* parasitant des plantes sauvages reste donc à faire. Toutefois, les espèces inféodées aux solanacées peuvent être considérées comme originaires des deux Amériques (figure 1.3). Ils forment un clade monophylétique. Ces espèces américaines se divisent en deux clades monophylétiques regroupant *G. rostochiensis* et *G. tabacum* d'un côté et *G. pallida* et *G. mexicana* de l'autre [sur la base de données personnelles et de l'étude de Subbotin *et al.* (2000) ; figure 1.2]. Dans le berceau d'origine (la Cordillère des Andes) *G. pallida* se distribue plus au nord et *G. rostochiensis* plus au sud avec une zone de sympatrie vers le lac Titicaca (Evans et Stone 1977 ; figure 1.3).

Le premier signalement du nématode à kyste de la pomme de terre est de Kühn (1881), qui l'identifie comme le nématode à kyste de la betterave (*Heterodera schachtii*). Wollenweber (1923) est le premier à distinguer le nématode à kyste de la pomme de terre de celui de la betterave. Il le nommera *Heterodera rostochiensis* à partir d'individus provenant de Rostock (Allemagne). Une seconde espèce (*H. pallida*) a été décrite par Stone (1972) créant le sous-genre *Globodera* que
Behrens (1975) élèvera au rang de genre. Jusqu'en 1972, ces deux espèces étaient donc confondues. Comme les caractères morphologiques permettant l'identification de chaque espèce sont ténus, une confusion restait possible jusqu'à l'établissement d'une identification moléculaire spécifique (Thièrry et Mugniéry 1996). Le travail rapporté dans cette thèse permet d'affiner encore plus la systématique de ce groupe en s'intéressant à la variabilité intra-spécifique de *G. pallida*.

<figure><img src="image_3.png" /><figcaption>**Figure 1.2** : (A) Synthèse schématique des relations phylogénique entre des espèces du genre *Globodera* et *Punctodera* séquencées avec l'ITS ou le gène cytochrome b (d'après Subbotin *et al.* 2000 et données personnelles) ; (B) Rangs taxonomiques conduisant au genre *Globodera* (du règne au genre).</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Figure 1.3** : Distribution géographique des espèces américaines de *Globodera* selon leur lieu probable d'origine. Toutefois, ces espèces ont été échantillonnées essentiellement en zone de culture et leur distribution sur des plantes sauvages reste inconnue.</figcaption></figure>
## 1.2. Principales caractéristiques biologiques pouvant influencer le patron génétique

G. pallida est un parasite obligatoire, dont le cycle se déroule principalement dans la racine des pommes de terre. Afin d'appréhender les éléments de la biologie de ce parasite pouvant influencer la structuration génétique de ses populations, il nous faut d'abord décrire son cycle parasitaire et sa capacité de dispersion. Pour estimer la capacité de dispersion de G. pallida, nous décrirons des études sur la capacité de dispersion des organismes du sol. Comme ces études sont peu nombreuses, nous les aborderons de façon quasiment exhaustive.

### 1.2.1. Interactions fines entre le parasite et l'hôte, reproduction amphimictique et forme de conservation

Comme chez tous des nématodes, G. pallida a quatre stades juvéniles et un stade adulte (figure 1.4). Le second stade juvénile (J2) éclot de l'œuf, stimulé par des substances chimiques émanant de la racine et particulièrement de la zone d'élongation (Rawsthorne et Brodie 1986). Les exsudats radiculaires de la pomme de terre peuvent provoquer plus de 80% d'éclosion (Fenwick 1949). Il est attiré jusqu'à la racine par le gradient de ces substances chimiques, probablement capté par les amphides, organes sensoriels pairs situés sur la tête. Ces stimulations sont spécifiques des plantes de la famille des Solanaceae. Des tests en laboratoire ont montré que G. pallida et G. rostochiensis peuvent parasiter une gamme d'hôte réduite aux Solanum tubéreux sauvages et cultivés, ainsi que Lycopersicon esculentum (la tomate).

Le nématode pénètre dans la racine essentiellement grâce à l'action mécanique de son stylet et migre de manière intracellulaire dans le cortex. Au niveau du cylindre central, il s'immobilise et perce la paroi d'une cellule cible unique. Les secrétions des glandes œsophagiennes sont injectées dans la cellule. Une interaction spécifique plante-parasite induit la formation d'un large syncytium par rupture des parois des cellules adjacentes (Gheysen et Fenoll 2002). Cette structure métaboliquement très active joue le rôle de site nourricier. Le nématode reste au niveau de ce site jusqu'à la fin de son développement qui peut durer trois mois dans les conditions naturelles (Jones et Northcote 1972). Si les interactions entre l'hôte et le parasite ne sont pas optimales, les qualités nutritives insuffisantes du syncytium entraînent alors un arrêt et un retard du développement et/ou une masculinisation. En fonction de leur capacité à induire et maintenir dans leur plante hôte un syncytium de taille suffisante pour leur nutrition, les juvéniles évoluent en mâles ou en femelles. La
détermination du sexe femelle se fait dans un milieu riche en nutriments tandis que des conditions de stress alimentaire conduisent à la formation de mâles (Trudgill 1967). La femelle adulte a une forme sphérique et fait saillie à la surface de la racine (figure 1.4). Le mâle est vermiforme (~ 1 mm) et migre à l'extérieur de la racine pour féconder les femelles (reproduction amphimictique). Par la suite, la femelle meurt, sa cuticule se durcit, devient brune et l'ensemble donne un kyste contenant jusqu'à mille œufs ou plus (figure 1.5). Ce kyste est la forme de conservation de l'espèce. Il permet aux œufs de rester viables plus de dix ans. Même si, au fil des années le nombre d'œufs viables décroît.

<figure><img src="image_5.png" /><figcaption>**Figure 1.4** : Cycle parasitaire du nématode à kyste de la pomme de terre (dessin original de Pauline Casters-Picard).</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 1.5** : Kystes (~ 0,5 mm de diamètre) de *Globodera pallida* flottant dans l'acétone au niveau du goulot d'un erlenmeyer (dernière étape extraction des kystes du sol).</figcaption></figure>

### 1.2.2. Capacité de dispersion du nématode à kyste

Le nématode à kyste peut se disperser activement lors de deux phases de son cycle (le stade mâle et le stade J2 ; figure 1.4). Cependant, cette dispersion active est très limitée dans le temps et dans l'espace, puisque les deux phases libres ont une durée de vie limitée à quelques jours et se déplacent uniquement dans la phase aqueuse du sol par des mouvements locomoteurs sinusoïdaux (Crofton 1971). Dès lors ces stades se déplacent tout au plus de quelques centimètres.

Pour ces raisons, la communauté scientifique pensait que les flux géniques de ce parasite du sol étaient négligeables (Bakker *et al.* 1993 ; Blok *et al.* 1997a ; Folkertsma *et al.* 2001 ; Bakker 2002). Des études sur différents organismes telluriques (voir ci-dessous) mettent en évidence qu'à longue distance, c'est la dispersion passive et non la dispersion active qui joue un rôle prépondérant dans la dissémination. Concernant les nématodes à kyste, on estimait que la dispersion passive était trop faible pour induire, à partir de quelques kystes, un flux génique conséquent entre parcelles de part les effectifs considérables au sein d'une parcelle cultivée en pomme de terre (jusqu'à dix milliards kystes par hectare ou sept kystes par gramme de sol ; Franco 1977). Des études sur la microfaune libre dans le sol révèlent des capacités importantes de dispersion passive à longue distance. Par exemple, chez deux espèces de collemboles (*Gomphiocephalus hodgoni* et *Orchesella cincta*), d'importants flux de gènes sur des distances supérieures à plusieurs kilomètres sont observés (Stevens et Hogg 2003 ; van der Wurff *et al.* 2003). Finlay (2002) généralise l'hypothèse d'une possible dissémination
à l'échelle mondiale à tous les organismes eucaryotes libres ayant une taille inférieure à 2 mm (pour mémoire le diamètre d'un kyste est de l'ordre de 0,5 mm). Des phénomènes semblables ont été observés aussi pour des nématodes. Lors d'une expérience originale menée par Wardle *et al.* (2003), des paniers remplis d'humus vierge de tout être vivant, sont disposés sur la canopée de la forêt tropicale de Nouvelle-Zélande. Six mois plus tard, les auteurs s'aperçoivent que de nombreux nématodes (bactériophages, mycophages, omnivores et prédateurs d'autres nématodes) ont colonisé ce milieu. Pour le nématode libre du sol *Caenorhabditis elegans*, la capacité à se disperser passivement atteint de telles proportions qu'elle empêche une structuration génétique entre la France, l'Allemagne, la Grande Bretagne, l'Australie, le Canada et les États-Unis d'Amérique (Koch *et al.* 2000).

Les nématodes à kystes ne sont pas des organismes libres du sol et doivent nécessairement rencontrer une plante hôte pour se développer, ce qui limite évidement leur potentiel d'une dissémination effective (c'est-à-dire d'une dissémination suivie d'une reproduction). Cependant, en plus de sa taille, le kyste possède d'autres atouts pour se disperser de façon efficace : les œufs dans un kyste peuvent survivre plus de 10 ans et un seul kyste transporte suffisamment d'individus (jusqu'à 1.000 œufs par kyste) pour fonder une nouvelle population.

De nombreux facteurs abiotiques et biotiques permettent une dispersion passive des nématodes. Une étude a été menée sur la dissémination éolienne des nématodes dans le bassin arachidier du Sénégal en piégeant le sable et les poussières dans des pots affleurant la surface du sol (Baujard et Martiny 1994). De nombreux nématodes libres du sol dont certains en état d'anhydrobiose (figure 1.6), ainsi que des nématodes phytoparasites (ordre des Tylenchida et ordre de Dorylaimida) ont été piégés. Ces nématodes transportés par le vent conservent leurs facultés biologiques de reproduction malgré les stress subis. White (1953) décrit qu'à la suite d'une tempête, des kystes contenant des J2 viables ont été récupérés sur trois assiettes disposées à un mètre du sol près d'un champ de pomme de terre moyennement contaminé (8 kystes pour 25 grammes de sol) par *Globodera rostochiensis*. Bien qu'aucune étude n'ait été menée sur l'eau de ruissellement, il est probable que celle-ci joue aussi un rôle dans la dissémination des nématodes. Les animaux sont aussi un des facteurs de dispersion, en déplaçant de la terre contaminée, mais aussi en transportant des nématodes dans leur tube digestif. En effet, des kystes contenant des J2 viables ont été retrouvés dans des déjections d'oiseaux et de rongeurs (Norton et Niblack 1991 ; Epps 1971).
<figure><img src="image_7.png" /><figcaption>**Figure 1.6** : Nématode libre (*Scutellonema cavenessi*) en état d'anhydrobiose, collé sur une particule de sable (d'après Baujard et Martiny 1994).</figcaption></figure>

L'homme contribue aussi à la dispersion passive des kystes. Tout d'abord, la culture de la pomme de terre augmente considérablement les effectifs du nématode parasite et les surfaces de sol contaminé ce qui, par conséquent, accroît la quantité de kystes pouvant être dispersée par les animaux, l'eau de ruissellement ou le vent (surtout dans les parcelles où la terre reste à nu entre deux cultures). Ensuite, l'homme peut être plus directement un facteur de dissémination. En effet, à l'échelle d'une exploitation, différentes pratiques agricoles peuvent être incriminées telles que le travail de la terre (Marshall et Brain 1999), le drainage de parcelles ou le déplacement d'outils agricoles souillés. À une échelle plus large, le transport de tubercules associés à de la terre contaminée est une voie de dissémination possible bien qu'actuellement des mesures de quarantaine (prises dans la plupart des pays du monde) doivent limiter son impact. Toutefois, à l'intérieur du Pérou, les services phytosanitaires ne pratiquent aucun contrôle sur *G. pallida* ou *G. rostochiensis*.

Nous terminerons cette partie consacrée à la capacité de dispersion des nématodes avec des travaux menés au sein de l'équipe dans le nord de la France sur un autre nématode à kyste, *Heterodera schachtii*, le nématode de la betterave. Les résultats obtenus montrent une faible différenciation génétique, entre des parcelles séparées jusqu'à 175 kilomètres ce qui illustre la forte capacité de dissémination des kystes dans un contexte agronomique (Plantard et Porte, 2004). Néanmoins, il faut noter que l'épandage en champ des rejets contaminés de sucrerie favorise considérablement la dispersion des kystes.
## 1.3. La plante hôte et l'homme

### 1.3.1 Le contexte évolutif : la plante hôte (Solanum sect. petota)

Les interactions fines existant entre l'hôte et le parasite (paragraphe 1.2.1), ainsi que leur longue histoire commune soulignent l'intérêt qu'il y a à retracer l'histoire des pommes de terre sauvages pour mieux comprendre celle de son parasite.

Le Genre *Solanum* est composé d'approximativement 1400 espèces réparties à travers le monde (Kellogg 1998). L'un des principaux groupes de ce genre est la section *Petota* regroupant les pommes de terre sauvages et cultivées. La section *Petota* composée uniquement de plantes tubéreuses est associée à trois autres sections non tubéreuses phylogénétiquement proches : *Lycopersicum* (tomate), *Juglandifolium* et *Euberosum* (Spooner et Hijmans 2001). Ces quatre sections sont réparties sur les deux Amériques. La distribution des 225 espèces (Hawkes 1990) de la section *Petota* s'étend du sud-ouest des États-Unis jusqu'au sud du Chili (figure 1.7). La distribution du nombre d'espèces n'est pas uniforme. Il existe deux centres de biodiversité majeurs. Le principal se situe au niveau des hautes altitudes entre les 10ème et 20ème degrés de latitude sud englobant le sud du Pérou et la Bolivie (figure 1.7). Le second, de moindre importance, se situe au cœur du Mexique (Hijmans et Spooner 2001).

La section *Petota* se divise en 21 séries dont la plus importante est la série *Tuberosa* (30 espèces) comprenant le complexe *Solanum brevicaule* d'où sont issues les neuf espèces et sous-espèces de pommes de terre cultivées (Spooner *et al.* 1999 ; figure 1.8). Mais, la systématique des pommes de terre sauvages diffère, à tous les niveaux taxonomiques (séries, espèces et sous-espèces ou variétés), d'un taxonomiste à l'autre. La systématique de ce groupe apparaît complexe et n'est toujours pas résolue, malgré les efforts de nombreux systématiciens pour ces plantes d'intérêt agronomique. Compte tenu des possibilités d'échanges génétiques entre les espèces et des faibles différences qui existent entre les génomes, il semble que la diversification soit plus la conséquence d'un isolement éco-géographique que de barrières cytogénétiques (Rossignol et Rousselle-Bourgeois 1996). Le complexe *Solanum brevicaule* illustre la complexité de la systématique de cette section. En effet, il existe de nombreux échanges génétiques entre les espèces du complexe, notamment entre le compartiment cultivé et le compartiment sauvage, avec des retours probables d'espèces cultivées à l'état sauvage. Spooner *et al.* (1999) se pose même la question de savoir s'il est possible de résoudre la systématique du complexe. Malgré tout, Hawkes (1990) propose une hypothèse pour l'origine des espèces cultivées (figure 1.8).
<figure><img src="image_8.png" /></figure>

**Figure 1.7** : Carte de la distribution du nombre d'espèces de *Solanum sect. petota* ; (a) répartition du nombre d'espèces en fonction de la latitude, (b) en fonction de l'altitude (d'après Hijmans et Spooner 2001).
<figure><img src="image_9.png" /><figcaption>**Figure 1.8** : Hypothèse de l'origine des espèces cultivées (d'après Hawkes 1990).</figcaption></figure>

1.3.2. Le contexte agronomique et culturel

Les conditions climatiques difficiles expliquent pourquoi les hommes n'ont pas colonisé plus rapidement les hautes terres des Andes. Il y a 18.000 ans, les glaciers qui recouvrent les hautes altitudes commencent à reculer. Toutefois, ce processus est lent et sa progression dépend du relief. Dans les Andes centrales, la fonte des glaces ne s'est produite qu'il y a 12.000 ans environ. À cette époque, les températures andines sont inférieures de cinq à huit degrés à celles qui règnent aujourd'hui (Aldenderfer 2004). Sur les flancs occidentaux des Andes, la limite des neiges éternelles se trouve 500 mètres plus bas que son niveau actuel (4.800 mètres), et les plantes adaptées au froid comme la plupart des Solanacées tubéreuses poussent à des altitudes plus basses qu'aujourd'hui (Vuilleumier et Simberloff 1980 ; Foster 2001 ; Garcia-Moreno et al. 2004). Le réchauffement de la terre permet ensuite aux premiers hommes de coloniser ces hautes terres vers -10.000 ans (figure 1.9). Les données archéologiques de cette période suggèrent que l'homme ne séjourne que temporairement à ces hautes altitudes (Aldenderfer 2004). L'agriculture et la domestication du lama auraient été développées dans la montagne vers 4.000 ans avant notre ère à la suite d'une période
assez longue de 2.000 à 3.000 ans pendant laquelle, de façon continue, est née l'agriculture sud-américaine (Jensen et Krantz 1974). Les pommes de terre diploïdes sans alcaloïde ont probablement été l'une des premières étapes de l'agriculture andine (Spire et Rousselle 1996).

<figure><img src="image_10.png" /></figure>

**Figure 1.9** : Illustration des 1ères trace de présence humaine dans la Cordillère Andine (d'après Aldenderfer 2004). Le peuplement des Andes a débuté vers la fin du dernier âge glaciaire, il y a une dizaine de milliers d'année. Monte Verde, le plus ancien site répertorié des Amériques, date d'il y a 13 500 ans. Il est intéressant de noter sur cette carte en trois dimensions l'élargissement des Andes au niveau de l'Altiplano, signe géologique suggérant que l'érection de la Cordillère des Andes a commencé dans cette région.

Le développement des pratiques agricoles culmine à la période Inca. Un site archéologique de cette période appelé Moray (figure 1.10) près de Cuzco aurait été l'un des premiers centres expérimental de recherche en agronomie. En effet, chaque étage est constitué d'un mètre d'empierrement pour obtenir un bon drainage sur lequel deux mètres de terre arable ont été ajoutés. L'ensemble de ces terre-pleins soutenus par des murs lithiques était irrigué par un système de canaux appropriés. L'héritage agronomique de la civilisation Inca reste riche en dépit de l'élimination de cette civilisation par les conquistadors. Poma de Ayala (1987) a listé 32 types de sols agricoles incluant les sols pour jachère et les sols pour rotations courtes dans une classification qu'utilisaient les Incas. Ils n'avaient probablement pas connaissance de la présence du nématode à kyste de la pomme de terre, mais leurs pratiques agricoles permettaient d'en limiter l'incidence. Une à trois cultures en rotation dont la première année était réservée à la culture de tubercules, suivies de huit ans ou plus de jachère étaient une pratique répandue à l'arrivée des espagnols, leur donnant l'impression de terres agricoles abandonnées (Thurston 1994, livre potato pest). Si de telles rotations étaient appliquées en Europe, on estime que ce mode de culture qui est encore pratiqué
dans certaines régions reculées au Pérou (cf. Annexe 1), permettrait de maintenir les populations du
nématode à kyste en dessous de son seuil de nuisibilité (Jones 1970 ; Jones 1972). L'héritage des
Incas et de leurs prédécesseurs ne se limite pas à des pratiques culturales. De nombreuses variétés
andines génétiquement très diverses sont encore disponibles. On rencontre notamment la variété
Hualash, encore cultivée, qui s'est avérée résistante à G. pallida (com. Pers. de Maria Scurrah). Ces
pratiques agricoles et la culture de variétés résistantes peuvent avoir un impact considérable sur le
patron génétique du parasite.

<figure><img src="image_11.png" /></figure>

**Figure 1.10** : Site archéologique Inca de Moray considéré comme l'un des premiers centres expérimentaux de recherche en agronomie. Il y a un dénivelé de 150 mètres entre les terrasses les plus hautes et les plus basses.
CHAPITRE II

Description du fonctionnement génétique
des populations de *Globodera pallida* dans
son berceau d'origine (le Pérou) à partir d'un
échantillonnage hiérarchisé
Ce chapitre a fait l'objet d'une publication dans le journal « Molecular Ecology ».

Cette étude constitue la première description du patron génétique du nématode à kyste de la pomme de terre à différentes échelles spatiales. Grâce un échantillonnage hiérarchisé et le génotypage de 504 individus à l'aide de huit loci microsatellites, la partition de la variabilité génétique et des flux de gènes à l'échelle spatiale d'une parcelle, d'une région et du pays seront estimés. Par ailleurs, l'isolement par la distance sera testé à l'échelle la plus fine, c'est-à-dire entre quarante plantes espacées de dix mètres les unes des autres dans une même parcelle. L'hypothèse de l'origine sud-américaine du parasite sera testée par la comparaison de la richesse allélique entre le Pérou et l'Europe, ainsi qu'entre trois régions péruviennes.

Enfin, l'analyse de la consanguinité (révélant la façon dont les allèles s'associent dans la descendance), sera décrite afin de préciser les modalités de reproduction des populations péruviennes.
# Inbreeding and population structure of the potato cyst nematode (*Globodera pallida*) in its native area (Peru)

D. PICARD,* O. PLANTARD,* M. SCURRAH† and D. MUGNIÉRY*

*UMR INRA-ENSAR BiO3P, Domaine de la Motte, B.P. 35327, 35653 Le Rheu cedex, France, †Centro Internacional de la Papa (CIP), Apartado 5969, Lima, Perú

## Abstract

The dispersal abilities and the population genetic structure of nematodes living in the soil are poorly known. In the present study, we have pursued these issues in the potato cyst nematode *Globodera pallida*, which parasitizes potato roots and is indigenous to South America. A hierarchical sampling regime was conducted in Peru to investigate gene flow on regional, field and plant scales. Multilocus genotypes of single individuals were obtained using eight polymorphic microsatellites markers. Large heterozygote deficiencies were observed at most loci. The limited active dispersal of larvae from their cyst, which favours mating between (half) siblings, could be responsible for this pattern. Within fields, as well as among fields within regions (even 35 km apart), low $F_{ST}$ values suggest extensive gene flow. Among fields within regions, only 1.5–4.4% genetic variability was observed. Passive dispersal of cysts by natural means (wind, running water, or wild animals) or by anthropogenic means (tillage, movement of infected seed tubers) is probably responsible for the results observed. Among regions, high $F_{ST}$ values were observed. Thus long-range dispersal (more than 320 km apart) is probably limited by major biogeographical barriers such as the mountains found in the Andean Cordillera. These results provide useful information for the management of resistant varieties, to slow down the emergence and spread of resistance-breaking pathotypes.

**Keywords:** gene flow, heterozygote deficiency, long-distance dispersal, microsatellite, phytoparasitic nematode, soil-dwelling invertebrates

Received 30 April 2004; revision received 28 May 2004; accepted 28 May 2004

## Introduction

Over the last few years there has been increasing interest in investigating the dispersal abilities of invertebrates and micro-organisms (Finlay 2002; Fenchel 2003; Stevens & Hogg 2003; van der Wurff *et al.* 2003). The use of molecular tools based on polymerase chain reaction (PCR) amplification now allows the estimation of gene flow for organisms that until now were too small to be investigated with alternative methods such as capture-recapture. For pest species, measurement of dispersal ability is of crucial importance to formulating control methods to limit the economic losses in agriculture (see for example Lenormand & Raymond 1998; Carriere *et al.* 2003). The use of population genetics to estimate dispersal also provides additional information concerning the inbreeding, the genetic structure and the level of gene flow at various spatial scales.

Correspondence: O. Plantard. Fax: 33 2 23 48 51 50; E-mail: plantard@rennes.inra.fr

*Globodera pallida* is a quarantine parasite in Europe and the USA (a quarantine parasite is a pest of potential economic importance to the area endangered where it is present but not widely distributed): distribution of seed tubers must be regulated to exclude all material that could be contaminated with this parasite. It is difficult to combat cyst nematodes with conventional control methods such as nematicides. Moreover, nematicidal products are among the most toxic agrochemicals and have been banned in several countries (e.g. USA, Switzerland). For these reasons, the use of resistant crops is currently the preferred control method. However, the existence of resistance-breaking pathotypes has already been reported for several nematode resistance genes (Janssen *et al.* 1990; Castagnone-Sereno 2002). In this context, a good knowledge of population genetics is essential to the development of efficient strategies for the durable deployment of resistant crops (McDonald & Linde 2002; van den Bosch & Gilligan 2003).

*Globodera pallida* (and the sibling species *G. rostochiensis*) was introduced into Europe from South America and is
probably native to the Andean Cordillera (Canto Saenz & Scurrah 1977; Stone 1985), the origin of its unique host genus *Solanum* (Hijmans & Spooner 2001). *Globodera pallida* is a sexually dimorphic amphimictic diploid species. Larvae of the second stage (hereafter referred to as J2) emerge from cysts and disperse actively in the soil, attracted to the plant root tips. While the females are sedentary, the males have an active dispersal stage in the soil, when they look for females and fertilize them. The fertilized females lay eggs within their body wall. When the female dies the body wall forms a cyst; this is the conservation stage containing several hundred J2s (up to 600) which can survive for many years (up to 10 years). The gene flow of *G. pallida* has been thought to be negligible (Bakker *et al.* 1993; Blok *et al.* 1997a; Folkertsma *et al.* 2001; Bakker 2002). However, contradictory observations also exist. In Europe, the two *Globodera* species occur in most regions where potatoes are cultivated (Turner & Evans 1998). A recent study (Plantard & Porte 2004) of population genetics of another cyst nematode (*Heterodera schachtii*) has also shown that cyst nematodes can have a high level of gene flow. However, the sugar beet cyst nematode is not dispersed on or with plant material (in contrast to *G. pallida* cysts which can be found in the soil attached to seed tubers), has a world-wide distribution (while *G. pallida* populations outside South America are considered as recent introductions), is polyphagous (being able to reproduce on numerous botanic families while *G. pallida* is restricted to *Solanum*) and have several generations a year.

The study of the population genetics of an introduced organism is more difficult because the current genetic structure will have been influenced by (i) number of individuals that were introduced, (ii) number of generations since introduction, (iii) origin, and thus genotypes, of the individuals introduced, and (iv) stochastic effect of genetic drift. Moreover, in the case of the potato cyst nematodes the history of the introduction events is poorly documented. A similar argument is at the heart of numerous studies of plant pathogens in their native area (Cattan-Toupance *et al.* 1998; Perez *et al.* 2001). To avoid the effect of bottlenecks and genetic drift as a consequence of one or more multiplication cycles in the laboratory (De Boer *et al.* 1992; Folkertsma *et al.* 1996, 2001; Blok *et al.* 1997b), we have only used individuals of *G. pallida* from cysts collected in South America without such multiplication.

The present study constitutes the first population genetic study of a *Globodera* species using microsatellite loci of individuals collected in South America. Our aim is to describe the genetic variability, gene flow at various spatial scales, and the reproductive mode (i.e. the precise modalities of mating that affect the way the alleles are re-associated in offspring) of Peruvian populations of this species. The consequences of our findings for control methods of *G. pallida* are discussed.

## Materials and methods

### Sampling design, field collection of nematodes and DNA sample preparation

During summer 2002, a hierarchical sampling programme was conducted to allow the investigation of three spatial scales: the geographical region/the field/the plant. Three large agronomic regions of potato and seed tuber production were sampled (Fig. 1). The distance between regions varied between 326 km (from Huancayo to Andahuaylas) to 832 km (from Andahuaylas to Otuzco). In each region, three fields separated from each other by 3–35 km were sampled. Approximately 500 g of soil was taken with a semi-cylindrical corer in an area of 2 m². Cysts were extracted from each soil sample using the Fenwick method. Each J2 was extracted from a different cyst.

For the investigation at the field scale, 40 J2s were used, each coming from a soil sample taken at a regular distance (10 m) from others, to maximize the area of the field investigated. For the investigation at the plant scale, four soil samples separated from each other by 10–70 m (Fig. 1) were taken from one field (Andahuaylas field 3). For each soil sample, 40 J2s from 40 different cysts were analysed.

DNA preparation and PCR were performed with only one J2 juvenile, as described in Plantard & Porte (2003). The 20 µL of DNA extract were diluted with 20 µL of water. From the 40 µL obtained for each juvenile, 10 µL were used for species identification based on rDNA PCR-restriction fragment length polymorphism (RFLP). Indeed, *Globodera*

<figure><img src="image_12.png" /><figcaption>**Fig. 1** Map of Peru showing locations of regions and fields (flower) where *Globodera pallida* were sampled. The sampling within a field is schematized with the 40 samples (• and +). The crosses symbolize plants where 40 cysts were analysed.</figcaption></figure>
pallida and its sibling species G. rostochiensis can be found in sympatry in some parts of Peru. However, they exhibit a distinct restriction profile by rDNA PCR-RFLP (Thiéry & Mugniéry 1996). The remaining part of the DNA extract was used for the microsatellite genotyping, using 2 µL for each locus. The microsatellite primers used were those described by Thiéry & Mugniéry (2000). Eight loci (GP20, -32, -33, -34, -36, -38, -42 and -46) were analysed. Allele size was determined with a LI-COR Global IR² DNA Analyzer, using fluorescent forward primers at IRDye 700 and IRDye 800. To avoid artefacts caused by poor amplification, all individuals with more than two amplification failures among the eight loci were excluded. Finally, the correspondence of alleles between gels was checked by reloading on the same gel different individuals arranged by allele size.

## Data analysis

The observed and expected heterozygosities were calculated using *GENETIX* (version 4.04; Belkhir *et al.* 2001). An unbiased estimate of expected heterozygosity was based on Nei (1978). Hardy-Weinberg exact tests were performed using *GENEPOP* (version 3.4; Raymond & Rousset 1995) assuming that the alternative hypothesis H1 is heterozygote deficiency. To determine without bias the exact *P*-value of this test, a Markov chain method was used, using the default options as the dememorization number (10 000), the number of batches (100) and the number of iterations per batch (5 000). Estimates of the parameter *F*<sub>IS</sub> were also calculated with *GENEPOP* based on Weir & Cockerham (1984). Significance of *F*<sub>IS</sub> for each population was tested using the permutation by population procedure of *GENETIX*. To generate a distribution of *F*<sub>IS</sub> under the null hypothesis (assuming a Hardy-Weinberg equilibrium within populations), 10 000 permutations were used and the observed value of *F*<sub>IS</sub> was compared to this distribution.

Pairwise *F*<sub>ST</sub> estimates were obtained from *F-STAT* (version 2.9.3; Goudet 2001), according to Weir & Cockerham (1984). Because uneven numbers of nucleotides were observed between different alleles of the same locus (despite being all based on dinucleotide repeats), we consider that they do not mutate according to a strict single-step mutation (SMM) model. This is possibly because of mutations in the flanking regions — see Ortì *et al.* (1997) or Delmotte *et al.* (2003) for example — as already observed for numerous invertebrate species in which SMM was not respected (see Estoup *et al.* 1995 for example). Thus we decided not to use allele size-based measures of differentiation such as Slatkin's *R*<sub>ST</sub> that hypothesize a strict SMM model and have a high variance (Gaggiotti *et al.* 1999; Balloux & Lugon-Moulin 2002).

We have also examined the population genetic structure by means of a hierarchical analysis of molecular variance (AMOVA) using *ARLEQUIN* (version 2.0; Excoffier *et al.* 1992;

Schneider *et al.* 2000): among regions, among fields within regions, within fields, among plants and within plants sources of variations were investigated. A two-way AMOVA (for region and fields) reflecting the hierarchical design of samples was conducted; *F*<sub>SC</sub> (i.e. permuting individual genotypes among populations but within groups) and *F*<sub>CT</sub> (i.e. permuting populations among groups) were calculated using *ARLEQUIN*. Three one-way AMOVA were also conducted by considering each region separately, to examine if the partitioning of genetic variability among vs. within fields was similar in each region. Because the four 'plant' samples were from only one field, only a one-way AMOVA was conducted to partition the genetic variability among vs. within plants.

Isolation by distance, in a subdivided or in a continuously distributed population, can be investigated with the help of spatial autocorrelation analysis and Mantel tests (Hardy & Vekemans 1999). Such analyses were performed using the software *GENALEX* (Peakall & Smouse 2001) on the data sets relating to the investigation of the field scales, for which GPS (Global Positioning System) coordinates of each soil sample had been recorded.

## Results

### Polymorphism at the eight loci

Among the 504 individuals genotyped, 134 alleles were found, with seven to 36 alleles per locus (Fig. 2). Over all loci, the Andahuaylas region had the highest allelic richness, with 86 alleles; there were 59 alleles in the Huancayo region and 46 alleles in the Otuzco region. This pattern remains true for each locus separately, except for locus GP36 which had more alleles in the Huancayo region.

Many alleles were region specific (52% for Andahuaylas, 37% for Huancayo and 28% for Otuzco). Few alleles (14%) were shared between the three regions. In each region, few alleles were field specific (for the example of Andahuaylas region: 10% for field 1 and field 3, 18% for field 2). In the Andahuaylas region, 52% of the alleles were shared between the three fields. In field 3 of the Andahuaylas region, few alleles were plant specific (3% for plant 1 and plant 4, 7% for plant 2, 8% for plant 3), while 48% of the alleles were shared between the four plants. The allelic diversity observed at the plant scale was very similar to those observed at the field scale (allelic richness in Andahuaylas field 3 was 60 alleles and mean allelic richness at the plant scale was 59 ± 3.5 alleles with 40 individuals per sample) (Fig. 2).

### Heterozygosis

Heterozygote deficiencies were observed for most loci in most fields and plants (Table 1). A significant deviation from Hardy-Weinberg equilibrium was found in 31 out of
<table><caption>**Table 1** Genetic diversity of *Globodera pallida* sampled at the field and plant scale</caption><thead><tr><th rowspan="2">Field:</th><th colspan="3">Otuzco</th><th colspan="3">Huancayo</th><th colspan="3">Andahuaylas</th><th colspan="4">Andahuaylas - Plant</th></tr><tr><th>1</th><th>2</th><th>3</th><th>1</th><th>2</th><th>3</th><th>1</th><th>2</th><th>3</th><th>1</th><th>2</th><th>3</th><th>4</th></tr></thead><tbody><tr><td>n:</td><td>37</td><td>38</td><td>40</td><td>38</td><td>40</td><td>37</td><td>37</td><td>40</td><td>39</td><td>40</td><td>40</td><td>39</td><td>39</td></tr><tr><td colspan="14">Locus GP20</td></tr><tr><td>H<sub>E</sub></td><td>0.6905</td><td>0.7257</td><td>0.6636</td><td>0.1562</td><td>0.2345</td><td>0.0796</td><td>0.7848</td><td>0.8272</td><td>0.7802</td><td>0.6959</td><td>0.8582</td><td>0.7686</td><td>0.7812</td></tr><tr><td>H<sub>O</sub></td><td>0.4865</td><td>0.4167</td><td>0.5500</td><td>0.1622</td><td>0.2000</td><td>0.0811</td><td>0.8056</td><td>0.7436</td><td>0.6410</td><td>0.5750</td><td>0.8500</td><td>0.6667</td><td>0.6923</td></tr><tr><td>P-val</td><td><strong>0.0007</strong></td><td><strong>0.0000</strong></td><td>0.0587</td><td>1</td><td>0.1051</td><td>1</td><td>0.2606</td><td><strong>0.0000</strong></td><td><strong>0.0029</strong></td><td>0.0971</td><td>0.4951</td><td>0.1714</td><td>0.4223</td></tr><tr><td>F<sub>IS</sub></td><td>+0.298</td><td>+0.429</td><td>+0.173</td><td>-0.038</td><td>+0.149</td><td>-0.008</td><td>-0.027</td><td>+0.102</td><td>+0.180</td><td>+0.176</td><td>+0.010</td><td>+0.134</td><td>+0.115</td></tr><tr><td colspan="14">Locus GP32</td></tr><tr><td>H<sub>E</sub></td><td>0.3543</td><td>0.2695</td><td>0.4443</td><td>0.0519</td><td>0.0494</td><td>0.1311</td><td>0.7360</td><td>0.6320</td><td>0.6895</td><td>0.6703</td><td>0.6383</td><td>0.6620</td><td>0.5937</td></tr><tr><td>H<sub>O</sub></td><td>0.3514</td><td>0.2632</td><td>0.4000</td><td>0.0526</td><td>0.0500</td><td>0.0833</td><td>0.7297</td><td>0.5750</td><td>0.6842</td><td>0.6750</td><td>0.6000</td><td>0.6410</td><td>0.6154</td></tr><tr><td>P-val</td><td>0.5211</td><td>0.6425</td><td>0.3842</td><td>1</td><td>1</td><td>0.1378</td><td>0.5664</td><td>0.3470</td><td>0.4643</td><td>0.7284</td><td>0.1440</td><td>0.6007</td><td>0.6315</td></tr><tr><td>F<sub>IS</sub></td><td>+0.008</td><td>+0.024</td><td>+0.101</td><td>-0.014</td><td>-0.013</td><td>+0.367</td><td>+0.009</td><td>+0.091</td><td>+0.008</td><td>-0.007</td><td>+0.061</td><td>+0.032</td><td>-0.037</td></tr><tr><td colspan="14">Locus GP33</td></tr><tr><td>H<sub>E</sub></td><td>0.5454</td><td>0.5582</td><td>0.5123</td><td>0.7000</td><td>0.6710</td><td>0.6598</td><td>0.7260</td><td>0.7263</td><td>0.7719</td><td>0.7845</td><td>0.7592</td><td>0.7869</td><td>0.7539</td></tr><tr><td>H<sub>O</sub></td><td>0.5676</td><td>0.4474</td><td>0.4750</td><td>0.3947</td><td>0.6410</td><td>0.5135</td><td>0.5946</td><td>0.6750</td><td>0.6216</td><td>0.5641</td><td>0.5897</td><td>0.7179</td><td>0.6410</td></tr><tr><td>P-val</td><td>0.5788</td><td>0.1298</td><td>0.3677</td><td><strong>0.0000</strong></td><td>0.3065</td><td>0.0904</td><td><strong>0.0261</strong></td><td>0.0641</td><td><strong>0.0100</strong></td><td><strong>0.0013</strong></td><td><strong>0.0252</strong></td><td><strong>0.0333</strong></td><td><strong>0.0017</strong></td></tr><tr><td>F<sub>IS</sub></td><td>-0.041</td><td>+0.201</td><td>+0.074</td><td>+0.439</td><td>+0.045</td><td>+0.224</td><td>+0.183</td><td>+0.071</td><td>+0.197</td><td>+0.284</td><td>+0.226</td><td>+0.089</td><td>+0.151</td></tr><tr><td colspan="14">Locus GP34</td></tr><tr><td>H<sub>E</sub></td><td>0.3803</td><td>0.3800</td><td>0.0731</td><td>0.0000</td><td>0.0497</td><td>0.0000</td><td>0.2980</td><td>0.4358</td><td>0.6004</td><td>0.5177</td><td>0.3737</td><td>0.6530</td><td>0.5837</td></tr><tr><td>H<sub>O</sub></td><td>0.2778</td><td>0.3947</td><td>0.0750</td><td>0.0000</td><td>0.0500</td><td>0.0000</td><td>0.1892</td><td>0.4000</td><td>0.4872</td><td>0.5250</td><td>0.2500</td><td>0.6667</td><td>0.5641</td></tr><tr><td>P-val</td><td>0.1142</td><td>0.7444</td><td>1</td><td>—</td><td>1</td><td>—</td><td><strong>0.0325</strong></td><td>0.0652</td><td><strong>0.0190</strong></td><td>0.5328</td><td>0.0641</td><td>0.5316</td><td>0.5018</td></tr><tr><td>F<sub>IS</sub></td><td>+0.272</td><td>-0.039</td><td>-0.026</td><td>—</td><td>-0.006</td><td>—</td><td>+0.368</td><td>+0.083</td><td>+0.191</td><td>-0.014</td><td>+0.334</td><td>-0.021</td><td>+0.034</td></tr><tr><td colspan="14">Locus GP36</td></tr><tr><td>H<sub>E</sub></td><td>0.7485</td><td>0.7409</td><td>0.6830</td><td>0.7629</td><td>0.7912</td><td>0.8230</td><td>0.4627</td><td>0.4002</td><td>0.5498</td><td>0.3983</td><td>0.4799</td><td>0.5544</td><td>0.5591</td></tr><tr><td>H<sub>O</sub></td><td>0.4375</td><td>0.2903</td><td>0.1818</td><td>0.2813</td><td>0.4211</td><td>0.3784</td><td>0.1613</td><td>0.2973</td><td>0.3243</td><td>0.3077</td><td>0.1795</td><td>0.4359</td><td>0.2564</td></tr><tr><td>P-val</td><td><strong>0.0013</strong></td><td><strong>0.0000</strong></td><td><strong>0.0000</strong></td><td><strong>0.0000</strong></td><td><strong>0.0000</strong></td><td><strong>0.0000</strong></td><td><strong>0.0000</strong></td><td>0.0564</td><td><strong>0.0006</strong></td><td>0.0982</td><td><strong>0.0000</strong></td><td>0.0528</td><td><strong>0.0001</strong></td></tr><tr><td>F<sub>IS</sub></td><td>+0.419</td><td>+0.612</td><td>+0.737</td><td>+0.635</td><td>+0.471</td><td>+0.544</td><td>+0.655</td><td>+0.260</td><td>+0.413</td><td>+0.230</td><td>+0.629</td><td>+0.216</td><td>+0.545</td></tr><tr><td colspan="14">Locus GP38</td></tr><tr><td>H<sub>E</sub></td><td>0.6335</td><td>0.4881</td><td>0.6364</td><td>0.2116</td><td>0.0731</td><td>0.2003</td><td>0.7438</td><td>0.6696</td><td>0.7289</td><td>0.7231</td><td>0.7389</td><td>0.7346</td><td>0.6633</td></tr><tr><td>H<sub>O</sub></td><td>0.5946</td><td>0.4474</td><td>0.5500</td><td>0.1842</td><td>0.0250</td><td>0.1667</td><td>0.6486</td><td>0.5750</td><td>0.6667</td><td>0.7000</td><td>0.5500</td><td>0.7179</td><td>0.6410</td></tr><tr><td>P-val</td><td>0.3798</td><td>0.1717</td><td>0.1080</td><td>0.4142</td><td><strong>0.0380</strong></td><td>0.3528</td><td>0.0911</td><td><strong>0.0293</strong></td><td>0.1192</td><td>0.2176</td><td><strong>0.0002</strong></td><td>0.3000</td><td>0.3162</td></tr><tr><td>F<sub>IS</sub></td><td>+0.062</td><td>+0.084</td><td>+0.137</td><td>+0.131</td><td>+0.661</td><td>+0.170</td><td>+0.129</td><td>+0.143</td><td>+0.086</td><td>+0.032</td><td>+0.258</td><td>+0.023</td><td>+0.034</td></tr><tr><td colspan="14">Locus GP42</td></tr><tr><td>H<sub>E</sub></td><td>0.6761</td><td>0.7014</td><td>0.6503</td><td>0.8382</td><td>0.8955</td><td>0.8381</td><td>0.8282</td><td>0.9101</td><td>0.9131</td><td>0.8509</td><td>0.9025</td><td>0.8848</td><td>0.8888</td></tr><tr><td>H<sub>O</sub></td><td>0.5833</td><td>0.6053</td><td>0.4103</td><td>0.7297</td><td>0.7778</td><td>0.7714</td><td>0.6944</td><td>0.8750</td><td>0.7436</td><td>0.8000</td><td>0.9000</td><td>0.8718</td><td>0.8462</td></tr><tr><td>P-val</td><td>0.0513</td><td>0.1256</td><td><strong>0.0000</strong></td><td>0.0601</td><td><strong>0.0015</strong></td><td><strong>0.0037</strong></td><td><strong>0.0184</strong></td><td>0.0890</td><td><strong>0.0000</strong></td><td><strong>0.0438</strong></td><td>0.4031</td><td>0.3198</td><td>0.1465</td></tr><tr><td>F<sub>IS</sub></td><td>+0.139</td><td>+0.139</td><td>+0.372</td><td>+0.131</td><td>+0.133</td><td>+0.081</td><td>+0.163</td><td>+0.039</td><td>+0.188</td><td>+0.061</td><td>+0.003</td><td>+0.015</td><td>+0.049</td></tr><tr><td colspan="14">Locus GP46</td></tr><tr><td>H<sub>E</sub></td><td>0.5520</td><td>0.5713</td><td>0.4902</td><td>0.7053</td><td>0.6506</td><td>0.6412</td><td>0.7397</td><td>0.7366</td><td>0.7058</td><td>0.8098</td><td>0.7361</td><td>0.7890</td><td>0.7916</td></tr><tr><td>H<sub>O</sub></td><td>0.4054</td><td>0.4324</td><td>0.4250</td><td>0.2895</td><td>0.4000</td><td>0.2162</td><td>0.2647</td><td>0.3333</td><td>0.5000</td><td>0.2632</td><td>0.3158</td><td>0.5946</td><td>0.5526</td></tr><tr><td>P-val</td><td><strong>0.0030</strong></td><td>0.0947</td><td>0.2487</td><td><strong>0.0001</strong></td><td><strong>0.0000</strong></td><td><strong>0.0000</strong></td><td><strong>0.0000</strong></td><td><strong>0.0000</strong></td><td><strong>0.0045</strong></td><td><strong>0.0000</strong></td><td><strong>0.0000</strong></td><td><strong>0.0003</strong></td><td><strong>0.0000</strong></td></tr><tr><td>F<sub>IS</sub></td><td>+0.268</td><td>+0.228</td><td>+0.134</td><td>+0.593</td><td>+0.419</td><td>+0.666</td><td>+0.646</td><td>+0.551</td><td>+0.295</td><td>+0.678</td><td>+0.574</td><td>+0.249</td><td>+0.305</td></tr><tr><td colspan="14">All loci</td></tr><tr><td>H<sub>E</sub></td><td>0.5726</td><td>0.5544</td><td>0.5192</td><td>0.4283</td><td>0.4269</td><td>0.4216</td><td>0.6649</td><td>0.6672</td><td>0.7175</td><td>0.6813</td><td>0.6859</td><td>0.7292</td><td>0.7019</td></tr><tr><td>H<sub>O</sub></td><td>0.4630</td><td>0.4122</td><td>0.3834</td><td>0.2618</td><td>0.3206</td><td>0.2763</td><td>0.5110</td><td>0.5593</td><td>0.5836</td><td>0.5512</td><td>0.5294</td><td>0.6641</td><td>0.6011</td></tr><tr><td>P-val</td><td><strong>0.0000</strong></td><td><strong>0.0000</strong></td><td><strong>0.0000</strong></td><td><strong>0.0000</strong></td><td><strong>0.0000</strong></td><td><strong>0.0000</strong></td><td><strong>0.0000</strong></td><td><strong>0.0000</strong></td><td><strong>0.0000</strong></td><td><strong>0.0000</strong></td><td><strong>0.0000</strong></td><td><strong>0.0004</strong></td><td><strong>0.0000</strong></td></tr><tr><td>F<sub>IS</sub></td><td>+0.1936</td><td>+0.2593</td><td>+0.2642</td><td>+0.3921</td><td>+0.2514</td><td>+0.3477</td><td>+0.2341</td><td>+0.1637</td><td>+0.1886</td><td>+0.1929</td><td>+0.2304</td><td>+0.0903</td><td>+0.1452</td></tr></tbody></table>

$H_E$ and $H_O$, expected and observed heterozygosity; *P-val*, significance of heterozygote deficiencies (in bold: significant departure from Hardy-Weinberg expectation); $F_{IS}$ values, estimate of heterozygote deficiencies; *n*, sample size.
<figure><img src="image_13.png" /><figcaption>**Fig. 2** Allele size (in base pairs) and allele frequencies at the eight microsatellite loci of *Globodera pallida*. The circle areas are proportional to the frequencies of the respective alleles in given population (1–3: three fields of the Otuzco region; 4–6: three fields of the Huancayo region; 7–9: three fields of the Andahuaylas region; 10–13: four plants in the field Andahuaylas 3).</figcaption></figure>

72 tests for field samples and in 12 out of 32 plant samples [Hardy-Weinberg exact test based on the alternative hypothesis (H1) of heterozygote deficiency; Table 1]. A heterozygote deficiency was observed with a highly significant deviation from Hardy-Weinberg equilibrium in all fields and all plants when global tests across loci were conducted. Furthermore, the high values of *F*<sub>IS</sub> are indicative of a high heterozygote deficiency (between 0.16 and 0.39 among the nine fields and between 0.12 and 0.23 among the four plants). One hundred per cent of the values of *F*<sub>IS</sub> obtained by permutation (GENETIX: 10 000 iterations) were lower than the observed *F*<sub>IS</sub> value. To test for the presence of null alleles in the data set, the relationship between the number of nonamplifying samples for each locus and *F*<sub>IS</sub> was examined (Beaumont *et al.* 2001). A positive relationship would indicate that *F*<sub>IS</sub> is affected by amplifying failure

hence suggesting that homozygous individuals are the result of null alleles. For each locus, this relationship was never significant (*P* > 0.05). Thus, we considered that the loci used were not greatly affected by null alleles.

*Partition of the genetic variability and gene flow*

The analysis of nine fields in three regions reveals that 33% of the genetic variability was observed among regions and 65% of the genetic variability was observed within fields (Table 2). The fixation index reflecting the nested design of the samples indicated that genetic differentiation was weak among fields within regions (F<sub>SC</sub> = 0.023, i.e. permuting individual genotypes among fields but within regions) while genetic differentiation was strong among regions (F<sub>CT</sub> = 0.319, i.e. permuting fields among regions). The high
<table><caption>**Table 2** Partition of genetic variability of *Globodera pallida* populations</caption><thead><tr><th>Source of variation</th><th>d.f.</th><th>Sum of squares</th><th>Variance components</th><th>Percentage of variation</th></tr></thead><tbody><tr><td colspan="5"><strong>At the regional scale</strong></td></tr><tr><td>Among regions</td><td>2</td><td>430.578</td><td>0.90835</td><td><strong>32.80</strong></td></tr><tr><td>Among fields within regions</td><td>6</td><td>34.552</td><td>0.05138</td><td><strong>1.86</strong></td></tr><tr><td>Within fields</td><td>683</td><td>1236.239</td><td>1.81001</td><td><strong>65.35</strong></td></tr><tr><td>Total</td><td>691</td><td>1701.370</td><td>2.76975</td><td></td></tr><tr><td colspan="5"><strong>At the field scale</strong></td></tr><tr><td colspan="5">Three fields in the region of Otuzco</td></tr><tr><td>Among fields</td><td>2</td><td>16.371</td><td>0.08338</td><td><strong>4.44</strong></td></tr><tr><td>Within fields</td><td>227</td><td>407.742</td><td>1.79622</td><td><strong>95.56</strong></td></tr><tr><td>Total</td><td>229</td><td>424.113</td><td>1.87960</td><td></td></tr><tr><td colspan="5">Three fields in the region of Huancayo</td></tr><tr><td>Among fields</td><td>2</td><td>3.840</td><td>0.01375</td><td><strong>1.56</strong></td></tr><tr><td>Within fields</td><td>227</td><td>196.582</td><td>0.86600</td><td><strong>98.44</strong></td></tr><tr><td>Total</td><td>229</td><td>200.422</td><td>0.87975</td><td></td></tr><tr><td colspan="5">Three fields in the region of Andahuaylas</td></tr><tr><td>Among fields</td><td>2</td><td>11.151</td><td>0.04503</td><td><strong>2.10</strong></td></tr><tr><td>Within fields</td><td>229</td><td>479.715</td><td>2.09483</td><td><strong>97.90</strong></td></tr><tr><td>Total</td><td>231</td><td>490.866</td><td>2.13986</td><td></td></tr><tr><td colspan="5"><strong>At the plant scale</strong></td></tr><tr><td colspan="5">Four plants in the field 3 Andahuaylas</td></tr><tr><td>Among plants</td><td>3</td><td>27.808</td><td>0.08255</td><td><strong>2.92</strong></td></tr><tr><td>Within plants</td><td>312</td><td>857.454</td><td>2.74825</td><td><strong>97.08</strong></td></tr><tr><td>Total</td><td>315</td><td>885.263</td><td>2.83080</td><td></td></tr></tbody></table>

$F_{ST}$ values (Table 3) between regions (> 0.2) are related to the relatively high level of genetic variation observed among regions (33%). So, both results suggest weak gene flow between regions.

In each region, a small part of the genetic variation is observed among fields (from 1.6% to 4.4% in the three one-way AMOVA treating each region separately, and 1.9% in the two-way AMOVA taking into account nine fields without considering regions). In Andahuaylas field 3, a small part of the genetic variation was observed among plants (3.8%) and consequently, a large part of the variation was observed within fields (between 96% and 98%) and within plants (96%) (Table 2). Inside each region, the weak $F_{ST}$ values between fields ($0.0001 < F_{ST} < 0.05$) were associated with the small part of genetic variation observed among fields (Table 3). In the Andahuaylas field 3, the small genetic differentiation between plants revealed by low $F_{ST}$ values ($0.01 < F_{ST} < 0.06$) was associated with a small part of the genetic variation observed among plants. This suggests extensive gene flow among plants and among fields within regions.

### Isolation by distance

Inside each field (i.e. between samples separated by 8–200 m), isolation by distance could not be detected using spatial autocorrelation analysis (when examining the genetic correlation as a function of distance, over all nine fields, among the 68 distance classes investigated, *r*-values lay

<table><caption>**Table 3** Genetic differentiation ($F_{ST}$) among *Globodera pallida* populations (a) between field among regions (in bold: $F_{ST}$ between field within each region) and (b) between plants</caption><thead><tr><th>Region:</th><th colspan="3">Otuzco</th><th colspan="3">Huancayo</th><th colspan="3">Andahuaylas</th></tr><tr><th>Field:</th><th>1</th><th>2</th><th>3</th><th>1</th><th>2</th><th>3</th><th>1</th><th>2</th><th>3</th></tr></thead><tbody><tr><td colspan="10"><strong>(a) between fields among regions</strong></td></tr><tr><td rowspan="3">Otuzco</td><td>1</td><td rowspan="3"><strong>0.0260</strong></td><td><strong>0.0298</strong></td><td>0.3808</td><td>0.3895</td><td>0.3883</td><td>0.2521</td><td>0.2374</td><td>0.2049</td></tr><tr><td>2</td><td><strong>0.0485</strong></td><td>0.3487</td><td>0.3573</td><td>0.3621</td><td>0.2543</td><td>0.2371</td><td>0.2057</td></tr><tr><td>3</td><td>0.4185</td><td>0.4273</td><td>0.4241</td><td>0.2995</td><td>0.2837</td><td>0.2393</td></tr><tr><td rowspan="3">Huancayo</td><td>1</td><td rowspan="3"></td><td rowspan="3"><strong>0.0152</strong></td><td><strong>0.0179</strong></td><td>0.3511</td><td>0.3450</td><td>0.3284</td><td></td><td></td></tr><tr><td>2</td><td><strong>0.0001</strong></td><td>0.3661</td><td>0.3588</td><td>0.3372</td><td></td><td></td></tr><tr><td>3</td><td>0.3693</td><td>0.3598</td><td>0.3406</td><td></td><td></td></tr><tr><td rowspan="2">Andahuaylas</td><td>1</td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td><td><strong>0.0059</strong></td><td><strong>0.0284</strong></td><td><strong>0.0197</strong></td></tr><tr><td>2</td><td></td><td></td><td></td><td></td></tr></tbody></table>

### (b) between plants of the Andahuaylas field 3

<table><thead><tr><th>Region:</th><th colspan="4">Andahuaylas - Plant</th></tr><tr><th>Plant:</th><th>1</th><th>2</th><th>3</th><th>4</th></tr></thead><tbody><tr><td>1</td><td></td><td>0.0453</td><td>0.0163</td><td>0.0075</td></tr><tr><td>2</td><td></td><td></td><td>0.0414</td><td>0.0451</td></tr><tr><td>3</td><td></td><td></td><td></td><td>0.0083</td></tr></tbody></table>
between the upper and the lower confidence limits in
all except four distances classes; 95% confidence interval;
results not shown) or Mantel tests (P was between 0.260
and 0.750 for the three fields of the Andahuayljas region,
between 0.080 and 0.330 for the three fields of the
Huancayo region, and between 0.320 and 0.620 for the
three fields of the Otuzco region). Among the three fields
inside each region and among the three regions, the spatial
autocorrelation or the Mantel tests could not be performed
because there were only three distance classes.

Discussion

Polymorphism and origin area

The hypothesis that *Globodera pallida* is native in the Andean
Cordillera, like its host, the *Solanum* genus (Hijmans &
Spooner 2001), is widely accepted (Canto Saenz & Scurrah
1977; Stone 1985; Bendezu *et al.* 1998; Blok *et al.* 1998;
Folkertsma *et al.* 2001). Thus, we expected that the allelic
richness level in Peru would be higher than in Europe
because of the founder effect and genetic drift following
introduction events. The analysis of 120 individuals from
five countries in Europe for the same seven loci (namely
GP20, -32, -34, -36, -38, -42 and -46) gave a total of 25 alleles
(Thiéry & Mugniéry 2000) while our study revealed 63
alleles in only 40 individuals from one field in Andahuaylas
(117 alleles for the data set). However, the genetic drift as
a result of consecutive laboratory population multiplications
could also be responsible for the lower allelic richness
observed in the European samples analysed by Thiéry
& Mugniéry (2000). Bendezu *et al.* (1998), using random
amplified polymorphic DNA markers, also found consid-
erably less genetic diversity in samples from the UK than
in those from Peru, but laboratory strains were also used in
this study.

Although only three regions have been investigated in
the present study, a higher allelic richness was observed
while sampling nearer Lake Titicaca. As *G. rostochiensis* is
also found in this area, our study highlights how interest-
ing this region is for the investigation of genetic variability
of both *Globodera* species.

Reproduction mode

Globodera pallida is characterized by a high deficit of
heterozygotes with high F<sub>IS</sub> values observed at both plant
and field scales. Because there is 10-m separation between
samples at the field scale and because the active dispersal
is less than several decimetres for juveniles and males, the
sampling could have generated a Wahlund effect responsible
for the heterozygote deficit observed. However, a similar
heterozygote deficit is observed at the localized plant scale,
allowing rejection of the Wahlund effect (Table 1). Moreover,

heterozygote deficiency is observed in all loci, as would be
expected when it is the result of inbreeding, while with a
Wahlund effect caused by population subdivision, the
frequency of heterozygotes would differ from panmictic
proportions in a way that depends on the co-variances of
allele frequencies across populations (Hartl & Clark 1989).
Artefacts as a result of null alleles are very unlikely to
account for the results because all eight loci were affected
and because there is no positive relationship between
nonamplification and F<sub>IS</sub>.

We hypothesize that the heterozygote deficiency is the
result of the particular biology and life cycle of this soil-
dwelling organism. Indeed, nematodes are able to move
only in the water phase in soil because of their sinusoidal
wave locomotor progression in surface tension films (Crofton
1971). Thus, active dispersal of larvae and males is prob-
ably limited to a few centimetres or decimetres. All larvae
from a given cyst are siblings, or half-siblings if several
males have fertilized the female (a case reported in labor-
atory studies by Thiéry *et al.* 1997). Also, at hatching, there are
several hundred sibling or half-sibling larvae that actively
move short distances to invade the roots of their host
plant. These larvae will develop into males and females
that will have a high probability of mating with each other
because of the small dispersal range of males. This life cycle
and behaviour should thus favour mating between siblings
or half-siblings enhancing the production of homozygotes.
Furthermore, this phenomenon occurs at each generation.

The assortative mating (tendency of like to mate with
like) could also explain the heterozygote deficiency observed.
Additional experiments should be conducted to invest-
igate those two hypotheses as there are no data on these
topics in the nematological literature.

A two-dimensional gel electrophoretic (2-DGE) study
of *G. pallida* did not show any significant deviation from
Hardy-Weinberg equilibrium (De Boer *et al.* 1992). This
result could be because of the difficulty of determining the
heterozygote state with 2-DGE. Furthermore, one cyst
containing several hundred (half)sibling J2 juveniles were
used for the protein extraction; the admixture of (half)
siblings could increase the number of heterozygotes. In another
study using 2-DGE, low heterozygosity was observed
(Folkertsma *et al.* 2001). However, because this study was
conducted on populations from an area (the Netherlands)
where *G. pallida* had been introduced, the relative role of
genetic bottlenecks since introduction relative to inbreeding
could not be evaluated in the assessment of heterozygosity.

In conclusion, *G. pallida* can be considered as a highly
inbred species as a result of the limited active dispersal of
larvae and males. Such a pattern has recently been observed
in another cyst nematode species, *Heterodera schachtii*
(Plantard & Porte 2004). However, the present study is
based on more loci (eight vs. five) and with greater poly-
morphism (36 alleles per loci vs. 11 alleles per loci). In
two studies of *Heterodera avenae* with enzymatic markers (Gillois *et al.* 1981; Lasserre *et al.* 1996), a heterozygote deficit was also observed but not with the entire data set. Thus, this reproductive mode could be a more general feature of those organisms characterized by restricted active dispersal and aggregated clutch (i.e. cysts containing several hundred eggs).

### Gene flow among regions

A large genetic differentiation was observed at the regional scale (distance: 326–832 km). The allelic diversity and frequency pattern are different from one region to another (Fig. 2). In fact, there is a high number of alleles specific to each region. This is congruent with the values of $F_{ST}$ ($F_{ST}$ = 0.2–0.4) and with a fixation index based on the nested design of the samples ($F_{CT}$ = 0.318) indicating a large genetic differentiation between regions. The *amoVA* results also suggest low gene flow at the inter-regional scale with a large part of the genetic variation being observed among regions (33%).

The three regions sampled correspond to the largest potato and seed tuber production areas at high altitudes (3000–3800 m) in Peru. These regions import few seed tubers. Moreover, these agronomic regions are isolated by large areas without *G. pallida* (tropical valleys and very high altitude areas, 4400 m, where potatoes are not grown). These facts could explain the genetic structure observed and the barrier to gene flow. Such a large genetic differentiation was not observed in the study of the sugar beet cyst nematode by Plantard & Porte (2004). However, in this latter study the largest distance between regions (150 km) was less than the inter-region distances of the present study. Moreover, no major barrier to gene flow exists between the French regions sampled, in contrast to the present study.

### Gene flow inside regions and inside fields

Within regions, *G. pallida* exhibits a low genetic differentiation from field to field even 35 km apart (distance: 3–35 km; $F_{ST}$ = 0.00–0.05; $F_{SC}$ = 0.023; partition of genetic variation: 1.6–4.4%). Within Andahuaylas field 3, *G. pallida* exhibits a low genetic differentiation from plant to plant (distance: 10–50 m; $F_{ST}$ = 0.01–0.06; partition of genetic variation: 2.9%). A large gene flow at both spatial scales is suggested by the $F_{ST}$ and the *amoVA* results. Moreover, no isolation by distance is observed when considering the geographical localization of each sample inside each field.

The origin of the gene flow within regions could be the passive dispersal of cysts. The cyst has biological characteristics that are well adapted to a long-distance effective dispersal. The eggs are protected inside a cyst and can survive for *c.* 10 years. One cyst contains several hundred eggs. The considerable number of cysts per hectare that *G.*

*pallida* can reach in soils (9 billion cysts per hectare or 3 cysts per gram of soil) enhances the probability of an effective long-range passive dispersal. Passive dispersal of cysts might have an anthropogenic or natural origin. Wind dispersal has been reported for cyst nematodes (White 1953; Norton & Niblack 1991; Gavassoni *et al.* 2001) and for other soil nematodes (Baujard & Martiny 1994). The water can carry some cysts with runoff, irrigation and drainage. Farm implements transport cysts from one part of a field to another or from field to field in the soil stuck on the wheels and tools. Seed tubers could also disperse cysts (Steiner *et al.* 1951), especially inside the three Peruvian regions sampled because of the exchange of many seed tubers within each region, without any phyto-sanitary control of cyst nematodes. In the Netherlands, a weak genetic structure was also observed, even for fields ±150 km apart (Folkertsma *et al.* 2001). However, because populations of *G. pallida* in Europe are introduced, this structure could either be the result of high gene flow inside regions or of a limited number of introduction events.

Recently, other studies on soil-dwelling organisms have observed large gene flow over long distances using a population genetics approach (see for example van der Wurff *et al.* 2003 in relation to the collembolan *Orchesella cincta*; Stevens & Hogg 2003 with regard to the collembolan *Gomphiocephalus hodgoni*; and Plantard & Porte 2004 with reference to the nematode *Heterodera schachtii*). Furthermore, for the free-living soil nematode *Caenorhabditis elegans*, Koch *et al.* (2000) suggest long-range dispersal to explain the low genetic differentiation at the global scale. The gene flow at long distances is not the only similarity between *G. pallida* and other soil-dwellings species such as *H. schachtii* or *Go. hodgoni* or *O. cincta*. A deficit of heterozygotes (at least for some loci) was also observed for all those three species. In this study, active dispersal of larvae and males at a very short distance leads to inbreeding, while passive dispersal of cyst leads to long-distance gene flow and lack of genetic differentiation within regions, as observed for *H. schachtii* (Plantard & Porte 2004).

### Implications for the control of cyst nematodes

According to our results, *G. pallida* is highly inbred, has a high capacity for dispersal and a large part of the genetic variation observed at the intra-regional scale was also observed at the plant scale. These results have consequences for the management and deployment of resistant varieties. The inbred reproductive mode favours the production of homozygotes. According to the gene-for-gene relationship theory (Flor 1955), because most plant disease-resistance genes are dominant while the corresponding pathogen-avirulence genes are recessive, only the pathogen individuals being virulent homozygotes (avr/avr) have a resistance-breaking phenotype. Thus, the reproductive mode of *G.*
*pallida* could facilitate the emergence of such homozygous virulent individuals.

Finally, our finding that gene flow is extensive not only at the intra-field scale but also among fields within the same regions, suggests that prophylactic measures such as the disinfection of seed tubers used for planting, the cleaning of farm machines and restricting the spread of cysts through irrigation water should be promoted.

According to the criteria used by McDonald & Linde (2002) *G. pallida* should be considered a parasite with a high capacity to overcome resistance because of its high capacity for dispersal and its large genetic and effective population sizes. The observations of frequent resistant-breaking pathotypes reported in the literature (Llontop *et al.* 1989; Janssen *et al.* 1990; Turner & Fleming 2002) follow this interpretation.

Van den Bosch & Gilligan (2003) have shown that population dynamics and population genetics of a pest should be linked to estimate the durability of resistance within an epidemiological model. The present study, by giving new information on the dispersal abilities of *G. pallida* and its genetic variability, should constitute a valuable step towards the building of epidemiological models to combat this pest.

## Acknowledgements

We would like to thank the 'Servicio Nacional de Sanidad Agraria' (SENASA, Peru), 'Instituto Nacional de Investigación Agraria' (INIA, Peru) and particularly Dr Elsa Carbonell Torres and Ing Susana Chumbiauca for all their help in the sampling of potato cyst nematodes in Peru. This work was financially supported by the INRA department 'Santé des Plantes et Environnement' and 'la Région Bretagne'. The French 'Ministère des affaires étrangères' provided funding for the trip. We would also like to thank two anonymous referees and Paul Sunnucks for useful comments on a previous version of the manuscript. Jean-Christophe Simon, Eric Petit and Mark Phillips made helpful suggestions. Finally, we thank Mrs Sylvie Valette, Mrs Pauline Casters-Picard, Mr Abou Bakari-Kouassi, Mr Marcelo Trebejo and Mr Mario Curasi-Nina for their help.

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The authors are members of the team 'Biology and Genetics of phytoparasitic nematodes' of the UMR BiO3P that focuses on the management of sedentary nematodes by the use of resistant varieties. This study is part of the PhD work of Damien Picard which is focused in population genetics and phylogeography of *Globodera pallida* and the systematics of the *Heteroderidae* family. Olivier Plantard is a researcher interested in population genetics of plant nematodes and other evolutionary aspects of those organisms. Maria Scurrah is a geneticist working on potato cyst nematodes in Peru. Didier Mugniéry is a senior research scientist interested in nematode control.
CHAPITRE III

Patron d'« isolement par la distance » le long
d'un transect de 1.500 km dans la cordillère
des Andes et structuration génétique chez un
organisme du sol de 500 micromètres
Dans le chapitre précédent, les populations de *G. pallida* analysées se sont avérées très consanguines. Le déficit en hétérozygotes observé peut s'expliquer par une reproduction préférentielle entre individus apparentés ou par la faible dispersion active de centaines de frères et sœurs lors de l'éclosion d'un kyste. Les principaux résultats concernant la partition de la variabilité génétique à différentes échelles spatiales et la structure génétique obtenus dans le chapitre II, sont résumés ici de façon synthétique :

* La quasi-totalité de la variabilité génétique observée dans une région (bassin de production de la pomme de terre) se retrouve à l'échelle de la rhizosphère d'une plante.

* Aucune structuration génétique n'est observée à l'intérieur des trois régions analysées, ce qui reflète des forts flux de gènes à cette échelle. Nous faisons l'hypothèse que ceux-ci sont dus à une forte dispersion passive des kystes.

* Une importante structuration génétique est observée entre les trois régions analysées, attestant de l'absence de flux de gènes à cette échelle, absence due probablement aux puissants reliefs géographiques de la Cordillère des Andes.

Dans ce chapitre III, la description du patron génétique à l'échelle intra et inter-région va être approfondie. Rappelons qu'à ces deux échelles géographiques, nous avons observé dans le chapitre II une absence de structuration génétique entre parcelles séparées jusqu'à 35 km à l'intérieur d'une région et une très forte structure génétique entre régions séparées de plus de 320 km. Se pose alors la question : Où, pourquoi et comment passe t'on de tout à rien ? Autrement dit : qu'est ce qu'une population pour un nématode à kyste ? Pour répondre à cette question centrale en génétique des populations, nous avons réalisé un échantillonnage spécifique qui a été analysé par deux approches.

Dans trois bassins agricoles, où la production de pommes de terre est importante et les parcelles sont contiguës, en plus des parcelles déjà analysées dans le chapitre II, de nouvelles parcelles ont été échantillonnées. Par ailleurs des parcelles de plus en plus isolées géographiquement entre elles ont été aussi ajoutées. L'analyse a concerné un total de 703 individus répartis sur 23 parcelles qui ont été génotypés à l'aide des huit loci microsatellites utilisés précédemment.

La première approche a consisté à établir le patron d'isolement par la distance (Isolation By Distance en anglais ; IBD). Deux méthodes ont été utilisées : le calcul du rapport *F*<sub>ST</sub>/(1 - *F*<sub>ST</sub>) *versus* la distance entre les parcelles et l'analyse de l'autocorrélation spatiale. La deuxième approche a quant à elle consisté à estimer le niveau de structure génétique entre individus ou entre groupes d'individus grâce à un algorithme bayésien de regroupement.
Isolation by distance only above 50 km for a 500 µm long soil nematode

Damien Picard, Olivier Plantard,

UMR INRA-ENSAR BiO3P, Domaine de la Motte, B.P. 35327, 35653 Le Rheu cedex, France.

*Corresponding author:* Olivier Plantard, UMR INRA-ENSAR BiO3P, Domaine de la Motte, B.P. 35327, 35653 Le Rheu cedex, France. Fax number: 33 2 23 48 51 50; olivier.plantard@rennes.inra.fr

**Abstract**

Although numerous species are distributed in discrete populations easily recognized by geographical barriers, continuous populations are a common feature of plants or marine organisms. This is particularly true for soil organisms as their habitat is continuous and their range cannot easily be assessed as they are buried below ground. In the case of organisms for which standard methods such as Markage/Capture/Recapture cannot be used, population genetics provide a straightforward approach to delimitate populations. In this study, we have pursued this topic with a soil-dwelling nematode (*Globodera pallida*), which parasitizes potato roots and is indigenous to South America. Potential barriers to gene flow were identified using the analysis of the *F*<sub>ST</sub>/(1- *F*<sub>ST</sub>) ratio against geographical distance and spatial autocorrelation combined with model-based clustering algorithm. Inside regions, neither genetic differentiation nor isolation by distance (IBD) occur among fields less than 50 km distant. We hypothesize that the large amount of gene flow revealed by the absence of genetic structure of this tiny organism could be due to large passive dispersion inside an agronomic area where *G. pallida* has a continuous distribution and is found at high density. The first evidence of genetic differentiation appeared when a field was separated from others by area without farming (where *G. pallida* is absent). Among regions, a high genetic structure coupled with an IBD pattern occurs as the consequences of the limitations of passive dispersal due to deep valleys or high mountains. To our knowledge, this is the first study identifying the spatial limit of a population for a plant nematode parasite.

**Keywords:** microsatellite, phytoparasitic nematode, soil dwelling invertebrates, spatial autocorrelation, IBD, clustering analysis, genetic structure, passive dispersion.
## 3.2. INTRODUCTION

Unlike the species definition, which has been the topic of extensive debate, the definition of a population is rarely evocated, even in population genetic studies. Classically, terrestrial animals have a discontinuous distribution due to habitat fragmentation. They disperse actively among those patches, which are used as the sampling unit for population studies. To measure the genetic structure of populations, Wright (1978) proposed to use a hierarchical sampling design that corresponds to spatial subdivisions of natural populations. In such a context, the zoologist decides *a priori* what a population is according to the biology of the organism (dispersal abilities...) and its landscape. But for plants and small marine organisms, the passive dispersion in a more or less continuous distribution does not allow *a priori* decisions to be made about what a population is. For nematode parasites of vertebrates, the population genetic structure is largely due to host movement (Blouin et al., 1995; Fisher and Viney, 1996; Hoekstra et al., 1997; Fisher and Viney, 1998; Peng et al., 1998; Viney, 1998; Müller-Graf et al., 1999; Otsen et al., 2000; Hawdon et al., 2001). For plant parasitic nematodes, which of course can not be dispersed by their hosts, intra- and interpopulation genetic variability as well as gene flow at various spatial scales are largely unknown. A common way to determine what a population is, for such organisms, is to study if the allele frequencies exhibit a spatial pattern (Palumbi, 1994; Vekemans and Hardy, 2004). Are they randomly distributed over space, or do they show any spatial association or correlation? To solve this task, the relationship between Wright's *F*-statistics (Wright, 1951) against spatial distance or spatial autocorrelation (Sokal and Wartenberg, 1983; Slatkin, 1993; Epperson and Li, 1997; Rousset, 1997) could be studied as specified by particular models of population structure. Some of these indirect methods consist in plotting the genetic distance (Sokal et al., 1986) or the *F*<sub>ST</sub>/(1- *F*<sub>ST</sub>) ratio (Rousset, 1997) against the spatial distance between individuals or groups of individuals. A classical genetic pattern is the “isolation by distance (IBD)” due to a process by which geographically-restricted gene flow generates, under migration-drift, a substantial increase of genetic divergence with the geographical distance among populations (Wright, 1943). More recently, a new and powerful method has been developed to assign individuals in groups according to their multilocus genotype (Structure software, Pritchard et al., 2000). Indeed, the model-based clustering algorithm identifies groups that have distinctive allele frequencies and enables inferences to be made about the level of genetic structure between groups of individuals.

The soil fauna, which is made up of thousands of nematoda, acari, insects, crustacean or annelids species, is a poorly known part of the global biodiversity. In these biota, numerous plant-, invertebrate- (insect-, mollusc-), or vertebrate-parasites are found (Silva et al., 2003). Numerous life-history traits of the species constituting this fauna are still unknown. Although active dispersion is restricted by the weak permeability of the soil habitat, some soil-dwelling organisms can disperse passively over a long distance [for examples, collembolans like *Orchesella cincta* (van der Wurff et al.,
2003) or *Gomphiocephalus hodgoni* (Stevens and Hogg, 2003) and nematodes like *Caenorhabditis elegans* (Koch et al., 2000) or *Heterodera schachtii* (Plantard and Porte, 2004) or *Globodera pallida* (Picard et al., 2004)]. Even though the passive dispersion was responsible for high gene flow at long distance, the weak active dispersion for individual buried in the soil and the egg clustering was responsible for the inbreeding (Stevens and Hogg, 2003; van der Wurff et al., 2003; Picard et al., 2004; Plantard and Porte, 2004). For *Caenorhabditis elegans*, the passive dispersion appears to prevent a genetic structure at the global scale (Koch et al., 2000), while a restricted gene flow was observed for *G. pallida* among only 3 regions separated by geographical barriers (deep valley and high mountains) in the Andean Cordillera (Picard et al., 2004). In all the cases, the delimitation of what a population is for a soil organism is still unanswered. We will address this issue by studying population genetics of *Globodera pallida*, a cyst nematode parasiting potato roots. *G. pallida* is a sexually dimorphic amphimictic diploid species. Juveniles (500 µm long) and males (1 mm long) are the two free stages of the parasiting cycle and can disperse actively over few centimetres (Norton and Niblack, 1991). When the female dies, the body wall forms a cyst, which is the conservation stage containing several hundred juvenile (up to 600) which can survive for many years (up to 10 years). *G. pallida* is probably native to the Andean Cordillera (Canto Saenz and Mayer de Scurrah, 1977; Stone, 1985), the origin of its main host genus *Solanum* (Hijmans and Spooner, 2001). In a given field, no genetic differentiation was observed among samples of nematodes associated to individual plants, in a previous analysis of the genetic structure of *Globodera pallida* populations using a hierarchical sampling (Picard et al., 2004). A field could therefore be considered as an homogeneous genetic unit and will be the smallest genetic entity considered in this article.

The aim of this study was to combine the complementary analysis of IBD patterns and clustering analysis to determine i) at which spatial scale a genetic structure is observed? ii) at which spatial scale an IDB pattern is observed? iii) what a population is for *G. pallida*? Finally, the usefulness of this approach for all organisms that disperse passively and do not show a priori evidence of spatial population subdivisions will be discussed.

**3.3. MATERIALS AND METHODS**

3.3.1. *Sampling design, field collection of nematodes and DNA sample preparation:*

Sampling program was conducted between 2,700 m and 4,200 m along 1,500 km of the Andean Cordillera in Peru (from the Equatorial to the Bolivian border) from June to August 2002.
Soil containing *Globodera pallida* cysts were collected from 342 fields sampled. Fields were located precisely by GPS (Global Positioning System).

Nine different regions were identified along the Andean Cordillera (Figure 1 & Table 1). In
the interest of clarity, our use of the term “region” pertains to a set of fields within a defined
geographic area rather than implying any hierarchical structure or analyses. The regions are
separated by major geographic relief such as deep valleys with tropical climate or high mountains (more than 4,300 m), acting as a geographic barrier to *Globodera pallida* passive dispersion (Picard et al., 2004). For example, the Huaraz region and the Huanuco region were considered as belonging to two different regions as they are separated by the Cordillera Blanca with Huascarán top (6,768 m) and the wide and deep valley of the Marañón river. The Arapa region (40 km from the Titicaca lake border) and the Amantani island region (an island in the Titicaca lake) were also considered as belonging to distinct regions as they are separated by water. Six regions are represented by one field (Table 1).

<figure><img src="image_14.png" /><figcaption>**Figure 1:** Map of Peru showing the field locations in the 9 regions. The stars symbolize the fields representing each region (in the subset data with one field for each region).</figcaption></figure>

The Otuzco and Andahuaylas regions are represented by 5 fields and the Huancayo region by 7 fields (Table 1). Inside these regions, we recognized (i) “agronomic areas” where *G. pallida* could be considered to have a continuous distribution due to the close proximity of highly infected fields and (ii) “isolated fields” separated from others by areas without farming where *G. pallida* is absent (figure 1). Finally, a total of 23 highly infected fields were analyzed (figure 1) including the nine already studied in Picard et al. (2004).

For each field, forty cysts were randomly taken from a soil sample that was collected with a semi-cylindrical corer. A total of 703 individuals were analyzed with 8 polymorphic microsatellite
markers. The protocols of extraction of cyst from the soil, DNA amplification and the specific identification of *G. pallida* were described in Picard et al. (2004). As clustering analysis has been developed to assign individuals in groups according to their multilocus genotype (Structure software, Pritchard et al., 2000), all individuals with only one locus without amplification were excluded from the data set to have the best assignation for each individual.

<table><caption>**Table 1:** Sampling sites, altitude and number of genotyped nematodes (n). In bold : fields representing each region (in the subset data with one field for each region); * fields analyzed in Picard *et al.* 2004.</caption><thead><tr><th colspan="2">Region</th><th>Field</th><th>Altitude (m)</th><th>n</th></tr></thead><tbody><tr><td>I</td><td>Cutervo</td><td></td><td>2786</td><td>28</td></tr><tr><td rowspan="5">II</td><td rowspan="5">Otuzco</td><td>1 Cajamarca</td><td>3583</td><td>29</td></tr><tr><td>2 Contumaza</td><td>2949</td><td>30</td></tr><tr><td>3 Otuzco*</td><td>3318</td><td>30</td></tr><tr><td>4 Otuzco*</td><td>3401</td><td>29</td></tr><tr><td><b>5 Otuzco*</b></td><td>3174</td><td>32</td></tr><tr><td>III</td><td>Huaraz</td><td></td><td>3993</td><td>26</td></tr><tr><td>IV</td><td>Huanuco</td><td></td><td>3585</td><td>35</td></tr><tr><td rowspan="7">V</td><td rowspan="7">Huancayo</td><td>1 Huasahuasi</td><td>3352</td><td>27</td></tr><tr><td>2 Jauja*</td><td>3549</td><td>31</td></tr><tr><td><b>3 Concepcion*</b></td><td>3652</td><td>34</td></tr><tr><td>4 Jauja*</td><td>3784</td><td>34</td></tr><tr><td>5 Huancayo</td><td>4041</td><td>28</td></tr><tr><td>6 Huancayo</td><td>4089</td><td>33</td></tr><tr><td>7 Huancavelica</td><td>4174</td><td>27</td></tr><tr><td rowspan="5">VI</td><td rowspan="5">Andahuaylas</td><td><b>1 Ayacucho</b></td><td>3456</td><td>36</td></tr><tr><td>2 Andahuayla*</td><td>3870</td><td>28</td></tr><tr><td>3 Andahuaylas*</td><td>3484</td><td>29</td></tr><tr><td>4 Andahuaylas*</td><td>2778</td><td>31</td></tr><tr><td>5 Andahuaylas</td><td>4118</td><td>31</td></tr><tr><td>VII</td><td>Cusco</td><td></td><td>3726</td><td>28</td></tr><tr><td>VIII</td><td>Arapa</td><td></td><td>3915</td><td>34</td></tr><tr><td>IX</td><td>Amantani island</td><td></td><td>4059</td><td>33</td></tr></tbody></table>

### 3.3.2. Population differentiation and IBD

Two complementary methods were used to test the association between genetic divergence and geographical distance. Firstly, the regression of $F_{ST}$ /(1- $F_{ST}$) estimates on logarithm of distance. Under IBD in a two-dimensional space, pairwise $F_{ST}$/ (1- $F_{ST}$) ratios are expected to increase approximately linearly with the logarithm of spatial distance (Rousset, 1997). Pairwise $F_{ST}$ estimates were obtained from FSTAT (version 2.9.3; Goudet, 2001), according to Weir and Cockerham (1984). Secondly, an unidirectional Mantel correlogram - correlation between genetic divergence and geographic distance - (Sokal et al., 1986, see also Arnaud, 2003) was performed with PASSAGE software (Rosenberg, 2001). To assess the genetic divergence among fields, the allele-sharing genetic distance $D_{AS}$ (Jin and Chakraborty, 1994) was estimated using
POPULATION version 1.2.28, a program written by O. Langella and available at
http://www.pge.cnrs-gif.fr/bioinfo. The geographical variable was assessed simply by the linear
geographical distance measured "as the crow flies", which is a straight-line distance between any
pair of locations. The distance classes were designated to achieve approximately equal frequencies
of field pairs. In relation to the mutation model of microsatellite loci, the IAM model was chosen
(Wright's *F*-statistics and allele-sharing genetic distance) for the reasons explained in Picard *et al.*
(2004).

3.3.3. *Genetic model-based clustering*

We used the model-based clustering method (Pritchard et al., 2000) to infer population
structure and assign individuals to populations using multilocus genotype data by a Bayesian
approach, as implemented in the Structure software. The model assumes K populations (or
clusters) modeled each by its own set of allele frequencies at each locus. The results of the
assignation process can be illustrated by colored graphs in which each individual is represented by a
vertical bar. At each cluster (K), the model gives a posterior probability of the data. We chose the
value of K clusters that maximizes the posterior probability of the data (PPD). We considered the
successive increase of the PPD for increasing values of K, which can be regarded as the gain of
information obtained after each addition of one cluster. Nevertheless, it should be emphasized that
the PPD is not an accurate estimate and should be regarded as an heuristic guide to determine the
most consistent model (Pritchard et al., 2000).

Five independent runs were conducted to assess the consistency of the results across runs
and the PPD was the mean of the five independent runs. All runs were based on 100,000 iterations
after a burn-in period of 20,000 iterations. Between the regions (using one field representing each
region), we tested if K was equal to the number of regions (K varying between 1 and 15). Inside
the regions, we tested if K > 1 to detect a genetic structure at the smallest scale (K varying between
one and the number of fields). Choosing a K value that maximizes the PPD can be difficult to
apply for complex data sets including many groups (Rosenberg et al., 2002). Therefore, in the
interest of clarity, the data set was split in four subsets (a subset with one field representing each
region, and three subsets corresponding to the Otuzco, Huancayo and Andahuaylas regions).

**3.4. RESULTS**

3.4.1. *Population differentiation and IBD*

The curve of the spatial autocorrelation analysis with 23 fields (figure 2a) decreased with a
monotonic trend characterizing the IBD. This pattern is due to positive values of mantel r for the
smallest geographical distances (because locations close to each other tend to have similar allelic
frequencies), while locations far from each other tend to have very different allelic frequencies and
thus are characterized by negative mantel r coefficients. The mantel r coefficients were significantly
different from zero for most distance classes. When using the 23 fields, the $F_{ST}/(1-F_{ST})$ ratio
against the logarithm of geographical distance (figure 2b) could be split in two parts. Below 50 km,
the $F_{ST}/(1-F_{ST})$ ratio between fields pairs remained constant and very low (< 0.06). The ratio was
not correlated with the geographical distance (y = 0.007x + 0.01, r = 0.37, ddl = 14, not
significant). Above 50 km, the ratio increased with geographical distance (y = 0.158x - 0.46, r =
0.54, ddl = 235, highly significant). These results suggested an IBD above 50 km and an absence of
IBD below 50 km.

Within the Otuzco and Huancayo regions, the spatial autocorrelation analysis (figure 3a,b) detected an IBD. Indeed, the curves decreased with a monotonic trend (significant positive mantel r coefficients for the smallest distance class and significant negative mantel r coefficients at the end of the curve). For the Huancayo region, if the two outlying fields (field 1 and field 7) were excluded of the analysis, the autocorrelation coefficients were no longer significant (figure 3c). For the Andahuaylas region, none of the autocorrelation coefficients was significant (figure 3d).

3.4.2. Genetic model-based clustering and hierarchical population structure

Using only one field representing each region, the best assignation was observed for K=9,
corresponding to the 9 regions we had defined (figure 4a). The PPD was maximum for this value
of K; moreover the standard error of the PPD was higher for greater values of K (figure 5a).
Structure software failed to calculate an homogeneous PPD between each iteration for K > 9. At
K=9, all individuals were always assigned in the same cluster among the 5 independent runs. One
of the clusters of individuals obtained for a given value of K was split into two clusters for K+1,
until K=9 (figure 4a). At K=9, 97% of the individuals were assigned to their clusters at more than
80% (see figure 4a). Only one individual out of 286 was assigned incorrectly (an Andahuaylas
individual was assigned to the Cusco cluster at 82%). When K=10, only 7 individuals (all in the
region of Huancayo) were in majority assigned to the new cluster (black color; cf field V, figure 4a).

In the Otuzco, Huancayo and Andahuaylas regions, for K equal to the number of fields
sampled, the clusters recognized by structure didn't match the fields sampled (figure 4b). For
example, although 5 fields have been sampled in the Otuzco region, the 5 clusters recognized by
Structure (when K is set to 5) did not correspond to them. The individuals were more correctly
assigned when they belonged to the outlying fields (figure 4b; see fields 1 & 2 for Otuzco and fields
1 & 7 for Huancayo). The PPD weakly increased in the three regions (figure 5b). In the Otuzco
region, when K goes from 3 to 5, still 3 groups (field 1, field 2 and fields 3, 4 & 5) could be
distinguished. In the Huancayo region, a similar pattern was observed. At K=3, the outlying fields
(field 1 and 7) could be distinguished. At K=7, no other group could be distinguished. In the
Andahuaylas region at K=5, only the field 5 could be weakly distinguished.
<figure><img src="image_15.png" /><figcaption>**Figure 2** IBD on all the data set (23 fields); (a) mantel correlograms depicting the relationship between estimates of genetic divergence (DAS) and geographical distance (straight-line), \* *p* < 0.05; (b) regression of *F*<sub>ST</sub>/(1 - *F*<sub>ST</sub>) estimates on logarithm of distance, two linear regressions were calculated below 50 km (*p* > 0.05) and above 50 km (*p* < 0.001).</figcaption></figure>
<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

Figure 3. Mantel correlograms depicting the relationship between estimates of genetic divergence and geographical distance inside regions; \* *p* < 0.05; (a) Otuzco region, (b) Huancayo region (7 fields), (c) Huancayo region (5 fields, *i.e.* without the peripheral field V1 and Field V7), (d) Andahuaylas region.
<figure><img src="image_20.png" /><figcaption>(b)</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>**Figure 4:** Estimated population structure; (a) analysis of the subset data with one field for each region; (b) analyses of the Otuzco region with 5 fields, the Huancayo region with 7 fields and the Andahuaylas region with 5 fields. Each individual is represented by a thin vertical line, which is partitioned into K colored segments that represent the individual's estimated membership fraction in K clusters (designated on the Y axis as a percentage). Black lines separate individuals of different populations. Populations are labeled below each figure.</figcaption></figure>
<figure><img src="image_22.png" /><figcaption>**Figure 5:** Posterior probability of the data (PPD) against the maximum number of clusters (K) considered; (a) analysis of the subset data with one field for each region, (b) analyses of the Otuzco, Huancayo and Andahuaylas regions.</figcaption></figure>
**3.5. DISCUSSION**

To our knowledge, this is the first study that clearly defined the limit of a population for a
soil-dwelling nematode. This goal was achieved by the complementary approach of both analysis of
Isolation by Distance pattern and assignation methods of multilocus genotypes of the potato cyst
nematode *Globodera pallida*.

3.5.1. *No evidence of genetic differentiation for fields less than 50 km apart.*

Inside a continuous potato production area (agronomic area; figure 1), nematodes found
inside fields located at less than 50 km apart could be considered as belonging to the same
population.

An absence of IBD below 50 km for the regression of F<sub>ST</sub>/(1- F<sub>ST</sub>) against the logarithm of
distance was observed and F<sub>ST</sub> values for such pairs of fields were very low (< to 0.057). This result
was consistent with the absence of IBD observed in the spatial autocorrelation of the Andahuaylas
and Huancayo regions (when the most distant fields were excluded from the data set of the
Huancayo region). The use of the assignation method confirmed this interpretation: in the Otuzco
 fields 3, 4 & 5), the Huancayo (fields 2, 3, 4, 5 & 6) and the Andahuaylas regions, the clusters
(K=5 or K=7) did not match the fields sampled, revealing very weak or no genetic structure
among fields. For another cyst nematode species (Heterodera schachtii, a parasite of sugar beet)
studied in a large sugar beet production area of the north of France, a low genetic differentiation
among fields (even 175 km apart) was observed, suggesting a significant gene flow at such a scale
(Plantard and Porte, 2004).

The absence of genetic structure could be due to large passive dispersion inside an
agronomic area where *G. pallida* has a continuous distribution and is found at high density (until 9
billion cysts/ hectare). The efficiency of passive dispersion should be enhanced by the fact that the
cyst is a conservation form containing hundreds of individuals that can stay viable more than 10
years. Passive dispersion could be due to natural factors (wind, irrigation, wild animal...) or human
activities (transport of cysts in soil stuck to seed tubers, wheels or tools). The impact of human
activities can for example be observed when comparing fields 7 of Huancayo and 5 of Andahuaylas
(respectively 4,174 m and 4,118 m, the maximum altitude for potato culture) to their neighboring
fields (which are not geographically isolated and belong to the same continuous production area). A
higher genetic differentiation was observed than among other fields at similar distance of the same
agronomic area. This differentiation could be due to lower tuber exchange, as at such an altitude,
farmers live in self-sufficient communities (self-production of seed tubers and no exportation of
seed tubers). Moreover, their extensive agriculture system with long rotations could favor recurrent
bottlenecks in nematode populations, increasing genetic differentiation.
### 3.5.2. Increasing genetic differentiation among fields with geographic distance above 50 km.

Above 50 km, an IBD pattern (i.e. increase of genetic differentiation correlated with that of geographical distance) was detected among fields. We could notice that in our sample set, 53 km corresponded to the weakest distance between two fields without farming (between the field 1 and the field 4 in the Huancayo region; figure 1). The two methods [$F_{ST}$/(1-$F_{ST}$)] and autocorrelogram] used to detect IBD pattern with 23 fields exhibited the same pattern of IBD among regions. The IBD pattern among regions could be explained by the increase of genetic differentiation between populations due to the increasing impact of the geographic relief (acting as a geographic barrier to *Globodera pallida* passive dispersion) with the increase of geographical distance between regions. However, the end of the spatial autocorrelation curve (23 fields) stops decreasing indicating that above the distance class [357 – 456 km] the genetic distance stops increasing with the geographical distance. At the same level of geographical distance, the $F_{ST}$/(1-$F_{ST}$) ratio exhibits a high variance. These observations could be explained by the fact that alleles from very divergent populations were submitted to homoplasy, a feature already described for the microsatellite markers (Goldstein and Pollock, 1997; Ortí et al., 1997; Estoup et al., 2002).

The individuals were strongly assigned to each of the 9 clusters (K=9) that matched clearly with the 9 regions (using one field representing each region). The same data set (9 fields for 9 regions) analyzed by a neighbor-joining clustering model with the $D_{AS}$ genetic distance also showed groups clearly matching regions, with few individuals assigned to another region (data not shown). In the Andean Cordillera, as *G. pallida* was found between the altitudes of 2,700 m and 4,300 m, the populations are largely isolated by the high geographic relief (such as the Cordillera Blanca reaching 6,768 m at Huascarán top). These obstacles could prevent a passive dispersion due to natural factors (wind, wild animals). Commercial exchanges, thus transport of cysts due to human activities are also limited by such relief.

### 3.5.3. What is a population?

The figure of 50 km as the limit prior to detect IBD should be considered as a rough estimation. Indeed, inside a given region, the first evidence of genetic differentiation appeared when an isolated field was separated from others by area without farming (where *G. pallida* is absent). For example, the IBD pattern in the Huancayo region was mainly due to the presence in this data set of the field 1 (isolated by area without farming), as the IBD pattern disappears when the field was excluded (data not show). The use of the assignation method was consistent with this result: in the Otuzco and Huancayo regions, the individuals from fields which are isolated by area without farming (fields 1 & 2 of Otuzco and field 1 of Huancayo), were more strongly inferred to a single group compared to individuals sampled in fields belonging to an agronomic area (figure 4b).
This effect could also be seen in the analysis of *F*<sub>ST</sub> values: in the Huancayo agronomic area, the genetic differentiation between fields 4 and 6, which are 86 km apart, was weak (*F*<sub>ST</sub> values = 0.01), while the genetic differentiation between fields 1 and 4, which are 53 km apart but are separated by an area without farming, was higher (*F*<sub>ST</sub> values = 0.13). All those results indicate a highest genetic differentiation for fields isolated by area without farming.

Finally, inside a given agronomic area, *Globodera pallida* does not behave as an animal with discrete populations but rather as an organism with continuous populations and substantial gene flow preventing the differentiation due to genetic drift over large areas. Quite similarly to plants (Hamrick and Godt, 1990; Hamrick et al., 1992), nematodes are characterized by their absence of (or very reduced) active dispersal coupled with long distance passive dispersal. In Peru, an IBD was observed, only above 50 km and is associated with areas without any culture of potato or major geographical barriers such as deep valleys or high mountains where found among the fields considered. Because we hypothesize that passive dispersal due to their small size is a key process to explain this pattern, the genetic structure observed for *G. pallida* could also be relevant to numerous other soil organisms. In the absence of any similar study, we can hardly extend our results to other soil organisms. However, we think that the combine analysis of IBD patterns and clustering methods constitute a powerful approach to determine the limit of populations of such organisms.
Acknowledgements

We would like to thank the “Servicio Nacional de Sanidad Agraria” (SENASA, Peru), “Instituto Nacional de Investigación Agraria” (INIA, Peru) and particularly Dr María Scurrah, Dr Elsa Carbonell Torres and Ing Susana Chumbiauca for all their help in the sampling of potato cyst nematode in Peru. This work was financially supported by the INRA department “Santé des Plantes et Environnement” and “la Région Bretagne”. The French "Ministère des affaires étrangères" provided fund for the trip. We would also like to thank Katell Jolivet, Éric Grenier and Didier Mugniéry for useful comments on a previous version of the manuscript. Finally, we thank Mrs Sylvie Valette, Mrs Pauline Casters-Picard for their technical help.
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CHAPITRE IV

Une colonisation du sud vers le nord de la
Cordillère Andine par le nématode à kyste de
la pomme de terre au cours des temps
géologiques suggère de multiples
changements d'hôtes des espèces sauvages
aux espèces cultivées
Dans le chapitre précédent lorsque l'on considère séparément chacune des trois régions agricoles étudiées, aucun IBD ni aucune structuration génétique (à l'exception des parcelles de hautes altitudes ; cf. paragraphe 6.1.2) n'ont été observés. Nous émettons donc l'hypothèse qu'il n'y a qu'une seule population à l'intérieur d'une région agricole où la distribution de *G. pallida* est continue de par la distribution continue de sa plante hôte. Par contre, un patron d'isolement par la distance émerge dès l'apparition de zones exemptes de cultures entre bassins de production mais ne devient plus détectable pour les régions séparées par des distances supérieures à ~ 400 km. L'absence de détection d'IBD peut être expliquée par le type de marqueur utilisé (loci microsatellites) qui serait soumis à l'homoplasie ou par des événements historiques (colonisation, fragmentation...) à l'origine de la mise en place de lignées entre lesquelles le flux génique serait nul ou négligeable. Dans ce chapitre IV, nous allons donc maintenant nous intéresser aux causes de la disparition de l'IBD à des distances supérieures à 400 km et aux relations historiques entre les populations.

Si l'approche de la génétique des populations, nous a permis de décrire le patron génétique actuel de *Globodera pallida* dans des parcelles cultivées de pommes de terre, ce n'est bien que parce que ce patron est issu de processus s'étant déroulés au plus au cours de quelques milliers de générations. En effet, la génétique des populations ne nous permet pas d'appréhender des processus évolutifs ayant eu lieu au cours de périodes beaucoup plus longues [dizaine de milliers d'années (glaciation) voire de millions d'années]. La phylogéographie intraspécifique fait le lien entre la génétique des populations et la systématique (Avise et al. 1987). La phylogéographie, nous permet donc de retracer l'histoire entre taxa à une échelle populationnelle.

Avant de développer le contenu de ce chapitre, nous allons brièvement introduire l'approche phylogéographique. La systématique hiérarchise le classement des organismes vivants en différents rangs taxonomiques (espèce, genre, famille...). Cette classification permet de mieux appréhender l'immense biodiversité de la planète, mais ne doit pas faire oublier que la diversité des êtres vivants est issue du hasard (mutation) et de la sélection suivant un processus continu dans le temps (Kupiec et Soniguo, 2000). Parmi les rangs taxonomiques utilisés, le classement en espèces (tout du moins lorsque celles-ci sont basées sur le concept biologique *i.e.* la possibilité de croissement engendrant une descendance viable et fertile) est certainement le moins arbitraire. Toutefois, le processus de spéciation est continu dans le temps et dans l'espace (figure 4.1B). Autrement dit, la phylogénie dans le sens macroévolutif est inéluctablement une extrapolation de la micro évolution (Avise et al. 1987). Le classement en espèces reste donc arbitraire dans le sens où il
impose de ranger les organismes en deux catégories à un temps donné alors qu'ils sont en
perpétuelle évolution. Il est donc plus ou moins facile de définir les limites d'une espèce
notamment en fonction du degré d'achèvement du processus de spéciation. Chez les vertébrés, la
mise en place de barrières à la reproduction et les fortes variations morphologiques facilitent le
classement en espèces. A l'opposé, certains organismes comme les nématodes dont la morphologie
varie peu (cf. paragraphe 1.1) et la lente mise en place de barrières à la reproduction (cf. paragraphe
6.3) rendent le classement en espèces plus ténu. La phylogéographie est à l'interface entre la
micro évolution et la macro évolution (figure 4.1A ; Avise 2000) en appréhendant la notion d'espèce
d'une manière évolutive. La phylogéographie est une approche du processus de spéciation qui
décrit sa mise en place à un moment précis du temps. Elle permet de retracer l'histoire de la
colonisation d'une espèce en confrontant les relations de parentés entre des populations ou entre
des dèmes (groupes de populations) avec leur distribution géographique.

<figure><img src="image_23.png" /><figcaption>**Figure 4.1** : (A) la phylogéographie est à l'interface de domaines scientifiques bien établis. (B) L'approche phylogéographique permet d'appréhender le processus de spéciation. Le processus de spéciation se met en place dans la durée ; dans ce schéma, la durée de spéciation a été estimée à partir de données de vertébrés (d'après Avise, 2000).</figcaption></figure>
Les relations historiques des populations de *G. pallida* ont été retracées dans ce chapitre.
Pour reconstruire cet arbre phylogénétique, trois distances génétiques ont été calculées avec huit
loci microsatellites entre 24 parcelles contaminées par *G. pallida*, plus une population de *Globodera
mexicana* pour enraciner l'arbre. Comme la résolution de l'arbre obtenu est faible, le gène
mitochondrial cytochrome b de 32 individus a été analysé pour confirmer les résultats obtenus avec
les marqueurs microsatellites. Les deux arbres analysés sont confrontés à la distribution
géographique des individus considérés. Par la suite nous chercherons à savoir s'il existe une
correspondance entre les arbres phylogénétiques et la distribution géographique des individus
considérés et si celle-ci reflète la route de la dispersion de la pomme de terre cultivée ou si elle est
antérieure ? Pour répondre à cette question, nous tenterons de calibrer une horloge moléculaire.
A South-to-North colonisation of the Andean Cordillera by the potato cyst nematode during geological times suggests multiple host shifts from wild to cultivated potatoes

D. PICARD & O. PLANTARD

UMR INRA-ENSAR BiO3P, Domaine de la Motte, B.P. 35327, 35653 Le Rheu cedex, France.

Date of receipt:

Keywords: microsatellite, cytochrome b, *Globodera pallida*, pest, phytoparasitic, potato cyst nematode, PCN, Peru, Phylogeography

Corresponding author: O. Plantard, UMR INRA-ENSAR BiO3P, Domaine de la Motte, B.P. 35327, 35653 Le Rheu cedex, France. Fax number: 33 2 23 48 51 50; plantard@rennes.inra.fr

Running title: potato cyst nematode phylogeography

All sequences have been deposited in GenBank under Accession Nos. AY851617 - AY851648 and AY8516
**4.1. Introduction**

The evaluation of human activities on the genetic variability of plant or animals is an old issue in evolutionary biology (Darwin, 1883). The recent analysis of polymorphic molecular markers that trace past events with the help of population genetics tools has allowed improvement of our understanding of such processes. After having placed those results in a geographical context, it has been possible to identify the centre of diversity of numerous crops (Heun *et al.*, 1997; Matsuoka *et al.*, 2002) and to retrace the route followed by men when they have spread such crops. In most cases, the relative impact of man (during domestication and spreading of a crop) is assessed relative to the natural variability of the wild plant from which the crop issued. This phylogeographical approach can also be applied to pests and has been especially fruitful to identify the origin of non-indigenous pests (Brasier *et al.*, 1999; O'Donnell *et al.*, 2000; Davies *et al.*, 1999). However, this phylogenetical approach is limited by the huge dispersal abilities of most pest organisms especially for wind dispersed fungi (but see Aguilar *et al.*, 2004 for the specific case of symbiotic bacteria associated with the roots of its host plant). For example, it has been shown that potato blight has spread across most of Europe from west to east in a few months (Austin Bourke, 1964). However, without any survey by man of this spread in "real time", it would not have been possible to mark out the routes followed by this pathogen because no molecular signature of such a rapid event could be kept in the fungal genome. Thus, the identification of the routes followed by a pest (both in space and time) can hardly be developed for crops or pests that have a long co-evolutionary history, prior to human domestication (but see Stahl *et al.*, 1999 - for an example of molecular signatures in the plant genome due to millions of years of interaction with its parasite).

Taking advantage of the still little pertubated phylogeographical patterns of plants in South America (numerous wild potatoes are still present in the Central Andes), we have followed a phylogeographical approach to a specialised pest with weak active dispersal abilities and have studied its genetic variability in its centre of origin (which is also the centre of origin of its host crop).

Native to the Central Andes (Evans & Stone, 1977), the potato cyst nematode *Globodera pallida* is an obligatory parasite of the genus *Solanum*, its unique host plant. Once inside the root, the nematode larva modifies the expression of some plant cells to produce a feeding cell (syncitium) to get nutrients from its host-plant (Davis *et al.*, 2004). Such a close association between the parasite and its plant suggest a long co-evolutionary history between *G. pallida* and *Solanum spp* (Stone, 1979). Moreover, numerous resistance genes have been found
in germplasm of wild *Solanum* species from South America (Castelli *et al.*, 2003), where a hotspot of species richness lies in Peru and Bolivia (Hijmans & Spooner, 2001).

*Globodera pallida* is a major pest of potatoes in South America, especially in the Andean Cordillera but also in Europe where it has been introduced since the XIX century. As the potato culture is estimated to have began 5000 years ago in the Andean Cordillera (Thurston, 1994), Peruvian growers must have been faced with the impact of potato cyst nematode for along time. It has been hypothesized that the 8 to 10 years long rotation of potato crops conducted by Incas avoided severe losses due to potato cyst nematodes. Indeed, nematode larvae are able to survive inside cysts in soils for almost 10 years, and only such long rotations allowed time for population levels to fall below the threshold of acceptable damage (Thurston, 1994). We have addressed two main questions on this plant-pathogen system: (1) is there any geographical structure in the genetic variability of *G. pallida* sampled in fields of its native area? (2) when did the phylogeographical pattern observed take place: following human domestication of potatoes (i.e. the spread of the nematodes would have been influenced by human activities) or before?

To address those issues, we have sampled populations along a 1500 km transect from the southern range of *G. pallida* (Titicaca lake) to the north border of Peru and genetically characterized them with two complementary molecular markers. Five clades where recognized, revealing a clear South-to-North pattern with decreasing genetic variability. The large genetic divergences among those clades suggest that the different clades diverge far before human settlement, and that those ancient populations of nematodes have independently made multiple host shifts from wild to cultivated potatoes. The consequences of this finding to control this pest both in its native and introduced areas will be discussed.

**4.2. Materials and Methods**

A sampling program was conducted in 2002 from 2,500 m to 4,300 m along the Andean Cordillera in Peru to collect *Globodera pallida* cysts (see Picard *et al.*, 2004 for a description of the methods to sample soil and to extract cysts from the soil). Forty two samples (41 from potato fields mainly cultivated with *Solanum tuberosum* subsp. *andigenum* and one, the Cajabamba sample, from a soil collected under a wild *Solanum* species - *S. chiquidenum* -) containing cysts were selected, covering a 1500 km North to South transect from the border with Ecuador to the Bolivian border (see figure 1). Although a few records of *G. pallida* from Bolivia has been reported, it seems that this species is replaced to the south of the 15.6° S in latitude by another potato cyst nematode, *G. rostochiensis* (Evans, Franco & Scurrah 1975, Stone 1979, Franco *et al.*, 1998). However, the species
identification of each individual sequenced or genotyped in the present study has been checked by
PCR-RFLP of the ITS region (see Picard *et al.*, 2004 for a description of this method).

<figure><img src="image_24.png" /></figure>

**Figure 1:** Location of *G. pallida* samples in Peru. Sample sites (symbolized by white points) are located at high altitude (from 2,570 m to 4174 m).
Two specific primers (INRAcytbL: 5'-GGGTGTGGCCTTGTTATTTC-3'; INRAcytbR: 5'-ACCAGCTAAAACCCCATCCT-3') were designed from the complete cytochrome b sequence of a Scottish population of *G. pallida* obtained by Armstrong *et al.* (2000). PCR products using those primers enabled the amplification of partial sequences (872 bp) of the cytochrome b gene (cytb) from 32 samples of the 42 mentioned above. Mitochondrial DNA preparation and PCR amplification were performed as described in Plantard & Porte (2003), except that mtDNA was extracted from one cyst (i.e. from 100 to 500 juveniles sharing the same mother instead of one larva). Five to ten PCR amplifications from the same DNA extract (i.e. from one cyst) were pooled to obtain enough DNA for sequencing. The amplified DNA was sent to Macrogen inc. for purification and sequencing. DNA sequences were aligned by eye. Because the sequences are protein-coding and have no introns or gaps, alignment was straightforward.

Model parameters for likelihood searches were estimated using Modeltest (Posada & Crandall, 1998), which compares goodness of fit of models using the likelihood ratio test. The best fitting model for the *Globodera* sequences was HKY85+G, with a ts/tv ratio=3.9338 and a gamma distribution shape parameter=0.2657. We used the base frequency values suggested by the model (pA=0.1659, pC=0.1391, pG=0.1907). Using PHYLIP package version 3.62 (Felsenstein, 2004), three methods of phylogenetic analysis were performed: maximum-likelihood (ML; Felsenstein & Churchill, 1996) using algorithm analogous to HKY85 model, maximum-parsimony (MP; Fitch, 1971; Eck & Dayhoff, 1966; Kluge & Farris, 1969) and neighbor-joining (NJ; Kishino & Hasegawa 1989; Felsenstein & Churchill 1996) using the F84 model. The robustness of the ML, MP and NJ results was assessed by bootstrap analysis (Felsenstein, 1985) involving 500 iterations. A sequence of *G. mexicana* (the sister group of *Gp*, producing viable hybrid with *G. pallida*, but only when using males of *G. mexicana* with females of *G. pallida* - Thiéry *et al.*, 1997 - ) was obtained from a sample collected in 1985 in Mexico by D. Mugniery on wild non-tuberous *Solanum*, and used as an outgroup in our phylogenetic analysis.

Twenty four populations (see Table 1) of the 42 mentioned above were analyzed with 8 polymorphic microsatellite makers (9 populations were already used in Picard *et al.*, 2004; see this paper for the description of the DNA extraction method, the PCR conditions and the allele identification method). All individuals genotyped with the 8 microsatellite loci have at least one individual from the same field or from neighboring fields in the cytochrome B analyses, excepted for the Huanuco population. Thirty nine individuals of *G. mexicana* were genotyped with 6 microsatellite markers (as GP36 and GP46 loci failed to amplify *G. mexicana*) and used as an outgroup in our phylogenetic analysis. To avoid artefacts caused by poor DNA extract quality, all individuals with more than two amplification failures among the eight loci were excluded.
<table><caption>**Table 1:** Sample size (N) and mean allelic richness (A) based on the rarefaction index method (n=26; ElMousadik & Petit, 1996) for each field population genotyped.</caption><thead><tr><th>Clade</th><th>Region</th><th>N</th><th>A</th></tr></thead><tbody><tr><td rowspan="8">Vm</td><td>Huancabamba</td><td>38</td><td>35</td></tr><tr><td>Cutervo</td><td>38</td><td>26</td></tr><tr><td>Cajamarca</td><td>39</td><td>34</td></tr><tr><td>Contumaza</td><td>38</td><td>32</td></tr><tr><td>Otuzco-1</td><td>38</td><td>22</td></tr><tr><td>Otuzco-2</td><td>37</td><td>20</td></tr><tr><td>Otuzco-3</td><td>40</td><td>27</td></tr><tr><td>Huaraz</td><td>38</td><td>36</td></tr><tr><td></td><td>Huanuco</td><td>40</td><td>35</td></tr><tr><td rowspan="7">IVm</td><td>Huasahuasi</td><td>37</td><td>45</td></tr><tr><td>Jauja-1</td><td>37</td><td>38</td></tr><tr><td>Jauja-2</td><td>38</td><td>39</td></tr><tr><td>Jauja-3</td><td>40</td><td>42</td></tr><tr><td>Huancayo-1</td><td>36</td><td>43</td></tr><tr><td>Huancayo-2</td><td>37</td><td>48</td></tr><tr><td>Huancavelica</td><td>33</td><td>47</td></tr><tr><td rowspan="5">IIIm</td><td>Ayacucho</td><td>39</td><td>51</td></tr><tr><td>Andahuaylas-1</td><td>39</td><td>51</td></tr><tr><td>Andahuaylas-2</td><td>40</td><td>61</td></tr><tr><td>Andahuaylas-3</td><td>37</td><td>57</td></tr><tr><td>Andahuaylas-4</td><td>37</td><td>62</td></tr><tr><td>IIm</td><td>Cusco-3</td><td>40</td><td>61</td></tr><tr><td rowspan="2">Im</td><td>Arapa</td><td>40</td><td>29</td></tr><tr><td>Amantani-2</td><td>40</td><td>52</td></tr></tbody></table>

To calculate genetic distances among populations, we considered that our microsatellite loci mutate according to the IAM mutation model (see Picard *et al.*, 2004 for a justification of this choice). For tree reconstruction, $D_A$ distances, a distance based on the IAM model (Nei *et al.*, 1983), were used as this method is estimated to be superior to the others in topology construction (Nei, 1995; Takezaki & Nei 1996; Goldstein & Pollock, 1997). We used $D_C$ (Cavalli-Sforza & Edwards, 1967) and $D_m$ (Nei, 1973) distances (both based on the IAM model) to compare the topology of the three trees obtained. All the genetic distances and the NJ trees were performed using Populations 1.2.28 (available at http://www.cnrs-gif.fr/pge). Statistical support estimates for phylogram nodes were obtained by resampling loci across 500 bootstrap iterations for each genetic distance. The allelic richness of each population was estimated by the rarefaction index (ElMousadik & Petit, 1996) using the Populations 1.2.28 software. This method avoids the biased error of the allele number variation due to different sample sizes among populations (sample size is fixed to the smallest number of individual genotypes in a field, i.e. n=26). In these analyses, all individuals with amplification failure at one locus were excluded because a null amplification is considered as an absence of an allele that can lead to an underestimation of the allelic richness.
**4.3. Results**

Prior to phylogenetic analysis, we looked for any evidence of saturation in our data set (cf
multiple substitutions at the same base that are especially frequent for transitions at the third
position of codons; Page & Charleston, 1998). With that aim, we partitioned our data set in to six
subsets (i.e. transitions and transversions among the three codon positions; see for example Groth,
1998). Then we examined the scatterplot of pairwise total divergence versus pairwise divergence
calculated from each data set. All six data sets did not exhibit any evidence of saturation as no
plateau was observed in the six scatterplots, even for pairwise comparisons involving the outgroup
(Annexe 6).

**4.3.1. Phylogeographical pattern based on the cytochrome B sequences**

ML, MP and NJ analyses of the cytb genes produced trees with similar topologies with
nodes well supported by high bootstrap values (figure 2a). Five well supported and monophyletic
clades were clearly differentiated with the most basal group corresponding to the southern clade
and the most derived group correponding to the northern clade (figure 2a). Thus, the northern
clade (clade Vc; Contumaza) is the sister group of the clade immediately to the south (i.e. clade IVc;
Huancayo); the group formed by the clades Vc and IVc is the sister group of the clade immediately
to the south (i.e. clade IIIc; Andahuaylas); the group formed by clades Vc, IVc and IIIc is the sister
group of the clade immediately to the south (i.e. clade IIc; Cusco); and finally the southern clade
(i.e. clade Ic; Titicaca Lake), is the sister group to all other clades. Thus, it is likely that the origin of
*G. pallida* is around the Titicaca lake in the central Andes (Altiplano), and that the species dispersed
subsequently northward. This South-to-North pattern is supported by robust nodes between clades
(except for the clade IIc with bootstrap values < 50%). Inside clades, sequences from the same
location (for example the 3 sequences from Amantani, the 2 sequences from Puno in clade Ic) are
always identical or form a monophyletic group (for example the 3 Cusco sequences from clade IIc),
except one sequence among the three from Puno (clade Ic) and Andahuaylas (clade IIIc). While the
largest geographical area is covered by the Northern clade (clade Vc), the largest genetic distance
among populations of a given clade is observed in the three Southern clades (maximum distance
between pair of populations: clade Ic: 0.07 > clades IIc and IIIc: 0.05 > clade IVc: 0.009 > clade Vc:
0.003; see also the branch lengths inside those clades in figure 2a). The pairwise genetic distances
among the three Southern clades (=0.05) were larger than between the two Northern clades
(=0.01). This pattern is congruent with the interpretation that the northern regions (i.e. clades IVc
and Vc) have been more recently colonized by *G. pallida* than southern regions (i.e. divergence time
is shorter between the two terminal clades than among southern clades).
<figure><img src="image_25.png" /><figcaption>**Figure 2:** Agreement between phylograms based on mitochondrial marker (a), and nuclear markers (b). The map indicates the limit of the five clades (numbered I to V from south to north) along the Andean Cordillera. (a) Maximum-likelihood analysis based on Cytochrome b partial gene (872 bp); Stars (*) symbolize haplotypes located outside the clade corresponding to their geographical origin (see text); bootstrap values (500 iterations) are indicated for the nodes common to the tree analyses (neighbor-joining /maximum parsimony /maximum likelihood). (b) Neighbor-joining analysis, with a IAM genetic distance $D_A$, based on 8 microsatellite loci; numbers by nodes represent bootstrap (500 iterations) for the node between clades common to the three IAM genetic distance Methods (Dm/Dc/Da).</figcaption></figure>
Because a multipartite mitochondrial genome has been described in *G. pallida* with at least two different subcircles carrying the cytochrome B gene in the nematode population studied independent evolution of duplicated sequences located on different subcircles could preclude the use of this marker for phylogeographical and population genetics investigations (Armstrong *et al.*, 2000). However, sequence similarity of two clones from two subcircles over 581 bp of comparable sequence containing part of the ND3 and the CytB genes were found to be 99% identical (Armstrong *et al.*, 2000). Without any homogenizing process of gene sequences from different subcircles such as gene conversion, we would not have obtained a clear and geographically structured pattern at both the intra and the interclade level; moreover, the 6 identical sequences observed among the 10 fields of the clade *Vc* or among the 7 fields of the clade *IVc* would not be observed.

### 4.3.2. Phylogeographical pattern based on the microsatellite loci

The analysis of microsatellite data allows recognition of 5 monophyletic clades among the 24 populations studied (figure 2b). When considering clade position relatively to each other, the tree topology obtained with the three genetic distances ($D_A, D_C, D_m$) is identical with the most basal clades in the south and the most terminal clades in the north of the transect. Inside clades, neighbouring fields (for example the three Otuzco fields in clade *Vm*, the three Jauja fields in clade *IVm*) always form monophyletic groups (except the 2 Huancayo populations that form a paraphyletic group). However, the microsatellite tree is poorly resolved with a few nodes well supported by high bootstrap values. A South-to-North pattern was also observed with allelic richness analysis: the allele numbers increase towards the South, with the allelic richness of the clades *Im* (except the Amantani population), *IIm* & *IIIm* nearly twice as high as in the clade *Vm* (Table 1). This decreasing South-to-North gradient is congruent with the hypothesis that the northern populations have been colonized more recently by *G. pallida*, after several allele losses due to genetic drift and *subsequent* bottleneck effects.

### 4.3.3. Comparison of the trees based on the mitochondrial and the nuclear markers

The topologies of the trees obtained using the cytochrome b sequences and the microsatellite loci were congruent (figure 2) and reveal the same phylogeographical regions. Only three cytb haplotypes do not belong to the same clade in both analysis: Cajabamba (which was not studied with the microsatellite loci but whose geographical location suggests as being part of clade V rather than IV), Otuzco [*IVc versus Vm*] and Ayacucho [*IIc versus IIIm*], whose both geographical location suggests that the most likely assignment to a given clade is deduced from microsatellite data rather than from cytochrome B sequences. Contrarely to the location of a population in the
microsatellite tree which is based on the multilocus genotype of more than 30 individuals, the location of a cytochrome B haplotype is only based on one female. As different sequences could be present in a single field or in closely located fields (see for example the cases of the Puno sequences in clade Ic or the Andahuaylas sequences in clade IIIc), this marker is more prone to exhibit drastic differences in the location of an haplotype in the tree from one individual to the other. Finally, recent migrations – due to human transport of potato tubers for example – could also explain this assignment of an individual to a clade that does not correspond to the geographical location where it has been sampled.

Apart from tree topology, a parallel between the mitochondrial and the microsatellite data can be made by the comparisons of the South-to-North pattern of reduction in allelic richness for the microsatellite loci with the South-to-North pattern of reduction in branch lengths among populations of the same clade in the cytochrome B tree. The decreasing divergence time among populations from the basal clades of the trees to the terminal ones could explain this similar pattern in both markers.

The uses of the two molecular markers provide complementary information. Due to its slower mutation rate, the cytochrome B tree is not affected by saturation and homoplasy and is thus more efficient to resolve clades positions relatively to each other (as they have diverged for a longer time) than to resolve populations positions inside clades. Bootstrap values on clade nodes are much weaker for the tree based on microsatellite data that are more subject to homoplasy due to their high mutation rates. However, in terminal and northern clades (such as V), which are constituted by populations that have diverged more recently, too few polymorphisms are observed among the cytochrome B sequences, preventing the resolution of the phylogenetical relationships among populations inside this clade. At this spatial and temporal scale, the microsatellite data are more efficient as illustrated by (1) the larger divergence between the Huanuco and Huaraz populations relatively to all the other populations from this clade Vm as expected due to their geographical isolation (figure 2b) (while the Huaraz haplotype does not diverge from other haplotypes of the Vc clade; figure 2a) and by (2) higher bootstrap values among populations of clade Vm than southern clades (data not shown).

**4.4. Discussion**

Our investigations on the phylogeography of the potato cyst nematode *G. pallida* in the Central Andes (Altiplano) clearly illustrate a northward expansion of populations from the South of Peru (around the Titicaca lake) to the north.
As all the nematodes genotyped and sequenced in the present study have been sampled in potato fields (except one), the South-to-North pattern identified among the five clades could result in two alternative hypothesis : (h1) a recent range expansion of the nematodes due to the spread of potato culture by man that has occurred during the last few thousand years (Jensen & Kantz, 1974) or (h2) recent and multiple host shifts from wild to cultivated potato of nematodes that were already present in the soil long before man cultivated potato in those areas.

The high level of cytb sequence divergence among the different peruvian clades of *Gp*
species (12%) is identical to the one observed between *Caenorhabditis briggsae* and *C. elegans* that are
considered to have diverged from each other between 23 to 120 My ago (Prasad & Baillie, 1989;
Heschl & Baillie, 1990, Lee et al., 1992; Kennedy et al., 1993; Thacker et al., 1999; Coghlan &
Wolfe, 2002). Studies in other animals provide similar estimations of the mutation rates of the
cytochrome B gene (insects: Diptera: 27%/~225 My, Coleoptera: 20%/~265 My, Lepidoptera:
20%/~195 My (Simmons & Weller 2001); rodents: mouse/rat: 19%/~9-12 My; Jaeger et al., 1986).
As the generation time of *Globodera pallida* is much longer than for *Caenorhabditis spp* (one year
versus a few days), this estimation of divergence time could be under-estimated for the plant-
parasitic nematode. However, the Tylenchida and Rhabditida are distant nematode orders among
which the calibration rate could be disparate. Alternatively, the mutation rate of the cytb gene of *G.
pallida* relatively to other nematodes could also be affected by the multipartite structure of its
mitochondrial genome. The cytochrome B mutation rate of *Meloidogyne* spp. (another genus of the
Tylenchida order, like *Globodera*) has also been reported as accelerated relatively to *C. elegans* (Power
et al., 1993). However, those species have a short generation time. The species are parthenogenetic
and crossing-over among mitochondrial molecules has been demonstrated (Lunt & Hyman, 1997).
To estimate divergent times among clades, we have also sequenced a nuclear gene, the ribosomal
ITS1-5.8S-ITS2, of individuals from three different clades (data not show). Using a calibration date
with our ITS1-5.8S-ITS2 sequences (cf. paragraph 5.3 of annexe 3), the divergence time observed
among the *G. pallida* clades corresponds to 16-11 My between "Amantani" and "Cusco-
Huancabamba" clades and 8.9-6.1 My between the "Cusco" and "Huancabamba" clades (sequence
divergence: Amantani *versus* Huancabamba = 1.9%; Amantani *versus* Cusco = 1.5%; Cusco *versus*
Huancabamba = 0.8%).

Despite the need for these two markers to be more precisely assessed (especially with the
help of precise calibration dates that are lacking for the whole nematode phylum), the divergence
times enable the rejection of the hypothesis that the *G. pallida* expansion has been
contemporaneous with the human spread of the cultivated potato during the 5000 last years.
The divergence time and the South-to-North pattern observed among the *G. pallida* clades match with the uplift of the Andean Cordillera that occurred firstly in central Andes (southern Peru and Bolivia) in the late Oligocene (~25My) followed by mountain uplift towards the north until now (Hartley, 2003; Sempere *et al.*, 1990; Gregory-Wodzicki, 2000; Rousse *et al.*, 2003; cf. annexe 3). A similar South-to-North pattern of dispersion (from Bolivia to Central America) has also been observed in another animal restricted to high altitude, the lizard genus *Proctoporus* (Doan, 2003).

Such a South-to-North pattern of decreasing genetic variability is frequently observed in Europe (see Taberlet *et al.*, 1998 for a review) or in North America (Avise, 2000) due to the recent colonization of northern latitudes from Southern refugia after the last ice ages. As similar climatic events also occurred during the Pleistocene in the Southern hemisphere, (Vuilleumier, 1969; Vuilleumier, 1971), a symmetric pattern is expected there. It has been observed at least once in a plant (*Solanum pimpinellifolium*) along the Peruvian coast (Caicedo & Schaal, 2004). Instead of such a North-to-South pattern of diversification, the opposite pattern is observed among the five *G. pallida* clades. This South-to-North pattern is not compatible with the hypothesis that the phylogeographical pattern observed for *G. pallida* is the result of warming following the last Pleistocene glaciations 12000 years ago. However, pleistocene glaciations may have affected population genetic variability of *G. pallida*. As in Peru this nematode is only found at high altitude (between 2000 and 4500 m; Evans *et al.*, 1975; Franco, 1977) and is adapted to cold climates (Evans & Stone, 1977), the range of this species could have moved down in altitude during periods of cooling, allowing gene flow among populations from different valleys that were previously isolated by the lower altitude limit of their range (Garcia Moreno et al., 2004).

With such an ancient diversification of *G. pallida* populations, it's tentative to speculate that it reflects a co-evolutionary process of this specialised and obligatory endoparasite with its host-plant (as already postulated by Stone 1979 or Franco 1977 or Bakker *et al.*, 1993). A geographical coincidence can be observed between the two groups, as the hot spot for the tuber-bearing *Solanum* diversity is observed from 2,000 m to 4,000 m in the south part of Peru and in Bolivia (Hijmans & Spooner, 2001). The systematics of the *Solanum* genus is particularly complex and its phylogeny is still poorly resolved (Spooner & Hijmans, 2001). However, some elements are in favour of a South-to-North diversification hypothesis. For example, a molecular phylogeny based on cpDNA restriction enzyme in the second species rich series (series *Conicibaccata*) of the *Solanum* genus and the analysis of ploidy level among species (with diploid being the ancestral state - Carputo *et al.*, 2003), suggests a South-to-North pattern from Bolivia to Mexico (Castillo & Spooner, 1997).

To observe such an ancient genetic divergence between clades of *G. pallida* that are now associated to cultivated potatoes, *G. pallida* should have shifted from wild to cultivated potatoes
independently in each area corresponding to each clade. For specialized parasites (such as these nematodes) that modify the expression of some root cells transforming them into a transfer organ on which they feed (Davis *et al.*, 2004), the individuals that are able to develop on a crop often exhibit a narrow genetic pool (Goodwin *et al.*, 1994), characterizing the fact that all the parasites are derive from a single host shift onto the crop. To our knowledge, this study is the first one illustrating that an ancient phylogeographical pattern of a plant parasite can be studied by the investigation of this parasite on cultivated plants. Although extensive gene flow among populations of *G. pallida* inside a given region or clade (but not among regions) has been previously demonstrated (Picard *et al.*, 2004), the ancient divergence of the five *G. pallida* clades attested by this molecular signature is probably due to the specific life history traits of those soil nematodes (poor active dispersal abilities, long conservation and huge effective population size) slowing the homogenizing effect of a few individuals passively transported among clades on the genetic structure of the populations.

The tremendous genetic diversity and phylogeographical pattern exhibited by the *G. pallida* clades should be taken into account to efficiently control this pest. Plant breeding efforts may benefit from including representatives of each lineage inside *G. pallida* species when testing new resistant varieties increasing the likelihood that broad-based resistance, effective against all lineages of the *G. pallida*, is achieved (O'Donnell *et al.*, 2000). If a co-evolution between *G. pallida* and its host has occurred, the discoveries of new resistance sources could be helped by taking into account the distribution of the different wild *Solanum* species and by selecting taxa or accessions endemic from the region where the targeted *G. pallida* clade is found (see for exemple Castelli *et al.*, 2003, Chavez *et al.*, 1988; Turner *et al.*, 1989). Finally, the characterisation of the five clades corresponding to five geographical regions in the Andes could enable the identification of the origin of the parasite imported by humans all over the world and to detect if there was a single or multiple importation(s) in each country.

Finally, this study illustrates that the poorly known, but ecologically important soil dwelling organisms (Wardle & Ettema, 2002; Ettema *et al.*, 1998; Mortimer *et al.*, 1999), could also be especially interesting to describe phylogeographical patterns. Indeed, the study of such species with poor active dispersal abilities could provide a unique and more accurate phylogeographical pattern for ancient events than organisms with larger dispersal ability.
Acknowledgements

We would like to thank the “Servicio Nacional de Sanidad Agraria” (SENASA, Peru), “Instituto Nacional de Investigación Agraria” (INIA, Peru) and particularly Dr Maria Scurrah, Dr Elsa Carbonell Torres and Ing Susana Chumbiauca for all their help in the sampling of potato cyst nematode in Peru. This work was financially supported by the INRA department “Santé des Plantes et Environnement” and “la Région Bretagne”. The French “Ministère des affaires étrangères” provided fund for the sampling trip. We would also like to thank Eric Grenier and Didier Mugniéry for useful comments on the manuscript and Miss Pauline Casters-Picard for the conception of the figures. Finally, we thank Miss Sylvie Valette, Mr Abou Bakari-Kouassi, Mr Marcelo Trebejo and Mr Mario Curasi-Nina for their help.
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