Thesis

The phylogeny and biogeography of an insular endemic moth radiation : the genus "Galagete" (Lepidoptera: Autostichidae) from the Galapagos Islands

SCHMITZ, Patrick

Abstract

Nos investigations sur la phylogénie et la biogéographie d'une radiation endémique insulaire de microlépidoptères nous ont permis d'apporter de nombreuses réponses et d'élargir considérablement nos connaissances sur la diversité, la répartition, l'écologie, l'histoire évolutive et les phénomènes de spéciation du genre Galagete des Iles Galapagos. Nos études morphologiques nous ont amené à décrire plusieurs nouveaux taxa utilisés par la suite dans nos reconstructions phylogénétiques. La biologie pour l'une des espèces de Galagete a été dévoilée et les stades immatures décrits en détail. Nos analyses moléculaires et investigations biogéographiques ont permis d'obtenir une solide phylogénie des espèces de Galagete et des différentes populations insulaires. Elles ont fourni de précieuses informations concernant l'histoire évolutive et la chronologie de la colonisation des Galapagos par cette radiation endémique insulaire et identifier, au sein de la radiation, un cas de spéciation cryptique.

Reference

SCHMITZ, Patrick. The phylogeny and biogeography of an insular endemic moth radiation : the genus "Galagete" (Lepidoptera: Autostichidae) from the Galapagos Islands. Thèse de doctorat : Univ. Genève, 2007, no. Sc. 3855

URN : urn:nbn:ch:unige-4793 DOI : 10.13097/archive-ouverte/unige:479

Available at: http://archive-ouverte.unige.ch/unige:479

Disclaimer: layout of this document may differ from the published version.

1 / 1 UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Département de zoologie et biologie animale Professeur Jan Pawlowski

Département d'entomologie MUSÉUM D'HISTOIRE NATURELLE DE GENÈVE Docteur Bernard Landry

The Phylogeny and Biogeography

of an Insular Endemic Moth Radiation:

The Genus Galagete (Lepidoptera: Autostichidae)

from the Galapagos Islands

THÈSE

présentée à la Faculté des Sciences de l'Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologique

par Patrick SCHMITZ de Genève (Suisse)

Thèse N° 3855

GENÈVE Atelier de reproduction de la Section de physique 2007

ii

“Seeing every height crowned with its crater, and the boundaries of most of the lava- streams still distinct, we are led to believe that within a period, geologically recent, the unbroken ocean was here spread out. Hence, both in space and time, we seem to be brought somewhat near to that great fact — that mystery of mysteries — the first appearance of new beings on this earth.”

Charles Darwin (The Voyage of the Beagle, 1845)

iii iv TABLE OF CONTENTS

REMERCIEMENTS ...... viii RÉSUMÉ ...... ix ABSTRACT ...... xix

GENERAL INTRODUCTION ...... 1

The Galapagos Islands...... 2 The genus Galagete Landry, 2002 ...... 2 Aims of the present study...... 3

CHAPTER 1: TWO NEW TAXA OF GALAGETE (LEPIDOPTERA, AUTOSTICHIDAE) FROM THE GALAPAGOS ISLANDS, ECUADOR ...... 5

ABSTRACT ...... 6

1. INTRODUCTION ...... 7

2. MATERIAL AND METHODS ...... 7

3. RESULTS...... 8

4. DESCRIPTIONS...... 9

5. ACKNOWLEDGEMENTS...... 14

CHAPTER 2: IMMATURE STAGES OF GALAGETE PROTOZONA (LEPIDOPTERA: GELECHIOIDEA: AUTOSTICHIDAE) FROM THE GALAPAGOS ISLANDS: DESCRIPTION AND NOTES ON BIOLOGY ...... 15

ABSTRACT ...... 16

1. INTRODUCTION ...... 17

2. MATERIAL AND METHODS ...... 17

3. DESCRIPTION OF THE LARVA AND PUPA...... 19

3.1. Larva and pupa ...... 19 3.2. Biology ...... 20 3.3. Relationships ...... 23 3.4. Notes...... 23

4. ACKNOWLEDGEMENTS...... 24

v CHAPTER 3: TWO NEW SPECIES OF CHIONODES HÜBNER FROM ECUADOR, WITH A SUMMARY OF KNOWN GALAPAGOS RECORDS OF GELECHIIDAE (LEPIDOPTERA)...... 25

ABSTRACT ...... 26

1. INTRODUCTION ...... 27

2. MATERIAL AND METHODS ...... 28

3. DESCRIPTIONS...... 29

4. ACKNOWLEDGEMENTS...... 36

CHAPTER 4: MOLECULAR PHYLOGENY AND DATING OF AN INSULAR ENDEMIC MOTH RADIATION INFERRED FROM MITOCHONDRIAL AND NUCLEAR GENES: THE GENUS GALAGETE (LEPIDOPTERA: AUTOSTICHIDAE) OF THE GALAPAGOS ISLANDS...... 37

ABSTRACT ...... 38

1. INTRODUCTION ...... 39

2. MATERIAL AND METHODS ...... 41

2.1. Specimen sampling...... 41 2.2. DNA extraction, PCR amplification, and sequencing...... 42 2.3. Phylogenetic analysis ...... 44 2.4. Molecular dating...... 45

3. RESULTS...... 46

3.1. Sequence data ...... 46 3.2. Phylogenetic analyses...... 47 3.3. Molecular dating...... 47

4. DISCUSSION ...... 49

4.1. Outgroups ...... 49 4.2. Galagete phylogenetic relationships ...... 49 4.3. Origin, dating, and biogeography...... 52

5. ACKNOWLEDGEMENTS...... 54

vi CHAPTER 5: EVIDENCE FOR CRYPTIC SPECIATION ON GALAPAGOS VOLCANOES IN THE MICROMOTH GALAGETE DARWINI (LEPIDOPTERA, AUTOSTICHIDAE)...... 57

ABSTRACT ...... 58

1. INTRODUCTION ...... 59

2. MATERIAL AND METHODS ...... 59

2.1. Samples and DNA methods ...... 59 2.2. Data analysis...... 60

3. DISCUSSION ...... 61

4. ACKNOWLEDGEMENTS...... 62

GENERAL CONCLUSIONS AND PERSPECTIVES ...... 65

LITERATURE CITED...... 67

APPENDIX ...... 81

vii REMERCIEMENTS

Je tiens ici à exprimer ma reconnaissance aux nombreuses personnes qui m’ont apporté leur aide, leur soutien, leurs compétences et leur amitié tout au long de cette étude. Je voudrais remercier tout particulièrement Bernard Landry qui durant toutes ces années m'a supervisé et fait confiance pour mener à bien ce travail. Durant cette période, il m'a transmis sa passion des lépidoptères à travers la préparation ardue de spécimens, l'art minutieux de la dissection de génitalia, et les heures passées ensemble les yeux rivés à un piège lumineux que se soit au sommet des volcans aux Galapagos, dans les vallées du Tirol du Sud, ou dans les marécages de Floride. Je remercie également Jan Pawlowski pour l'accueil chaleureux dans son laboratoire dès les premiers balbutiements du projet jusqu'à sa consécration, ainsi que pour son soutien inconditionnel dans mes nombreuses démarches administratives. Je suis très reconnaissant au professeur Louisette Zaninetti et au professeur Volker Mahnert pour m'avoir offert la possibilité de débuter cette thèse. Je tiens à remercier de tout coeur Lazaro Roque pour son hospitalité insulaire, son appui logistique, et sa compagnie sur le terrain. Un grand merci à Jackie Guiard qui m'a guidé à travers les protocoles de laboratoire et José Fahrni pour son expérience au profit de l'obtention des séquences nécessaires à ce travail. Je remercie aussi vivement Alice Cibois pour son aide avisée et ses nombreux conseils judicieux. Merci à Xav et Ben pour être ce qu'ils sont, Pat et Luca parce qu'ils sont sur le même étage, Fabien, Fred, JP, Juan, Delphine, Luca, Lydia, Joëlle, Délia, Béatrice, Yurika, Cédric, et David, pour les thérapies de groupe en systématique moléculaire indispensables à toute entente scientifique à long terme, Fabrice, Raphaël, Nicole, Alex, Pedro, Lionel, Giulio, Bernhard, Ivan, Aline, Corinne, Janine, Roselyne, Marie-Christine, Laurent, Manu, Michel, Janik, Andy, Giancarlo, Florence, André, Jean Wuest et Henri Dirickx, pour m'avoir supporté pendant toutes ces années au Muséum. Une belle pensée à tous les collègues et amis rencontrés au hasard aux quatre coins du monde autour d'un café, d'une bière, d'un encocado, d'une lampe UV, ou d'un simple réchaud à gaz, en particulier, Ana Maria Ortega, Alejandro Mieles, Henri Herrera, Valentina Cruz, Novarino Castillo, Christine Parent, Sarah Garrett, Bill Conner, Jill Key, Felix Sperling, Dan Rubinoff, Jean-François Landry, Matthias Nuss, Andras Kun, Béatrice et Jean-Thomas Bujard, pour les bons moments partagés ensemble. Je tiens également à remercier le Département des affaires culturelles de la Ville de Genève, la Baslerstiftung für biologische Forschung, l'Académie Suisse des Sciences Naturelles, la Société Académique de Genève, ainsi que les Fondations G. & A. Claraz, A. Lombard, et E. & L. Schmidheiny, pour leurs soutiens financiers généreux sans lesquels ce projet n'aurait été possible. Ces remerciements ne seraient pas complets, si je ne mentionnais pas ma famille et mes amis proches qui m’ont soutenu sans faille au cours de toutes ces années d’études et de recherches. A ma femme, Stefania, qui m'a accompagné, soutenu et encouragé durant ma thèse, et à mon fils, Elia, à qui j'ai souvent changé les couches durant la rédaction, je leur dédie ce travail.

viii RÉSUMÉ La phylogénie et la biogéographie d'une radiation endémique insulaire : le genre Galagete (Lepidoptera : Autostichidae) des Galapagos.

Introduction

Les archipels océaniques d’origine volcanique tels que les îles Galapagos sont souvent considérés comme des laboratoires naturels de l'évolution (Whittaker, 2002) du fait qu'ils offrent un environnement fertile pour l’étude du procédé de spéciation. Ceci pour six raisons différentes : (i) ce sont des entités géographiques restreintes délimitées par des barrières océaniques importantes ; (ii) le flux génétique entre les îles isolées est fortement réduit par les barrières océaniques qui les séparent ; (iii) la petite taille des îles permet de cataloguer plus facilement que sur les continents la flore et la faune ; (iv) bien que les îles soient de petite taille, elles hébergent un grand nombre d’habitats différents, et ; (v) ces archipels sont souvent géologiquement dynamiques avec une activité volcanique et érosive connue d’un point de vue historique et contemporain (Emerson, 2002). La présence de nombreuses îles, leur isolement et leur histoire géologique ont engendré un niveau élevé d’endémisme et ont fait des Galapagos un microcosme du processus évolutif. Ces îles possèdent encore de nos jours une mosaïque d’habitats quasiment intacts où la perturbation des écosystèmes par l’homme est restée faible, contrairement à d’autres archipels. Elles ont donc conservé leur caractère unique de zone d’endémisme avec une histoire évolutive et géologique complexe, une situation propice pour l’étude des mécanismes qui ont conduit à cette diversité biologique.

L'archipel des Galapagos

Découvert officiellement en 1535 par le cardinal de Panama et visité brièvement par Charles Darwin en 1835, l'archipel des Galapagos est traversé par l'équateur et se situe dans l'océan Pacifique à une distance d'environ 1000 km du continent sud-américain. Il se compose de 127 îles, dont 19 possèdent une superficie de plus de 1 km2. Ce sont des îles océaniques d'origine volcanique qui ont pris naissance au-dessus d'un point chaud fixe situé à l’emplacement actuel de l’île de Fernandina où le magma traverse la croûte terrestre pour

ix former d'imposants volcans-boucliers (Cox, 1983 ; White et al., 1993). Ces derniers se déplacent vers l'est sous l'effet du déplacement de la plaque de Nazca, deviennent inactifs, puis s'érodent, pour finalement se retrouver immergés sous les flots. Bien que les îles actuelles soient relativement récentes (environ 4 millions d'années), des vestiges d'îles submergées ont été découverts au large des Galapagos à l'est de l'île de San Cristobal et datés d’environ 9 millions d'années (Christie et al., 1992), ce qui prolonge considérablement le temps d'évolution disponible à la formation de nouvelles espèces chez les nombreux organismes ayant colonisés l'archipel. Le climat aride, atypique pour des îles océaniques situées dans la zone equatoriale, est influencé en grande partie par le courant marin froid de Humboldt. La zone aride qui recouvre la majorité des îlots et les régions côtières des grandes îles est caractérisée par une flore xérophytique. Cette zone importante laisse place vers l'intérieur des terres à une zone de transition, puis à une zone humide située aux sommets des volcans (McMullen, 1999). L'origine de la majorité des lignées de la flore et de la faune terrestres endémiques aux Galapagos se situe sur la côte sud-américaine (Grehan, 2001).

Les Galapagos se caractérisent par un nombre restreint d'espèces compensé par un taux d'endémisme important. Si l'on exclu les taxa introduits par l'homme, ce taux s'élève chez les plantes à 29% (McMullen, 1999), et, en ce qui concerne les animaux terrestres, à 53% chez les invertébrés (Peck, 2001), à 84% chez les oiseaux, et à 100% chez les mammifères et les reptiles (Tye et al., 2002). On distingue deux types d'endémisme : le paléoendémisme et le néoendémisme (Whittaker, 2002). Le premier concerne les taxa jadis largement répandus sur la surface du globe qui ont depuis trouvé refuge sur des îles inaccessibles, telle la tortue géante terrestre. Le second est souvent le résultat d'une radiation endémique suite à la colonisation par un ancêtre commun, tels les pinsons de Darwin. Quant à la composition de la faune insulaire, elle est dite disharmonique. Effectivement, la colonisation d'archipels lointains est avant tout un évènement accidentel qui implique le mode de dispersion et les chances de survie des organismes colonisateurs, ce qui entraîne une absence notable de certains groupes taxonomiques par rapport au continent (Whittaker, 2002). Ainsi aux Galapagos, on observe une domination des reptiles et des oiseaux sur les espèces de mammifères, et une absence complète d'amphibiens et de poissons d'eaux douce (Grehan, 2001). Un autre phénomène insulaire est l'absence partielle ou complète des organes du vol. Les vestiges alaires du cormoran aptère et la perte de l'aptitude à voler chez certains cafards et grillons (Peck, 2001) en sont de bons exemples.

x Comme pour d'autres archipels d'origine volcanique, plusieurs radiations endémiques insulaires ont été rapportées et documentées. Il a été démontré que la plupart de ces radiations sont le résultat d'un seul évènement de colonisation (Grant, 1999 ; Sato et al., 2001), voir parfois deux (Kizirian et al., 2004). La plus connue est évidemment celle des pinsons de Darwin, dont on connaît actuellement 15 espèces, dont une est endémique de l'île de Coco au large du Costa Rica (Grant, 1999). La plus diversifiée est celle des escargots terrestres du genre Bulimulus, qui compte de nos jours 71 espèces répertoriées dans l'archipel (Parent & Crespi, 2006). La plus hétéroclite, est celle des plantes à fleurs du genre endémique Scalesia qui comporte parmi ses 15 espèces des formes herbeuses, buissonnantes et arborescentes (Schilling et al., 2004). Ces radiations adaptatives sont des exemples de l'évolution de la diversité écologique et phénotypique à l'intérieur d'une lignée qui se différencie rapidement (Schluter, 2000).

Le genre Galagete Landry, 2002

Les microlépidoptères du genre Galagete (Lepidoptera : Autostichidae) sont un élément distinct de la faune des papillons de nuit aux Galapagos car ils représentent la plus grande radiation endémique insulaire de Lépidoptères connue à ce jour dans l'archipel, comparable en terme de nombre d'espèces à celle des pinsons de Darwin (Landry, 2002). Ils sont également présents sur toutes les îles principales et à toutes les altitudes, de la zone côtière aride aux sommets humides des volcans. Le genre comprend 12 espèces nominales (Figs 1-12) possédant des aires de répartition propres. Certaines espèces possèdent une distribution qui englobe l'intégralité de l'archipel, alors que d'autres sont endémiques d'une seule île en particulier (Landry, 2002 ; Schmitz and Landry, 2005 ; Schmitz et al., submitted). Dans une analyse cladistique basée sur des caractères morphologiques, la monophylie du genre ainsi que celle de trois groupes d'espèces ont été mis en évidence (Landry, 2002) : un clade incluant les trois grandes espèces G. gnathodoxa, G. protozona et G. seymourensis, le couple G. espanolaensis et G. turritella, et un clade incluant G. cinerea, G. consimilis et G. darwini. Des espèces comme G. consimilis, G. darwini et G. espanolaensis sont superficiellement similaires et l'unique moyen pour les déterminer correctement consiste à disséquer leurs génitalia. Certaines variations géographiques peuvent être parfois particulièrement frappantes. Par exemple, les nuances de coloration entre les différentes populations insulaires chez G. levequei représentent un cas intéressant de gradation linéaire à

xi travers l'archipel. Dans cette étude, Taygete sphecophila (Meyrick) aussi connu des Galapagos, a été utilisé entre autre comme outgroup sur la base de caractères morphologiques partagés entre les deux genres (Landry, 2002 ; Landry et al., 2006). Galagete a été placé dans les Autostichidae, Symmocinae sensu Hodges qui inclue aussi les Autostichinae et les Holcopogoninae (Hodges, 1998). Cependant une analyse morphologique récente a démontré que les Autostichidae sont paraphylétiques (Kaila, 2004). L'écologie des Galagete, quant à elle, est relativement peu connue à part les habitudes alimentaires détritivores des larves qui sont consistantes avec celles observées chez d'autres Autostichidae (Hodges, 1998 ; Landry, 2002).

FIGURES 1-12. Adultes d'espèces de Galagete (échelle non respectée ; Landry, 2002 ; Schmitz & Landry, 2005). 1. G. protozona ; 2. G. gnathodoxa ; 3. G. seymourensis ; 4. G. cinerea ; 5. G. pecki ; 6. G. turritella ; 7. G. consimilis ; 8. G. darwini ; 9. G. espanolaensis ; 10. G. levequei ; 11. G. cristobalensis ; 12. G. griseonana.

xii Plan et objectifs de cette étude

Dans ce projet de recherche qui propose une approche multidisciplinaire (morphologique, écologique et moléculaire) d’une radiation endémique insulaire, nous avons mené une étude phylogénétique des microlépidoptères nocturnes du genre Galagete afin de répondre à plusieurs questions en rapport avec les modèles de colonisation et les procédés de spéciation. Dans ce but, nous avons effectué un échantillonnage intensif sur les différentes îles de l'archipel des Galapagos afin d'obtenir du matériel pour complémenter l’inventaire et les données sur les aires de répartition des microlépidoptères du genre Galagete et décrire les taxa manquants (Schmitz & Landry, 2005).

Durant ces missions scientifiques nous avons apporté de nouvelles données écologiques et documenté les stades immatures pour l'une des espèces, G. protozona, qui étaient jusqu'alors inconnus pour le genre (Schmitz & Landry, 2007). L'une de ces missions nous a mené à prospecter sur la côte équatorienne située à plus de 1000 km des Galapagos à la recherche de possibles Galagete continentaux ou d'espèces proches. Deux nouvelles espèces du genre Chionodes appartenant à la superfamille des Gelechioidea, dont fait également partie le genre Galagete, ont été ainsi décrites (Schmitz & Landry, in press) afin de pouvoir inclure l'une d'elles en tant que outgroup pour enraciner nos arbres phylogénétiques.

Les spécimens récoltés et conservés en alcool ont été utilisés dans la reconstruction d'une solide phylogénie incluant toutes les espèces de Galagete et la plupart des différentes populations insulaires à l’aide de plusieurs gènes (2 gènes mitochondriaux : COI et COII, ainsi que 2 gènes nucléaires : EF-1α et Wingless). Nous avons appliqué à cette reconstruction phylogénétique une nouvelle méthode d’horloge moléculaire relaxée, calibrée grâce aux datations géologiques fournies pour les différentes îles (Schmitz et al., submitted). Enfin, nous avons mis en évidence un phénomène particulier à l'intérieur du clade de G. darwini suggérant une spéciation cryptique entre deux lignées distinctes (Schmitz & Landry, in prep.).

Ce travail est organisé en plusieurs chapitres de façon à répondre lors de notre recherche aux investigations successives mentionnées ci-dessus. Chacun de ces chapitres fait l'objet d'une publication.

xiii Chapitre 1 : Description de deux nouveaux taxa de Galagete

Pour permettre la détermination et la documentation des différentes espèces des microlépidoptères du genre Galagete, la description des nouveaux taxa a été nécessaire. Dans ce chapitre, nous décrivons deux nouveaux taxa à partir de matériel sec collecté en 2004 aux îles Galapagos. Le premier, G. griseonana, est une nouvelle espèce, présumée endémique de l'île de Santa Cruz, et qui, avec une envergure d'environ 6.5 mm, représente le plus petit membre du genre. Le deuxième, G. pecki flavofasciata, a été décrit comme une nouvelle sous- espèce sur la base de l'absence de différences notables dans les génitalia, mais également d'après les variations de taille et de coloration des ailes, et de l'aire de répartition allopatrique avec la sous-espèce nominotypique. La divergence des séquences de 2.7 % obtenue pour un fragment de 1,4 kb du gène COI entre G. p. pecki et G. p. flavofasciata, qui correspond pour ce gène à une divergence intraspécifique conformément aux résultats obtenu dans d'autres études sur les Lépidoptères, nous a conforté dans notre décision.

Chapitre 2 : Description des stades immatures de Galagete protozona

La biologie des différentes espèces de Galagete est très peu connue. Plusieurs spécimens de G. darwini et de G. levequei ont été élevés à partir de feuilles mortes de Scalesia, un genre endémique de plantes à fleurs de la famille des Asteraceae, et un seul spécimen adulte de G. gnathodoxa a été élevé à partir d'excréments de tortue géante des Galapagos. Cependant les stades immatures n'ont jamais été préservés ou étudiés. Lors de notre expédition aux Galapagos en 2005, nous avons eu l'occasion de récolter du matériel en alcool pour l'une des espèces de Galagete. Dans ce chapitre, la morphologie de la larve ainsi que de la chrysalide de G. protozona, endémique des îles Galapagos, est décrite et illustrée. Les immatures ont été observés se nourrissant dans les excréments d'iguane terrestre sur l’île de Fernandina.

Chapitre 3 : Description de deux nouvelles espèces de Chionodes

Les Gelechiidae représentent avec plus de 4500 espèces l'une des plus grandes familles de Lépidoptères. Ils appartiennent à la superfamille des Gelechioidea dont fait également partie le genre Galagete. Dans le but d'utiliser des espèces de microlépidoptères comme

xiv outgroup pour enraciner les arbres phylogénétiques obtenus pour la radiation, une nouvelle espèce de Chionodes, présumée endémique des îles Galapagos, a été décrite. De plus, suite à notre expédition aux Galapagos en 2006, nous avons prospecté et collecté de nombreux spécimens de papillons de nuit dans le parc national de Machalilla situé sur la côte équatorienne à environ 1000 km de l'archipel et qui possède un climat et une végétation similaires à ceux des Galapagos. En y cherchant des taxa possiblement apparentés au genre Galagete, nous avons découvert une autre espèce de Chionodes d'apparence identique à celle des Galapagos, bien que très différente au niveau des génitalia. Dans ce chapitre, ces deux nouvelles espèces de Chionodes sont décrites en détail.

Chapitre 4 : Phylogénie moléculaire et datation d'une radiation endémique insulaire

Afin de mieux comprendre la diversification de cette radiation endémique insulaire, de démêler les relations entre les différentes espèces et populations insulaires de Galagete, et de donner un aperçu des premiers stades de spéciation, nous avons effectué avec les spécimens en alcool collectés lors des trois expéditions aux Galapagos une reconstruction phylogénétique basée sur la concaténation des fragments de quatre gènes (2 gènes mitochondriaux : COI, COII, ainsi que 2 gènes nucléaires : EF-1α et Wingless). Nos résultats confirment la paraphylie des Autostichidae sensu Hodges ainsi que la parenté proche du genre Galagete avec Taygete sphecophila. La monophylie du genre est supportée et suggère un seul évènement de colonisation de l'archipel. La position basale de G. cinerea et G. consimilis suggère une espèce ancestrale possédant des analogies phénotypiques avec celles-ci. Deux cas de paraphylie ont été observés entre espèces. G. espanolaensis qui se retrouve à l'intérieur du clade de G. turritella représente probablement un cas de limite interspécifique imparfaite ou de variation géographique. Quant à la population de G. protozona de l'île de Santa Fe inclue dans le clade de G. gnathodoxa, il pourrait s'agir soit d'hybridation introgressive entre ces deux espèces proches, soit d'une rétention de polymorphisme ancestral.

L’étude de l’histoire évolutive du genre Galagete est très limitée du fait qu’il n’existe pas de trace de ces microlépidoptères dans le bilan fossile. Grâce à une nouvelle méthode d’horloge moléculaire relaxée et calibrée avec les données géologiques sur l’émergence des îles, nous avons proposé une séquence chronologique de la radiation insulaire afin d’estimer

xv l’origine de la divergence du genre ainsi que l’apparition des différents clades. Ceci nous a permis d'estimer le premier évènement de spéciation à l'intérieur du genre Galagete, qui s'est déroulé sur l'archipel il y a 3.3 ± 0.4 million d'années. Étant donné que l’arbre moléculaire ne tient compte que des espèces existantes à l’heure actuelle, ceci doit être considéré comme un âge minimum pour le genre Galagete. En comparaison, la radiation des pinsons de Darwin a été estimée aux alentours de 2 à 3 millions d’années. Le genre Galagete s'est diversifié relativement rapidement en à peu près 1.8 million d'années, ce qui donne une estimation du taux de spéciation de 0.8 espèces par million d'années. Ce scénario tient compte du fait que les opportunités pour une diversification allopatrique au départ étaient limitées étant donné le nombre restreint d’îles. Mais, par la suite, parallèlement à l’augmentation du nombre de celles-ci dans l’archipel, la radiation a subitement accéléré. Bien que le scénario de colonisation suggère un modèle de dispersion stochastique, l'arrivée de l'ancêtre commun et la diversification de la radiation coïncident avec l'émergence chronologique des îles principales.

Chapitre 5 : Spéciation cryptique au sommet des volcans des Galapagos

On rencontre sur les îles océaniques d'origine volcanique une grande diversité d'habitats. L'effet de l'isolement géographique couplé à un gradient d'humidité marqué en fonction de la zone altitudinale résulte en une compétition moindre et de nombreuses niches écologiques vides. Aux Galapagos, la plus grande diversité et le plus grand taux d'endémisme chez les insectes sont observés sur la côte aride, la plus vaste zone écologique de l'archipel. Cette zone est prédominante sur les petites îles, alors que sur les grandes îles, elle s'élève jusqu'à une altitude entre 120 à 300 m, voire plus haut, puis est remplacée graduellement par une zone humide. Dans ce chapitre, nous présentons et discutons des résultats surprenants obtenus pour le clade formé par les différentes populations de G. darwini, l'espèce de Galagete la plus répandue dans l'archipel. En effet, notre analyse phylogénétique de ce clade révèle que les populations de spécimens de G. darwini collectés au sommet des volcans des îles principales situées à l'ouest représentent une lignée distincte des populations rencontrées dans la zone côtière aride de ces mêmes îles. La présence d'au moins deux taxa cryptiques suggère que le processus d'isolation géographique est corrélé selon l'altitude, et dans une moindre mesure, selon la zone de végétation. La récente spéciation a probablement été initiée sur l'île de Floreana et de Santa Cruz par isolation allopatrique, puis par isolation marginale sur l'île de Santa Cruz dans les habitats humides situés à haute altitude par un ancêtre

xvi provenant de la zone aride. Un contact secondaire entre les lignées résultantes subdivisées selon l'altitude s'est ensuite produit sur les îles occidentales. Nos résultats mettent en évidence un mode de spéciation en fonction de l'altitude. Nos données supportent ainsi l'idée que ces microlépidoptères sont des organismes au mode de dispersion élevé dont la spéciation a été promue plutôt par des mécanismes reproductifs et par la préférence d'habitat que par des barrières géographiques à la dispersion, qui sont généralement acceptées comme jouant un rôle clé dans la formation et le maintien des espèces.

Conclusions et perspectives

Nos investigations sur la phylogénie et la biogéographie d'une radiation endémique insulaire de microlépidoptères nous ont permis d'apporter de nombreuses réponses et d'élargir considérablement nos connaissances sur la diversité, la répartition, l'écologie, l'histoire évolutive et les phénomènes de spéciation du genre Galagete. Premièrement, nos études morphologiques nous ont amené à décrire plusieurs nouveaux taxa utilisés par la suite dans nos reconstructions phylogénétiques (Chapitre 1 et 3). Deuxièmement, la biologie pour l'une des espèces de Galagete a été dévoilée et les stades immatures décrits en détail (Chapitre 2). Troisièmement, nos analyses moléculaires et investigations biogéographiques ont permis d'obtenir une solide phylogénie des espèces de Galagete et des différentes populations insulaires. Elles ont fourni de précieuses informations concernant l'histoire évolutive et la chronologie de la colonisation des Galapagos par cette radiation endémique (Chapitre 4). De plus, nos résultats ont identifié au sein de la radiation un cas de spéciation cryptique chez G. darwini dont deux lignées distinctes sont subdivisées en fonction de l'altitude sur les îles situées à l'ouest de l'archipel (Chapitre 5).

Néanmoins, les aspects suivants permettraient d'étendre ces investigations et d'ouvrir de nouvelles voies de recherche dans notre compréhension de cette radiation endémique insulaire :

1) Ajouter aux données phylogénétiques l'information provenant de microsatellites pour affiner les relations entre espèces et populations, et ainsi peut-être obtenir une meilleure résolution au sein de la radiation.

xvii 2) Complémenter et intensifier l'effort d'échantillonnage sur les côtes d'Amérique du Sud dont la richesse des microlépidoptères est actuellement encore largement sous- estimée. La recherche de Galagete continentaux ou d'espèces proches permettrait peut-être de trouver des candidats potentiels au statut d'espèce ancestrale.

3) Étudier l'écologie des différentes espèces de Galagete afin d'élucider les causes environnementales de la spéciation et d'apporter une confirmation à la nature adaptative de la radiation, ainsi qu'une meilleure résolution dans l'analyse cladistique des relations interspécifiques.

4) Planifier la conservation des espèces de microlépidoptères du genre Galagete grâce aux données écologiques, de répartition, et d’endémisme recueillies.

xviii ABSTRACT

Remote volcanic island archipelagos make incomparable natural laboratories for the study of patterns of diversification and species formation. The endemic autostichid moth genus Galagete from the Galapagos Islands, which comprises 12 nominate species, was used as a model in order to better understand the diversification of an endemic insular radiation, to unravel relationships among species and populations, and to get insight into the early stages of speciation. During this research project, we have produced a number of morphological and molecular studies to show that (1) the monophyly of the genus is strongly supported, thus suggesting a single colonization event, (2) the colonization scenario is following a stochastic dispersal pattern in the archipelago, (3) the first split occurring within the Galagete lineage on the archipelago is estimated at about 3.3 million years ago and the genus radiated relatively quickly in about 1.8 million years, (4) the arrival of the ancestor and the diversification of the radiation coincide with the chronological emergence of the major islands, (5) two cases of paraphyly between species are hypothesized to represent imperfect species limits, introgressive hybridization or incomplete lineage sorting, and (6) an event of cryptic speciation in one of the species occurred between two distinct populations that are subdivided along an elevational gradient on the western Galapagos volcanoes.

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xx

GENERAL INTRODUCTION

1 GENERAL INTRODUCTION

The Galapagos Islands Remote volcanic island archipelagos often considered as living laboratories enable the study of the process of speciation (Whittaker, 2002). Such island systems are attractive environments for studying evolution for a number of reasons: (i) they present discrete geographical entities within defined oceanic boundaries; (ii) gene flow between individual islands is reduced by oceanic barriers; (iii) their often small geographical size has made the cataloguing of flora and fauna easier than in continental systems; (iv) despite their small geographical size they can contain a diversity of habitats and; (v) they are often geologically dynamic with historical and contemporary volcanic and erosional activity. In combination the above factors have manifested themselves in typically high levels of endemism within oceanic islands, presenting a microcosm of the evolutionary process (Emerson, 2002). In addition, the Galapagos Islands are the world’s most pristine, best preserved and protected, tropical oceanic island ecosystem. In the archipelago there is a decrease in the age of the islands from west to east which is attributed to the activity of a mantle hotspot (Christie et al., 1992; White et al., 1993) present beneath the youngest island of Fernandina where magma extrudes through the crust to build huge shield volcanoes. The hotspot model postulates that each volcano first emerged where Fernandina is today, and has since been carried to its present position by the motion of the Nazca plate, San Cristobal and Espanola being the oldest islands. Although the emerged islands are relatively young (Cox, 1983; White et al., 1993), evidence of drowned seamounts east of San Cristobal about 9 Myr old (Christie et al., 1992), which might have been islands, extend the temporal window available for evolution in the Galapagos.

The genus Galagete Landry, 2002 The recently described endemic genus Galagete (Lepidoptera: Autostichidae) is a group of small moths that represents the largest endemic radiation of Lepidoptera so far found in the Galapagos Islands comparable in size to that of Darwin's finches (Landry, 2002). The genus comprises 12 nominate species with variable distributions encompassing the whole archipelago for some species to single-island endemics (Landry, 2002; Schmitz and Landry, 2005; Schmitz et al., submitted). In a cladistic analysis based on morphological characters (Landry, 2002), the monophyly of Galagete and that of three species groups were recovered: a clade formed by the three larger species G. gnathodoxa, G. protozona, and G. seymourensis, G. espanolaensis and G. turritella pair, and a clade composed of G. cinerea, G. consimilis, and G. darwini. Species like G. consimilis, G. darwini, and G. espanolaensis are superficially

2 GENERAL INTRODUCTION so similar that they can be identified reliably from their genitalia only (Landry, 2002). Galagete was placed by Landry (2002) in the Autostichidae, Symmocinae sensu Hodges (1998), which also includes the Autostichinae and Holcopogoninae. However Kaila's (2004) morphological analysis showed that the Autostichidae sensu Hodges (1998) are paraphyletic with regard to the Glyphidoceridae and Lecithoceridae placed in an Autostichid assemblage. Little is known about the ecology of Galagete apart from their scavenging feeding habits which are consistent with those of other Autostichidae (Hodges, 1998).

Aims of the present study During this research project which takes part in a multidisciplinary approach, we undertook a thorough molecular investigation on the species of the microlepidoptera genus Galagete in order to get insights in the early stage of speciation of this endemic insular moth radiation and to elucidate particular sequences of dispersal and colonization among the Galapagos Islands. In this perspective, we collected all of the 12 described species of the genus Galagete and sampled nearly all known populations on 13 islands during three expeditions in the Galapagos archipelago from 2004 to 2006. We first described the remaining missing taxa of the genus Galagete to allow suitable determination and distribution of the various species. Second, we documented the feeding habits and illustrated the morphology of the immature stages of G. protozona endemic to the Galapagos. Third, we described two new species belonging to the genus Chionodes to use one of these in the outgroup in order to root our phylogenetic trees obtained for the radiation. Fourth, we investigated the molecular phylogeny and the divergence timing in the Galagete radiation by applying phylogenetic analyses on the combined dataset composed of fragments of mitochondrial and nuclear genes. We proposed a biogeographic hypothesis for the colonization scenario of the Galapagos Islands by Galagete. Fifth, we finally revealed the mechanism of cryptic speciation observed between two distinct populations of G. darwini. The five studies mentioned above are detailed in respective order in the next chapters.

3

4

CHAPTER 1

TWO NEW TAXA OF GALAGETE (LEPIDOPTERA, AUTOSTICHIDAE) FROM THE GALAPAGOS ISLANDS, ECUADOR.

P. SCHMITZ & B. LANDRY

Muséum d’histoire naturelle, C. P. 6434, CH-1211 Geneva 6, Switzerland

Published in: Revue suisse de Zoologie, 112: 511–517, 2005

5 CHAPTER 1

ABSTRACT

A new species and a new subspecies of the genus Galagete (Lepidoptera, Autostichidae) from the Galapagos Islands are described and illustrated. Galagete griseonana sp. n., known only from males, is endemic to the island of Santa Cruz. Galagete pecki flavofasciata ssp. n., known from both sexes, occurs on the islands of Santa Cruz and Santiago, where it is also believed to be endemic.

6 CHAPTER 1

1. INTRODUCTION

Autostichidae are gelechioid moths characterised by the gnathos being an articulated band with an unarticulated mesial hook, and by the presence of spiniform setae in a band across the abdominal terga. The caterpillars feed on dead or decaying plant or animal tissue (Hodges 1998). With a total of 13 species (one of which is not described), the genus Galagete represents the first documented case of an extensive radiation of endemic Lepidoptera in the Galapagos Islands comparable in size to the radiation of the Darwin’s finches with some species confined to one single island (Landry 2002). The second largest radiation in Galapagos Lepidoptera found so far contains only three species (genus Utetheisa, Arctiidae). For the purpose of enabling recognition and documentation of the elements of the Galagete radiation, and because we were able to obtain sufficient material in early 2004, we are describing below a new species of Galagete and a new subspecies of Galagete pecki Landry.

2. MATERIAL AND METHODS

The 51 specimens forming the basis of this study were collected by ourselves mostly during one expedition to the Galapagos in March-April 2004. Three of the specimens were collected by the second author in 1989 and 1992. The moths were collected at light, either with an ultra-violet light or a mercury vapour light suspended next to a white sheet. In listing the label data of the holotypes, the information is copied as found on the labels with slashes to express changes of lines, and abbreviations spelled out in square brackets, except “m” for “meters.” As regard the lists of paratypes, the specimens' data are listed first in alphabetical order of island collected and then in order of dates collected, the information is recorded without indications of line changes, the abbreviations, except for distances, "GPS" (= Global Positioning System), and cardinal points, are spelled out only once at first encounter, collecting localities are reported without accented letters, dates are standardised, and collectors' information is standardised and placed in parentheses. For each species' holotype the data label is printed in black on white card stock while the holotype label is hand-written in black ink on red card stock. Other than the above mentioned, the following acronyms are used: BMNH for The Natural History Museum (London, England), CNC for Canadian National Collection of Insects, Arachnids, and Nematodes (Ottawa, Ontario,

7 CHAPTER 1

Canada), CDRS for Charles Darwin Research Station (Santa Cruz Island, Galapagos), MHNG for "Muséum d'histoire naturelle de Genève" (Geneva, Switzerland), and USNM for National Museum of Natural History (Washington, D. C., USA). Genitalia were dissected after the abdomen had macerated in a cold 20% KOH solution overnight. The dissected parts were kept in lactic acid stained with orange G for description purposes. They were subsequently stained with chlorazol black and mounted on slides in Euparal. The forewing lengths were measured with a reticule on a stereomicroscope. The illustrations of the moths and genitalia were made with the AutoMontage® system using a JVC® video camera mounted on a Leica MZ APO stereomicroscope or a Zeiss Axioskop compound microscope. Illustrations of all Galagete species can be viewed on the web site of the MHNG at www.geneva-city.ch/musinfo/mhng/.

3. RESULTS

Our expedition to the Galapagos in 2004 brought only one new island record for previously described Galagete species: G. turritella is also present on San Cristobal Island. Contrary to what is recorded in Landry (2002, p. 848) the holotype of Galagete consimilis Landry is not dissected and in the Diagnosis for G. consimilis (same page of Landry 2002), the 3rd line should read “G. darwini” instead of “G. consimilis”. One of the paratype females of G. seymourensis was wrongly associated with slide number BL 1344; the correct slide number was BL 1343.

FIGURES 1-2. Holotypes of Galagete species. 1. G. griseonana; 2. G. pecki flavofasciata.

8 CHAPTER 1

4. DESCRIPTIONS

Galagete griseonana P. Schmitz & B. Landry sp. n. Fig. 1, 3-5

Galagete sp.; Landry 2002: 819, 820, fig. 15.

MATERIAL EXAMINED: Holotype ♂, [1] “ECU[ADOR], Galápagos, Santa Cruz/ C[harles] D[arwin] R[esearch] S[tation]/ base of El Barranco/ GPS: S 00°44.305’ W 90°18.105’/ 18.iii.2004, u[ltra] v[iolet] l[ight]/ leg[it]. B[ernard]. Landry, P[atrick]. Schmitz”. [2] ”HOLOTYPE/ Galagete/ griseonana/ Schmitz & Landry”. Specimen in perfect condition deposited in the MHNG. Paratypes, Ecuador: 30 ♂, from the island of Santa Cruz, Galápagos Islands, collected at UVL by B. Landry and P. Schmitz, unless specified otherwise. 1 ♂ (dissected, slide MHNG 2703), CDRS, arid zone, 3.ii.1989, M[ercury] V[apour] L[amp] (B. Landry); 2 ♂, with same data as holotype; 7 ♂ (one dissected, slide BL 1596), CDRS wall of Invert[ebrate]s. Lab[oratory]., GPS: S 00°44.478’ W 90°18.132’, 19.iii.2004; 20 ♂ (one dissected, slide BL 1595), same locality but 6.iv.2004. Deposited in the BMNH, CNC, CDRS, MHNG, and USNM.

DIAGNOSIS: Among the species of Galagete, G. griseonana can be distinguished by its very small size (wingspan: 5.7-7.1 mm) and by its forewing appearing uniformly grey. This combination of size and colour is unique. Some specimens of Galagete darwini (wingspan: 7.0-9.0 mm) and Galagete pecki (wingspan: 7.5-8.7 mm) are only slightly larger, but their background colour is brown or beige respectively with distinct markings (see Landry 2002, figs 7, 8, and 13). Galagete cinerea is also a grey species, but it is much larger (wingspan: 8.5-11.2 mm) and has darker markings (see Landry 2002, fig. 14).

DESCRIPTION: MALE (n=31) (fig. 1): Head grey with whitish beige scales around eye. Haustellum and maxillary palpus whitish beige. Labial palpus grey on first segment; second segment whitish beige medially and dorsally, laterally grey except for apical whitish beige ring laterally and ventrally; third segment grey ventrally, whitish beige dorsally. Antennal scape with few whitish beige scales apically; flagellum grey. Thorax dorsally grey except for shining whitish grey metascutellum. Foreleg coxa grey at base, whitish beige apically; femur, tibia, and tarsomeres mostly grey with whitish beige at apex of tibia and apex of tarsomere I and V. Midleg femur whitish beige with grey on dorsal edge apically; tibia pale grey with

9 CHAPTER 1 whitish beige apically and on spurs; tarsomeres I-V mostly whitish beige with grey at base of each segment. Hindleg whitish beige. Male wingspan: 5.7-7.1 mm (Holotype: 6.8 mm). Forewing grey, with sometimes slightly darker grey markings as small spots submedially and in cubital fold and postmedially at the end of cell; fringe grey. Hindwing greyish white, fringe pale whitish beige. Abdomen whitish beige, without modified scales. Male genitalia (n=3) (figs 3-5). Basal half of uncus only slightly angled from second half; second half not produced dorsally, apical margin only slightly concave; arms slightly laterally compressed, triangular, short, apically rounded; dorsal crests broadly rounded, poorly demarcated. Median hook of gnathos rather short and thick, very slightly upturned and pointed apically. Dorsal connection of tegumen wide; pedunculi short and broad. Lateral arms of transtilla moderately long, broad, rounded, median surface with fan-shaped scales towards base and setae mostly apically, edges setose; median arm as long as or slightly longer than lateral arms, very narrow for whole length. Valva of medium length and width, dorsal margin angled ventrally very gently before apex; ventral margin angled dorsally from about 1/3 and with subbasal notch; apex broadly rounded; costa melanised from base to about 2/3; sacculus rather wide, of medium length, apically flattened and blunt. Juxta symmetrical, somewhat heart shaped, with rather shallow and broadly rounded median notch. Vinculum bulbous, short and rounded, not projecting dorsoapically. Aedeagus narrow, slightly arched, larger at base with very short coecum penis; apical 1/3 open ventrally, dorsal wall slightly bent to right, narrowly rounded; vesica with abundant spicules, without cornuti. FEMALE: Unknown.

ETYMOLOGY: From the Latin griseus, grey, and nanus, a dwarf, referring to the colour of the forewing and the size of this species.

BIOLOGY: Unknown although moths are attracted to light in the arid zone between February and April.

DISTRIBUTION: Currently known only from the Galapagos island of Santa Cruz; presumed to be endemic to the archipelago and possibly to Santa Cruz.

REMARKS: This species had been recognised as new by Landry (2002) but was not described then because only one damaged specimen was available.

10 CHAPTER 1

3

4

5

FIGURES 3-5. Male genitalia of Galagete griseonana. 3. Whole genitalia without aedeagus (slide BL 1595); 4. Aedeagus in dorsal view (slide BL 1595); 5. Aedeagus in lateral view (slide BL 1596).

Galagete pecki flavofasciata P. Schmitz & B. Landry ssp. n. Fig. 2

Galagete sp.; Landry 2002: 866, fig. 36 E, F.

MATERIAL EXAMINED: Holotype ♂, [1] “ECU[ADOR], Galápagos, Santa Cruz/ C[harles] D[arwin] R[esearch] S[tation], wall of Invert[ebrate]s. Lab[oratory]./ GPS: elev[ation]. 11 m, S 00°44./478’ W 90°18.132’ 6.iv.2004/ u[ltra] v[iolet] l[ight]/ leg[it]. B[ernard]. Landry, P[atrick]. Schmitz”. [2] ”HOLOTYPE/ Galagete/ pecki flavofasciata/ Schmitz & Landry”. Specimen complete except for small notch at apex of left forewing and few segments missing on left flagellum. Deposited in the MHNG.

11 CHAPTER 1

Paratypes, Ecuador: 9 ♂, 10 ♀ from the Galapagos Islands, collected at UVL by B. Landry and P. Schmitz, unless specified otherwise. Santa Cruz: 1 ♂, 3 ♀, transition zone, recently cut road, GPS: S 00°42.528’ W 90°18.849’, 12.iii.2004; 1 ♂, 1 ♀, low agriculture zone, GPS: S 00°42.132’ W 90°19.156’, 13.iii.2004; 6 ♂ (one dissected, slide BL1600), 2 ♀ (one dissected, slide BL 1601), CDRS base of El Barranco, GPS: S 00°44.305’ W 90°18.105’, 18.iii.2004; 1 ♂, 1 ♀, CDRS wall of Inverts. Lab., GPS: elev[ation]. 11 m, S 00°44.478’ W 90°18.132’, 19.iii.2004; 1 ♀ (dissected, slide BL 1306), Finca Vilema, 2 km W [of] Bella Vista, 1.iv.1992, M[ercury] V[apour] L[amp] (B. Landry); 1 ♀, with same data as holotype. Santiago: 1 ♀, Aguacate, 520 m elev., 7.iv.1992, M[ercury] V[apour] L[amp] (B. Landry). Deposited in the BMNH, CNC, CDRS, MHNG, and USNM.

DIAGNOSIS: Among the species of Galagete, G. pecki flavofasciata can be easily distinguished by its yellowish-orange (or whitish beige) forewing markings. Only Galagete pecki pecki, G. levequei, and Galagete cristobalensis are similar in wing markings (see Landry, 2002, figs 10-13), but the background colour of their forewings is white to beige or pale greyish brown.

DESCRIPTION: MALE (n=10) (fig. 2): Head whitish beige to yellowish-orange with few thin dark brown scales along posterior eye margin. Haustellum and maxillary palpus whitish beige. Labial palpus greyish brown, shining on first segment; whitish beige on second segment sometimes with brown scales laterally at base; third segment whitish beige except for pair of usually connected blackish brown spots on second half laterally and ventrally and with small blackish brown spot ventrally at base. Antennal scape and flagellum blackish brown. Thorax dorsally blackish brown at base to 1/5-1/3, less so on tegula, whitish beige to yellowish orange in middle including most of tegula and lateroposteriorly, dark greyish brown at apex medially; metascutellum greyish brown, shining. Foreleg coxa whitish beige; femur blackish brown with whitish beige at apex; tibia blackish brown with whitish beige at apex, base, and small patch medially; tarsomere I blackish brown with whitish beige at apex; tarsomeres II-IV blackish brown. Midleg femur and tibia as in foreleg, tibial spurs whitish beige; tarsomere I, II and V blackish brown with whitish beige at apex; tarsomere III and IV blackish brown. Hindleg femur whitish beige; tibia greyish brown with whitish beige apically, spurs whitish beige; tarsomere I, II, and V with whitish beige at apex; tarsomere III and IV blackish brown. Wingspan: 6.0-6.9 mm (Holotype: 6.5 mm). Forewing blackish brown with whitish beige to yellowish-orange as small basal spot, subbasal band of variable width sometimes separated by line of blackish brown scales in cubital fold, two small spots of

12 CHAPTER 1 variable shape, sometimes connected, one on coastal margin between 2/5 and 1/2 and one slightly larger on inner margin at 1/2, and a transverse band of variable width postmedially from costa to below cell or to inner margin; fringe dark greyish brown. Hindwing and fringe greyish brown. Abdomen greyish brown, shining, without modified scales, but whitish beige on valvae. Male genitalia (n=1) (fig. 30 A-D in Landry 2002) as in Galagete pecki pecki. FEMALE. (n=10): Colour as in males but often with more extensive whitish beige to yellowish orange markings. Antenna slightly thinner than those of males. Forewing length: 6.4-8.2 mm. Female genitalia (n=2) (fig. 36 E, F in Landry 2002) as Galagete pecki pecki (fig. 32 A, B in Landry 2002). Other dissected specimen differs from that illustrated on fig. 36 E, F (Landry 2002) in having more broadly rounded lobes of sternum VIII, more broadly emarginated apical margin of tergum VIII, and more distinct scobination on membrane posterad ostium. Both dissected specimens differ from G. p. pecki in having a more simple signum, without smaller spines at base of lateral spines, and no scobination on corpus bursae.

ETYMOLOGY: From the Latin flavus, yellow, and fascia, band, referring to the colour and pattern of the forewings.

BIOLOGY: Unknown except for the fact that adults come to light and fly from March to April in habitats between near sea level to 520 m in elevation.

DISTRIBUTION: Presently known only from the islands of Santa Cruz and Santiago, the subspecies is presumed to be endemic to the Galapagos.

REMARKS: The male (6.0-6.9 mm) and female (6.4-8.2 mm) wingspans of Galagete pecki flavofasciata are smaller than those of the respective sexes (7.5-8.7 and 8.2-8.3 mm) of G. pecki pecki, which is known only from Isabela Island. These differences in wing size and those of the forewing coloration and details of female genitalia were the reasons we decided to describe as a distinct subspecies (G. pecki flavofasciata) the G. pecki specimens collected on the islands of Santa Cruz and Santiago. We were also comforted in this decision when we found that a specimen of G. pecki flavofasciata had a 2.7 % divergence in a 1.4-kb fragment of the Cytochrome Oxidase I gene with G. p. pecki (P. Schmitz, unpublished). This amount of intraspecific divergence in this gene is within the range of results obtained in other Lepidoptera studies (Landry et al. 1999).

13 CHAPTER 1

5. ACKNOWLEDGEMENTS

We are very thankful to the authorities of the Galapagos National Park and those of the Charles Darwin Research Station for allowing fieldwork and for logistical support in 1989, 1992, and 2004. We are especially grateful to Lazaro Roque-Albelo and family for their hospitality and logistical help in 2004. The comments of L. Roque-Albelo and Ole Karsholt on our manuscript were also appreciated. This work was carry out with the financial support of a “Bourse Augustin Lombard,” the MHNG, and the University of Geneva to P. Schmitz, and the MHNG to B. Landry.

14

CHAPTER 2

IMMATURE STAGES OF GALAGETE PROTOZONA (LEPIDOPTERA: GELECHIOIDEA: AUTOSTICHIDAE) FROM THE GALAPAGOS ISLANDS: DESCRIPTION AND NOTES ON BIOLOGY.

P. SCHMITZ a, b & B. LANDRY a a Muséum d’histoire naturelle, C. P. 6434, CH-1211 Geneva 6, Switzerland. b Département de Zoologie et Biologie Animale, Molecular Systematic Group, Université de Genève, 30 quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland

Published in: The Canadian Entomologist, 139: 201–208, 2007

15 CHAPTER 2

ABSTRACT

The morphology of the larva and pupa of Galagete protozona (Meyrick) (Lepidoptera: Gelechioidea: Autostichidae), an endemic of the Galapagos Islands, is described and illustrated. The immatures were observed feeding within droppings of the Land Iguana Conolophus subcristatus (Gray) (Iguanidae) on the island of Fernandina in 2005.

16 CHAPTER 2

1. INTRODUCTION

Galagete protozona (Meyrick, 1926) was described in the genus Gelechia Hübner (Lepidoptera: Gelechiidae) on the basis of three specimens from the Galapagos Islands. Landry (2002) transferred the species to Galagete in the Autostichidae and provided a redescription of the adult. The genus, with a total of 12 species, represents an endemic insular radiation comparable in size to that of Darwin’s Finches. This group of microlepidoptera is a distinctive element of the moth fauna of the Galapagos Islands because they represent the largest endemic radiation of Lepidoptera in the archipelago and are present on all major islands at almost all elevations (Landry 2002). Several specimens of G. darwini Landry (2002) and G. levequei Landry (2002) were reared from dead leaves and branches of Scalesia species, an endemic genus of the Asteraceae, and one specimen of G. gnathodoxa (Meyrick, 1926) was reared from the droppings of the endemic Galapagos Giant Tortoise Geochelone nigra (Quoy & Gaimard) (Testudinidae) (Landry 2002). However, the immature stages have not been preserved or studied. Here, the morphology of the larva and pupa, and the scavenger feeding habit of the larvae of G. protozona are described.

2. MATERIAL AND METHODS

This study is based on 67 larvae and 4 pupae of G. protozona preserved in 70% EtOH and collected at elevations of 353 to 815 meters in February 2005 on the island of Fernandina, the youngest and most active volcano in the Galapagos Archipelago. The arid slopes of the island are home to an undisturbed and large population of the endemic Galapagos Land Iguana Conolophus subcristatus (Gray) (Iguanidae). Droppings of these iguanas were found with numerous small holes in them. It was later discovered that these holes were the result of the activity of larvae of G. protozona. Many adults of G. protozona were reared from these droppings (Fig. 1) and immature stages were extracted, preserved, and studied. For the electron microscopy study, larvae were cleaned in 10% EtOH with a hairbrush and dehydrated in increasing concentrations of EtOH to absolute EtOH. After dehydration, specimens were critical-point dried using a LWU critical point dryer, mounted on stubs, and coated with gold-palladium (40-60%) using a Cressington sputter coater. Images were taken with a Zeiss DSM940A scanning electron microscope. Gross morphological observations and measurements of the larvae and pupae were made using a dissecting microscope (reflected light) with a calibrated micrometer. Maps of

17 CHAPTER 2 the larval chaetotaxy and pupae were initially drawn using a camera lucida mounted on a WILD compound microscope. Terminology for chaetotaxy follows Stehr (1987). All vouchers of Galagete protozona (larvae, pupae, and reared adult specimens) are deposited in the Natural History Museum of Geneva (MHNG), Geneva, Switzerland. In order to certify that the larvae corresponded to the adults found we sequenced a fragment of the mitochondrial gene Cytochrome oxidase I (COI). Whole genomic DNA was extracted using the Nucleospin kit (Macherey-Nagel). The COI gene was amplified by PCR with two primers: Jerry (5’-CAACATTTATTTTGATTTTTTGG-3’), and Pat2 (5’- TCCATTACATATAATCTGCCATATTAG-3’). The thermal profile started with an initial denaturation at 95°C for 5 min, followed by 35 cycles at 94°C for 30 s, 47°C for 30 s, and 72°C for 1 min 30 s, and a final step at 72°C for 10 min. The purified PCR product was sequenced in an ABI 377 automated sequencer. The sequences are available from GenBank (Accession No. EF126758-EF126759).

FIGURE 1. Adults of Galagete protozona just after emergence on droppings of Galapagos Land Iguanas Conolophus subcristatus collected on the island of Fernandina.

18 CHAPTER 2

3. DESCRIPTION

3.1. Larva and pupa Larva. (Figs. 2-12): Length 5.0-11.6 mm (n = 51), < 5.0 mm (n = 16). Body pale gray, with microconvolutions; head capsule amber; prothoracic shield amber, gradually darkening posteriorly; pinacula pale brown; anal plate pale amber; setae with widened, circular, and slightly raised sockets. Head (Figs. 2-5, 12): hypognathous, granulose (Figs. 2-3); adfrontal sclerites widened distally, AF1 and AF2 short and equal in length, AF2 above apex of frons and AF1 below; F1, C1, and C2 about equal in length, at least 4 times longer than AF-setae; F1 slightly closer to AF1 than to C1; clypeus with 6 pairs of setae, 3 pairs on medial half, 3 on lateral half; mandible (Fig. 12) with rounded outer edge, with small apical dentition, and with one large, narrow dentition on inner surface, bearing pair of subequal setae on outer surface near condyle; sensilla types and arrangement on antenna (Fig. 4) and on maxillary palp (Fig. 5) similar to those of other gelechioids (for references see Landry et al. 2006). Three stemmata in genal area, an proximate pair dorsolateral to antenna, and 1 stemma below antenna (stemmata 1, 2, and 6 absent); substemmatal setae about equal in length, arranged as in Fig. 3; S1 and S2 short, about equal in length, S3 about 2 ½ times longer than S-setae; S3 ventral to S2, S2 proximate to stemma 3, and S1 proximate to stemma 5; A-group setae above gena, mesal to L1; P1 dorsolateral to AF2, P2 dorsomesal to P1. Thorax (Figs. 6, 9): T1 with L-group trisetose, on large pinaculum extended beneath and anterior of spiracle; setae anterior to spiracle; L1 proximate and posteroventral to L2, about 2 times length of L2 and 4 times length of L3; L2 about equal distance from L1 than to L3; SV-group setae on anterior part of elongate pinaculum; SV1 about 1/3 longer than SV2; SV1 in straight line with SV2; coxae nearly touching, V1s very proximate (not shown); segments of leg smooth textured, with minute spines dorsally on tibia, claw single (Fig. 6); shield with SD1 slightly anterior to and slightly longer than XD2 and XD1; XD2 and XD1 equal in lengths and about 1/3 longer than D1; D1 and SD2 about equal in lengths; XD2 about equal distance from XD1 than to SD1; D1 in straight line with XD1, slightly posterior to SD2 and D2; D2 about as long as SD1, in straight line with SD2. T2-T3 (Fig. 9): D2 about 4 times length of D1, both on small pinaculum; SD1 almost 4 times length of SD2, both on small pinaculum; L1 about 2 times length of L2, both on small pinaculum; L3 slightly shorter than L2, posterior to or in vertical line with SV1; MV1 on anterior margin of T2 and T3, slightly above SV1 (hard to see); V1s on T2-T3 about equal distance apart, at least 4 times distance between V1s on T1 (not shown). Abdomen (Figs. 7, 8, 10, 11): A1-A2 (Fig. 10): D2 about 2 ½-3 times longer than

19 CHAPTER 2

D1, MD1 on anterior part of segment anteroventral to D1; SD1 above spiracle, about 1/3 longer than D2, with minute SD2 (anterior part of pinaculum); a small opening on ventroposterior margin of pinaculum bearing SD1 and SD2; spiracle on A1 slightly larger than those on A2-A7; L1 2 times length of L2, both on same pinaculum, aligned with or slightly posterior of spiracle; L3 about 3 times length of L2, anterior to, in vertical line with, or posterior to D2; SV-group bisetose and separate on A1, trisetose on A2, and on same pinaculum; V1s equal distance apart (not shown). A3-A10 (Figs. 7, 8, 11): A3-A6 with 4 pairs of protuberant prolegs, crochets biordinal, in circle (Fig. 7); setae as A2; A7 as A2 except SV-group bisetose and on same pinaculum; A8 as A2 except with spiracle slightly larger and SV-group unisetose; A9 with D2 about 2 ½-3 times longer than D1; D1 anterior to D2, in straight line with it, in vertical line with and closer to SD1, SD1 more than 2 times length of D1; L1 and L2 on same pinaculum, slightly anterior to D1; L1 almost 5 times length of L2; L3 about as long as L2; L1 about 2 times length of SV1; V1s as previous segments; A10 (Figs. 8, 11): anal plate with SD2, SD1, and D2 roughly equal in lengths, about 3 times length of D1; crochets of proleg biordinal, in semicircle, gradually shortened mesally and laterally. Pupa. (Figs. 13-16): Length 5.0-6.7 mm (n = 4): amber, smooth, all setae apically hooked. Sclerites of antennae annulated, widely separated anteriorly, gradually convergent from beyond basal 1/3 of sclerites of maxillae, fused near apices of sclerites of maxillae, gradually divergent posteriorly, exposing distal part of sclerites of hindlegs; sclerites of midlegs not fused distally; paired nodular scars of prolegs on A5-A6 (Fig. 13); A6-A10 fused, rotating as a unit; cremaster dorsolaterally flattened, extended posterolaterally into 2 slightly divergent and elongate spinelike processes (Figs. 15, 16).

3.2. Biology The small pinkish colored eggs are laid underneath dry iguana droppings. The Galapagos Land Iguana is mainly herbivorous but feeds on animal matter if easily accessible (Werner 1982). In the dry fecal matter we found numerous seeds, leaves, and other plant material, but also remains of the large endemic painted locust Schistocerca melanocera (Stål) (Acrididae), and once also iguana skin (probably from moulting). Therefore, the larvae may feed on dead plant and animal material, which is consistent with the feeding habits of other Autostichidae (Hodges 1998). In February 2005, adults were attracted to light on Fernandina from sea level to the crater rim, which culminates at an elevation of 1341 meters.

20 CHAPTER 2

FIGURES 2-8. Scanning electron micrographs of the larva of Galagete protozona. 2. Frontolateral view of head capsule, Scale = 100 µm; 3. Ventrolateral view of head capsule, Scale = 100 µm; 4. Sensilla of antenna; 1 = sensilla basiconica, 2 = sensillum chaetica, 3 = sensillum styloconicum, 4 = sensillum trichodeum, Scale = 10 µm; 5. Sensilla of maxillary palpus; A2 = sensillum styloconicum, A1, A3, M1-2, L1-3 = sensilla basiconica, SD = sensillum digitiform, Scale = 10 µm; 6. Distal portion of left prothoracic leg showing claw, Scale = 100 µm; 7. Left proleg on A5, Scale = 100 µm; 8. Anal plate of A10, Scale = 100 µm.

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FIGURES 9-12. Chaetotaxy of the larva of Galagete protozona. 9. Thorax; 10. Abdominal segments 1-2; 11. Abdominal segments 6-10; 12. Mandible.

22 CHAPTER 2

3.3. Relationships Main differences between larvae of Galagete protozona Landry (2002) and those of the closely related, Taygete sphecophila (Meyrick) also known from the Galapagos (Landry et al. 2006), are as follows: mandible less angular, without subapical notch, and with smaller and narrower dentition on inner surface; position of S3 on the head capsule more mesal; microsculpture of larval head different (that of T. sphecophila has slightly raised polygonal ridges); thoracic legs less spiny; D2 longer than D1 on T2-T3; L3 much longer than L2 on A1-A7 (3 times longer versus 1/3 longer in T. sphecophila); A9 with SD1 more than 2 times length of D1 (versus about as long); SD1 closer to D1 and in vertical line with it (versus equidistant between D1 and D2, and in vertical line with D2); L1 almost 5 times length of L2 (versus 3 times). In both species, the larvae possess only three stemmata, a condition that is highly unusual in Gelechioidea (Landry et al. 2006). The pupae of G. protozona and T. sphecophila are also similar except for the presence of hooked setae ventrally as well as dorsally, whereas T. sphecophila has them only dorsally, the antenna and hindleg sclerites reach beyond the apical margin of A4, whereas they just reach A4 on T. sphecophila, and the longer and curved (versus straight) processes at the apex of the abdomen.

3.4. Notes Known only from the Galapagos, G. protozona is present on the islands of Baltra, Fernandina, Floreana, Isabela, Santa Cruz, Santa Fe, and Seymour Norte. The actual distribution of the Galapagos Land Iguana Conolophus subcristatus is on the islands of Baltra, Fernandina, Isabela, Plaza Sur, Santa Cruz, and Seymour Norte. It became extinct on Santiago, and very restricted to isolated localities on Santa Cruz. Another endemic species of Land Iguana, Conolophus pallidus Heller, occurs only on the island of Santa Fe. Although the presence of G. protozona matches closely the distribution of these iguanids in the Galapagos, an existing specificity is not confirmed yet. The sequences of a larva of G. protozona from Baltra and an adult from Seymour Norte showed no substitution, which clearly indicates conspecificity. These sequences have been added to a dataset that includes sequences of other Galagete species and that has being used to reconstruct the phylogeny of the species and populations of Galagete to get insight into the early stages of its speciation (unpublished).

23 CHAPTER 2

FIGURES 13-16. Pupa of Galagete protozona. 13. Ventral view; 14. Dorsal view; 15. Segments 8-10; 16, Lateral view.

4. ACKNOWLEDGEMENTS

We are grateful to the authorities of the Galapagos National Park for permits, to Lazaro Roque-Albelo and other staff of the Charles Darwin Research Station, Galapagos, for logistical support, and to L. Roque, Jill Key, and Novarino Castillo for companionship and help in the field. We thank André Piuz, manager of the Scanning Electron Microscopy Laboratory of the MHNG for preparing the larval specimens for study and for the electron micrographs (Figs. 2-8); and Florence Marteau, scientific illustrator at MHNG, for the digital illustrations and production of the plates of the larva and pupa (Figs. 9-16). Finally, we would like to thank the Augustin-Lombard Foundation, the E. and L. Schmidheiny Foundation, the Swiss Academy of Sciences, the Baslerstiftung für biologische Forschung, the Department of cultural affairs of the city of Geneva, and the MHNG for travel funds, and two anonymous reviewers for their comments on the manuscript.

24

CHAPTER 3

TWO NEW SPECIES OF CHIONODES HÜBNER FROM ECUADOR, WITH A SUMMARY OF KNOWN GALAPAGOS RECORDS OF GELECHIIDAE (LEPIDOPTERA).

P. SCHMITZ & B. LANDRY

Muséum d’histoire naturelle, C. P. 6434, CH-1211 Geneva 6, Switzerland

Published in: Revue suisse de Zoologie, 114: 175–184, 2007

25 CHAPTER 3

ABSTRACT

Two new species of the genus Chionodes Hübner, 1825 (Lepidoptera, Gelechiidae) from Ecuador are described and illustrated. Chionodes stefaniae sp. n. occurs in the Galapagos on the islands of Floreana, Isabela, Pinta, Pinzon, Rabida, San Cristobal, and Santa Cruz, where it is believed to be endemic. Chionodes manabiensis sp. n. occurs on the Ecuadorian coast in Machalilla National Park. The previous Galapagos records of Aristotelia howardi Walsingham, 1909, and Stegasta bosqueella Chambers, 1875 were erroneous. Stegasta zygotoma Meyrick, 1917 is reported from the Galapagos for the first time. Altogether, five species records of Gelechiidae are now considered valid for the Galapagos.

26 CHAPTER 3

1. INTRODUCTION

The Gelechiidae represent one of the largest families of Lepidoptera with more than 4,530 species described (Hodges, 1998). Kaila (2004: 322) reported that the monophyly of the group is supported by 12 synapomorphies of which one is unique, i.e. the presence on the forewing of a row of narrow scales ventrally on vein R in females only. Genus Chionodes Hübner, 1825 is found throughout the Holarctic region and in the Neotropics. The largest number of species occurs in North America and in the mountain zones of the Palearctic region including the Arctic (Huemer & Sattler, 1995). An apparent apomorphy for Chionodes is the presence of a caecum on the phallus (Hodges, 1999: 15). In his revision of the North American species, Hodges (1999: 20, 24, 25) recognized and characterized six species-groups for the 187 species occurring in America North of Mexico and the 21 species in the Neotropical region. The two species described here belong to the phalacrus-group which already contains five described species in the Neotropics (i.e., south of the U.S.A.): C. argosema (Meyrick, 1917) described from the Ecuadorian Andes, C. consona (Meyrick, 1917) described from Peru, C. donatella (Walker, 1864) described from Jamaica, C. eburata (Meyrick, 1917) described from Colombia, and C. phalacrus (Walsingham, 1911) described from Mexico. Two undescribed species from Brazil were also recorded to belong to this group by Hodges (1999: 165). The known host plants of the group are in the Malvaceae: Abutilon, Hibiscus, Malacothamnus, Malvastrum, Sida, Sidalcea, Sphaeralcea, and Wissadula (Hodges, 1999).

As part of a project to document the entire microlepidopteran fauna of the Galapagos, dissected specimens of the Gelechiidae species collected by Bernard Landry (BL) in the Galapagos were critically examined by him with the help of Dr. Klaus Sattler at the Natural History Museum, London, England (BMNH) in 2000. Among them was a new species of Chionodes which was used as an outgroup to study the evolution of the Galapagos endemic genus Galagete Landry (Autostichidae; Schmitz et al., submitted). And while searching for potential taxa possibly related to Galagete at Machalilla National Park, on the coast of continental Ecuador, north of Guayaquil, Patrick Schmitz (PS) came upon another new species of Chionodes that proved to be different, yet very similar to the Galapagos species and to a few other described species. These two new species are described below. So far only four valid species of Gelechiidae have been reported for the Galapagos. Schaus (1923) reported Aristotelia howardi Walsingham, 1909, and Stegasta bosqueella

27 CHAPTER 3

Chambers, 1875. However, we can report here that these two records were erroneous and respectively represent Aristotelia naxia Meyrick, 1926, described from the Galapagos, and Stegasta zygotoma Meyrick, 1917, described from Colombia, Ecuador, and Peru, and for which Clarke (1969) selected a lectotype from Ecuador, Huigra, 4,500 ft. The two erroneous records and A. naxia were subsequently listed also by Linsley & Usinger (1966) along with Gelechia protozona Meyrick, 1926 and G. gnathodoxa Meyrick, 1926, which Landry (2002) transferred to the Autostichidae. The other previous gelechiid records are those of the widespread invasives Sitotroga cerealella (Olivier, 1789) and Tuta absoluta (Meyrick, 1917) in Causton et al. (2006). Collections hold at least 12 more Gelechiidae species from the Galapagos, and they will be treated in a forthcoming paper.

FIGURES 1-2. Holotypes of Chionodes spp. 1. C. stefaniae; 2. C. manabiensis.

2. MATERIAL AND METHODS

The 57 specimens forming the basis of this study were collected mostly by BL during three expeditions to the Galapagos in 1989, 1992, and 2002. Other specimens were collected by both of us during two more expeditions on the archipelago in 2004 and 2005, and by PS in the Galapagos and in Machalilla National Park in 2006. Seven additional specimens come from the collection of the Invertebrates Department of the Charles Darwin Research Station, Santa Cruz Island, Galapagos (CDRS). In addition to this institution, specimens will be deposited in the Natural History Museum, London, England (BMNH), the Canadian National Collection of Insects, Ottawa, Canada (CNC), and the Muséum d’histoire naturelle, Geneva, Switzerland (MHNG).

28 CHAPTER 3

The manner of giving the label data of the holotypes and paratypes is presented in Landry (2006) and so are the methods used for specimen collecting, genital preparation, forewing length measurement, and illustrations. The terminology regarding genitalia follows Hodges (1999).

3. DESCRIPTIONS

Chionodes stefaniae sp. n. Figs 1, 3-8

MATERIAL EXAMINED: Holotype ♂, [1] “ECU[ADOR]., GALAPAGOS/ Isabela, V[olcan]. Darwin/ 630 m[eters] elev[ation]., 17.v.1992/ M[ercury]V[apour]L[amp], leg[it]. B[ernard]. Landry.” [2] “HOLOTYPE/ Chionodes/ stefaniae/ Schmitz & Landry”. Specimen in perfect condition except for small hole in left forewing. Deposited in MHNG. Paratypes, Ecuador: 19 ♂, 29 ♀, from the Galapagos Islands, collected with an ultra- violet light and by B. Landry, unless specified otherwise. – Floreana: 1 ♀, close to Loberia, G[lobal]P[ositioning]S[ystem]: S 01°17.002’, W 90°29.460’, 11.iv.2004 (P. Schmitz); 2 ♀, Las Cuevas, 23.iv.1992, M[ercury]V[apour]L[amp]; 1 ♂ (dissected, slide MHNG 3204), Zona arida, 300 m[e]t[er]s, Finca Las Palmas, 26.xii.1997, UVL-F.L. (L. Roque). – Isabela: 1 ♂, V[olcan]. Darwin, campamento base, 1.iii.2000, Malaise trap (L. Roque); 1 ♀, 1 km W [of] Puerto Villamil, 3.iii.1989, MVL; 1 ♀, 11 km N Puerto Villamil, 9.iii.1989, MVL; 2 ♀, 11 km N Pto Villamil, 13.iii.1989, MVL; 1 ♂ (dissected, slide MHNG 3201), NE slope [Volcan] Alcedo, Los Guayabillos camp, GPS: elev[ation]. 869 m, S 00°24.976’ W 91°04.617’, 2.iv.2004, 4h00-5h30 (B. Landry, P. Schmitz); 1 ♀, [Volcan] Alcedo, lado NE, 200 m, camp arida alta, 14.iv.2002 (B. Landry, L. Roque); 2 ♀, [Volcan] Alcedo, lado NE, 400 m, camp pega-pega, 15.iv.2002 (B. Landry, L. Roque); 1 ♀ (dissected, slide MHNG 3208), [Volcan] Alcedo, lado NE, low arid zone, bosq[ue]. palo santo, 18.iv.2002 (B. Landry, L. Roque); 3 ♂, V. Alcedo, 570 m elev., 11.x.1998 (L. Roque). – Pinta: 1 ♂ (dissected, slide MHNG 3202), 1 ♀, 200 m elev., 16.iii.1992, MVL; 1 ♀, 400 m elev., 17.iii.1992, MVL; 1 ♀, N 00°34.476', W 90°45.102', 372 m elev., 16.iii.2006 (P. Schmitz, L. Roque); 1 ♂, 400 m elev., 18.iii.1992, MVL; 3 ♀, N 00°34.591', W 90°45.137', 421 m elev., 18.iii.2006 (P. Schmitz, L. Roque). – Pinzon: 1 ♀ (dissected, slide PS028), 01.v.2003 (L. Roque). – Rabida: 1 ♀, Tourist trail, 3.iv.1992, MVL. –San Cristobal: 1 ♀ (dissected, slide MHNG 3209), antiguo botadero, ca. 4 km SE Pto Baquerizo, GPS: 169 m elev., S 00°54.800', W 089°34.574', 25.ii.2005; 1 ♂ (dissected, slide MHNG 3203), near Loberia, GPS: elev. 14 m, S

29 CHAPTER 3

00°55.149’, W 89°36.897’, 16.iii.2004 (B. Landry, P. Schmitz). – Santa Cruz: 1 ♂, Tortuga Res[erve]., W [of] Santa Rosa, 6.ii.1989, MVL; 1 ♀, NNW [of] Bella Vista, GPS: 225 m elev., S 00°41.293', W 090°19.665', 18.ii.2005 (B. Landry, P. Schmitz); 1 ♀, E[stacion]. C[ientifica]. C[harles]. D[arwin]., 7.iii.1992; 1 ♀, transition zone, recently cut road, GPS: S 00°42.528’, W 90°18.849’, 12.iii.2004 (B. Landry, P. Schmitz); 1 ♀, El Barranco, ECCD, 13.iii.2000, MVL Trap (L. Roque); 1 ♂, Finca S[teve]. Devine, 17.iii.1989, MVL; 1 ♂, ECCD, El Barranco, S 00°44.291', W 90°18.107', 22 m elev., 23. iii.2006 (P. Schmitz); 2 ♂, Finca Vilema, 2 km W [of] Bella Vista, 1.iv.1992, MVL; 1 ♀, C[harles] D[arwin] R[esearch] S[tation], [El] Barranco, 20 m elev., 30.iv.2002; 4 ♂ (one dissected, slide MNHG 3212), 3 ♀ (one dissected, slide MHNG 3207), Los Gemelos, 27.v.1992, MVL; 1 ♂, Barranco, CDRS, 23.x.2001 (L. Roque). Deposited in the BMNH, CDRS, CNC, and MHNG.

DIAGNOSIS: Among the species of Chionodes, C. stefaniae is similar in wing pattern to C. argosema, C. donatella, C. manabiensis sp. n., C. mariona (Heinrich, 1921), and C. petro Hodges, 1999 of the phalacrus-group of Hodges (1999). In male genitalia, C. stefaniae differs from C. argosema, C. donatella, C. mariona, and C. petro in having a shorter mesial projection of the uncus and short, curved and rather stout valval projections as opposed to long and thin projections in C. argosema (Clarke, 1969), and short and straight projections in the other three species (Hodges, 1999). Furthermore, C. stefaniae differs from C. donatella, C. mariona, and C. petro in having the male abdominal tergum VIII wider than long versus about twice as long as wide (Hodges, 1999: pl. W figs 16, 17, 20), and in the female, there is a modification at the posterior margin of tergum VII while modifications occur at the posterior margin of abdominal tergum VI and anterior margin of tergum VII in the other species (Hodges, 1999: pl. VV figs 11-13). Chionodes stefaniae differs from C. manabiensis in several characters mentioned in the Diagnosis and Description of this species, below.

DESCRIPTION: MALE (n=20) (Figs 1, 3–5): Head off-white with yellowish orange scales on forehead. Haustellum dark brown; maxillary palpus off-white, 4-segmented. Labial palpus dark brown on first segment; second segment off-white to yellowish orange with broad scale brush; third segment off-white to yellowish orange, slender, dark brown on distal 1/3 to 1/2. Antennal scape dark brown with white scales ventrally; flagellum dark brown. Thorax off-white, tegula and metathorax dark brown. Foreleg coxa, tibia, femur and tarsomeres dark brown with white at apices of tarsomeres I and V. Midleg and hindleg femora and tibiae dark brown at base, apices 1/3 off-white; spurs white; tarsomeres I-V mostly dark brown with white at apex of each segment. Wingspan: 9.3-11.0 mm (Holotype: 11.0 mm). Forewing dark brown with pair of prominent off-white patches with yellowish orange scales on costal margin

30 CHAPTER 3 at 3/4 and on inner margin at end of fold; sometimes with slightly darker brown markings visible as two small spots submedially in middle, one above the other, and another spot postmedially above second off-white patch; with some scattered off-white scales between patches, on termen, and around both small spots; fringe dark brown. Hindwing dark greyish brown, fringe pale greyish brown. Abdomen dark brown dorsally, off-white ventrally; tergum VIII with more thickly sclerotized anterior margin a broad inverted V, with posterior margin broadly rounded; sternum VIII with lateral margins slightly convex and with conspicuous striae, anterior margin bearing pair of submesial lobes, posterior margin with broad and short rounded lateral lobes (apically folded on Fig. 5). Male genitalia (n=4) (Figs 3, 4). Uncus with broadly rounded anterobasal lobes with stout setae ventrally and few hairlike setae on apical margins; with small, blunt, apicomesial projection. Median hook of gnathos rather long and thin, slender from base to apex, upturned and pointed apically. Dorsal connection of tegumen wide; pedunculi short and broad, shorter than vinculum. Valva with long and slender sicklelike projection of 1/2-1/3 X length of tegumen; with small recurved knob at lateral base of each projection; sacculus short, with few setae at base ventrally. Vinculum tapering to narrowly rounded saccus. Phallus narrow, with distinct, sclerotized rim around opening of ductus ejaculatorius, broadest at this point; slightly upturned at 5/6 of length; with long, rodlike caecum of about 1/3 X length of phallus; vesica without spines or cornuti.

FIGURES 3-5. Male genitalia of Chionodes stefaniae from specimen on slide MHNG 3201. 3. Whole genitalia without phallus; 4. Phallus; 5. Abdominal segment VIII.

31 CHAPTER 3

FEMALE (n=29): Colour as in male. Antenna slightly thinner than that of male. Female wingspan: 8.7-10.9 mm. Tergum VII apically modified, with low, median depression associated with thin canal directed proximally. Female genitalia (n=5) (Figs 6-8). Papillae anales slightly longer than wide. Posterior apophyses long, straight, very slightly enlarged at apex (about 2.4 X length of papillae), reaching base of antrum. Anterior apophyses partly fused with antrum, free proximal section arising from lateral margin of antrum near base, down curved with slightly enlarged apex. Antrum well developed with short, longitudinal sclerotized band in dorsal wall; also with heavily sclerotized lateral bands of about half length of ductus bursae and fused toward apex, with left band bent toward right one. Ductus bursae short, of medium girth (width = 0.2-0.25 X its length), slightly constricted at apex of lateral bands of antrum. Inception of ductus seminalis at base of corpus bursae. Corpus bursae elongate, widening and rounded at proximal end, with light scobination; signum situated posteriad middle of corpus bursae, triangular with heavily sclerotized, inwardly directed, large spine arising from each angle, sometimes with small extra spine.

FIGURES 6-8. Female genitalia of Chionodes stefaniae from specimen on slide MHNG 3207. 6. Whole genitalia; 7. Segment VII; 8. Signum.

32 CHAPTER 3

ETYMOLOGY: We are pleased to name this species in honour of Stefania Bertoli- Schmitz for her love and support to PS through the last seven years.

BIOLOGY: The moths were collected at light in February, March, April, May, October, and December from sea level to 869 m.

DISTRIBUTION: Currently known only from the Galapagos islands of Floreana, Isabela, Pinta, Pinzon, Rabida, San Cristobal, and Santa Cruz; presumed to be endemic to the archipelago.

REMARKS: This species and the following are apparently most closely related to each other than to any of the other Chionodes species on the basis of the short mesial projection of the uncus, the curved and rather short valval projections, the wide abdominal tergum VIII of the male, and the modified posterior margin of tergum VII of the female.

Chionodes manabiensis sp. n. Figs 2, 9-14

MATERIAL EXAMINED: Holotype ♂, [1] “ECU[ADOR], Manabi, Parque nacional/ Machalilla, Los Frailes/ S 01°29.340’, W 80°46.686/ 40 m[eters] elev[ation]., u[ltra] v[iolet] l[ight], 25.iv.2006/ leg[it]. P[atrick]. Schmitz.” [2] “HOLOTYPE/ Chionodes/ manabiensis/ Schmitz & Landry”. Specimen in perfect condition except for small notch in left hindwing. Deposited in MHNG. Paratypes, Ecuador: 4 ♂, 3 ♀, from Manabi, collected at uvl by P. Schmitz. 1 ♀, Puerto Lopez, Hosteria Mandala, S 01°32.955’, W 80°48.617’, 10 m elev., 24.iv.2006; 3 ♂ (one dissected, slide MHNG 3187), with same data as holotype; 1 ♀ (dissected, slide MHNG 3210), Parque nacional Machalilla, Agua Blanca, S 01°31.421’, W 80°46.081’, 45 m elev., 26.iv.2006; 1 ♀, Parque nacional Machalilla, Los Frailes, S 01°29.369’, W 80°46.805’, 45 m elev., 27.iv.2006; 1 ♂, Parque nacional Machalilla, Los Frailes, S 01°29.053’, W 80°47.064’, 86 m elev., 28.iv.2006. Deposited in the BMNH and MHNG.

DIAGNOSIS: Based on wing pattern, C. manabiensis is impossible to distinguish from C. argosema, C. donatella, C. mariona, C. petro, and C. stefaniae of the phalacrus-group of Hodges (1999), but the same diagnostic characters mentioned under the diagnosis for C. stefaniae can be applied to separate C. manabiensis from C. argosema, C. donatella, C. mariona, and C. petro. Chionodes manabiensis differs from C. stefaniae in the slightly darker ground colour of the forewing, in the presence of a pair of patches of modified scales on abdominal tergum VIII of the males, in the female modification of tergum VII, which is

33 CHAPTER 3 located more medially and is associated with a low, elongate crest, and in genital characters as mentioned below.

DESCRIPTION: MALE (n=5) (Figs 2, 9-11): As C. stefaniae, except forewing and abdomen darker; wingspan: 9.0-10.0 mm (Holotype: 10.0 mm); abdominal tergum VIII more narrowly rounded apically, with pair of patches of modified scales, with anterior margins more rounded; abdominal sternum VIII slightly longer, less striated laterally, with lobes of anterior margin less heavily sclerotized. Male genitalia (n=1) (Figs 9, 10). As in C. stefaniae, except uncus with more heavily sclerotized setae ventrally; tegumen longer than vinculum; long projection of valva less strongly bent and broader at base; small recurved lobe at base of projection slightly longer; sacculus shorter, with more setae ventrally; vinculum broader, especially at apex; phallus more broadly bent upward at 3/4 of length.

FIGURES 9-11. Male genitalia of Chionodes manabiensis from specimen on slide MHNG 3187. 9. Whole genitalia without phallus; 10. Phallus; 11. Abdominal segment VIII.

FEMALE (n=3): As in C. stefaniae, except colour as in male of C. manabiensis; wingspan: 9.5-11.9 mm; modification of tergum VII located more medially and associated with low, elongate crest. Female genitalia (n=1) (Figs 12-14). As in C. stefaniae, except papillae anales longer (1.5 X longer than broad); posterior apophyses longer (about 3.4 X length of papillae); antrum with sclerotized band in dorsal wall much longer, apically reaching beyond margin of sternum VII, proximally reaching apices of lateral bands and more thickly sclerotized; lateral bands

34 CHAPTER 3 narrow, tapering gradually, at least 2 X length of those of C. stefaniae, with left one bending over right one, free apically.

ETYMOLOGY: The name of C. manabiensis is derived from that of the type locality.

BIOLOGY: The adults were attracted to light in April from sea level to 86 m.

DISTRIBUTION: Currently known only from Machalilla National Park and the adjacent town of Puerto Lopez on the Ecuadorian coast (province of Manabi).

REMARKS: The divergence in a DNA fragment of the Cytochrome Oxidase mitochondrial gene (555 base pairs long) between C. stefaniae and C. manabiensis is 6.7 % (GenBank accession numbers EF423724 and EF423725).

FIGURES 12-14. Female genitalia of Chionodes manabiensis from specimen on slide MHNG 3210. 12. Whole genitalia; 13. Segment VII; 14. Signum.

35 CHAPTER 3

4. ACKNOWLEDGEMENTS

We are very thankful to the authorities of the Galapagos National Park and those of the Charles Darwin Research Station for allowing fieldwork and for logistical support in 1989, 1992, 2004, 2005, and 2006. We thank also Carlos Zambrano Bravo for the permission to collect in the Machalilla National Park. We are indebted to Lazaro Roque-Albelo of the CDRS for his collecting and rearing efforts, and for the loan of material. The comments of Ron Hodges, Lazaro Roque-Albelo, and Klaus Sattler on our manuscript were also appreciated. This work was carried out with the financial support of the Claraz, Lombard, and Schmidheiny Foundations, the Swiss Academy of Sciences, the Baslerstiftung für biologische Forschung, the Département des affaires culturelles of the City of Geneva, the MHNG, and the University of Geneva to PS, and S. B. Peck, the Galapagos Conservation Trust, and the MHNG to BL. We are most grateful to Klaus Sattler for critical information and Florence Marteau (MHNG) for producing the plates.

36

CHAPTER 4

MOLECULAR PHYLOGENY AND DATING OF AN INSULAR ENDEMIC MOTH RADIATION INFERRED FROM MITOCHONDRIAL AND NUCLEAR GENES: THE GENUS GALAGETE (LEPIDOPTERA: AUTOSTICHIDAE) OF THE GALAPAGOS ISLANDS.

P. SCHMITZ a, b, A. CIBOIS a & B. LANDRY a a Muséum d’histoire naturelle, C. P. 6434, CH-1211 Geneva 6, Switzerland. b Département de Zoologie et Biologie Animale, Molecular Systematic Group, Université de Genève, 30 quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland

Published in: Molecular Phylogenetics and Evolution, 2007

37 CHAPTER 4

ABSTRACT

Galagete is a genus of microlepidoptera including 12 nominate species endemic to the Galapagos Islands. In order to better understand the diversification of this endemic insular radiation, to unravel relationships among species and populations, and to get insight into the early stages of speciation, we developed a phylogenetic reconstruction based on the combined mitochondrial cytochrome oxidase I (555 bp) and II (453 bp), and the nuclear elongation factor-1α (711 bp) and wingless (351 bp) genes. Monophyly of the genus is strongly supported in the Bayesian and maximum likelihood analyses suggesting a single colonization event by a common ancestor. Two cases of paraphyly observed between species are hypothesized to represent imperfect species limits for G. espanolaensis nested within the G. turritella clade, and introgressive hybridization or lineage sorting in the case of the population of G. protozona from Santa Fe nested within the G. gnathodoxa clade. A geologically- calibrated, relaxed molecular clock model was used for the first time to unravel the chronological sequence of an insular radiation. The first split occurring within the Galagete lineage on the archipelago is estimated at 3.3 ± 0.4 million years ago. The genus radiated relatively quickly in about 1.8 million years, and gives an estimated speciation rate of 0.8 species per million years. Although the colonization scenario shows a stochastic dispersal pattern, the arrival of the ancestor and the diversification of the radiation coincide with the chronological emergence of the major islands.

38 CHAPTER 4

1. INTRODUCTION

Remote volcanic archipelagos, like the Canary, Hawaiian, and Galapagos Islands, have played a significant role in studies of speciation processes that frequently focus on the outcome of endemic insular radiations, the process by which one species evolves into multiple species over a relatively short time scale (i.e. Emerson and Oromi, 2005; Gillespie, 2004; Grant, 1999; Jordan et al., 2003; Shaw, 2002). True oceanic islands have been recognized as natural laboratories for studying evolution (Whittaker, 2002) and phylogenetic approaches have recently opened new insights into these systems (Emerson, 2002), particularly with regard to molecular time calibration in the Canary Islands (Emerson and Oromi, 2005), the Hawaiian Islands (Baldwin and Sanderson, 1998; Fleischer et al., 1998; Hormiga et al., 2003; Mendelson and Shaw, 2005), and the Galapagos Islands (Bollmer et al., 2006; Caccone et al., 1999; Rassmann, 1997; Sato et al., 2001; Sequeira et al., 2000). Molecular dating analyses have been criticized for a variety of reasons including calibrations using suspect fossil dates, violation of the molecular clock assumption, absence of confidence intervals, and use of inappropriate taxa (Graur and Martin, 2004). The reliability of the modern molecular clock has also been questioned in the case of explosive radiations (Bromham, 2003; Bromham and Penny, 2003), in particular in the case of island endemic radiations because they combine a number of factors, such as reduced population size, elevated speciation rate, adaptation to new niches and release from previous ecological constraints, which could influence rate evolution and make the molecular clock run faster. But Bromham and Woolfit (2004) found no support for a consistent increase in rates in island taxa compared to their mainland relatives. More recently, new methods enabling the incorporation of variable rates into molecular dating have become available (for a review, see Renner (2005)). Such techniques offer greater potential for insight into the history of lineages with poor or non-existent fossil records, and enable estimates of the time of origin of any biological lineage (Welch and Bromham, 2005). The Galapagos provide the best opportunity to examine the molecular dating of an endemic radiation because of their discrete geographical nature, the absence of historical connection between the archipelago and the mainland, and the known geological age of their component islands. Within the Galapagos archipelago, an increase in the age of the islands from west to east is attributed to the eastward displacement of the Nazca plate over a mantle hotspot present beneath the youngest island, Fernandina (Bailey, 1976; Cox, 1983; White et

39 CHAPTER 4 al., 1993). Although the emerged islands are young (3.3 million years old at the most), evidence of drowned seamounts east of San Cristobal, which might have been islands, extend the temporal window available for evolution in the Galapagos to about 9 million years (Christie et al., 1992). The microlepidoptera of the genus Galagete are a distinctive element of the moth fauna of the Galapagos because they represent the largest endemic radiation of Lepidoptera in the archipelago and are present on all major islands at almost all altitudes (Landry, 2002). The genus comprises 12 nominate species with variable distributions encompassing the whole archipelago for some species to single-island endemics (Landry, 2002; Schmitz and Landry, 2005). In a cladistic analysis based on morphological characters (Landry, 2002), the monophyly of Galagete and that of three species groups were recovered: a clade formed by the three larger species (G. gnathodoxa, G. protozona, and G. seymourensis), the G. espanolaensis and G. turritella pair, and a clade composed of G. cinerea, G. consimilis, and G. darwini. Species like G. consimilis, G. darwini, and G. espanolaensis are superficially so similar that they can be identified reliably from their genitalia only (Landry, 2002). Galagete was placed by Landry (2002) in the Autostichidae, Symmocinae sensu Hodges (1998), which also includes the Autostichinae and Holcopogoninae. However Kaila's (2004) morphological analysis showed that the Autostichidae sensu Hodges (1998) are paraphyletic with regard to the Glyphidoceridae and Lecithoceridae placed in an Autostichid assemblage. Little is known about the ecology of Galagete apart from their scavenging feeding habits which are consistent with those of other Autostichidae (Hodges, 1998). For example, the larva and pupa of G. protozona were discovered recently in droppings of the Galapagos Land Iguana Conolophus subcristatus (Schmitz and Landry, 2007a). Here, we present a phylogenetic analysis of all species and most known populations of Galagete based on genes from both mtDNA and nDNA in a combined analysis. We compare the resulting phylogeny to morphologically-based hypotheses (Landry, 2002) and examine important factors in the diversification of this insular endemic radiation in the Galapagos. We apply a relaxed molecular clock approach developed in a Bayesian framework incorporating geological estimates for the age of emergence of the islands. This provides a chronological template of evolution to investigate the time of divergence and colonization of the radiation. We compare the phylogenetic pattern and the age estimates of molecular divergence obtained for Galagete with other groups to elucidate particular sequences of dispersal and colonization among the Galapagos Islands.

40 CHAPTER 4

2. MATERIAL AND METHODS

2.1. Specimen sampling We sampled all of the 12 described species of the genus Galagete and analyzed 47 specimens collected in the Galapagos archipelago on 13 islands from 2003 to 2006 (Fig. 1). All specimens were collected alive and immediately stored in EtOH 100%, except for one dry and pinned specimen of G. levequei from Santa Cruz. Outgroup taxa were chosen from the Gelechioidea: one from the family Gelechiidae and seven from different lineages of the Autostichid assemblage of Kaila (2004) (Table 1).

Species Sample localities COI GenBank No. COII GenBank No. EF1α GenBank No. Wingless GenBank No. Outgroup: Autostichidae: Autostichinae Autosticha modicella JAP: Hokkaido, Sapporo (K. Sugisima) EF680572 EF680625 EF680680 EF680519 Autostichidae: Holcopogoninae Holcopogon bubulcellus CR: Island of Krk, Kampelje (G. Baldizzone) EF680568 EF680621 EF680676 EF680515 Autostichidae: Symmocinae Taygete sphecophila EC: Galápagos, Santa Cruz Landry et al., 2006 EF680619 EF680674 EF680513 Oegoconia novimundi CH: Zurich, Steinmaur (R. Fritschi) EF680569 EF680622 EF680677 EF680516 Lecithoceridae Odites leucostola JAP: Hokkaido, Sapporo (K. Sugisima) EF680570 EF680623 EF680678 EF680517 Rhizosthenes falciformis JAP: Honsyû, Isikawa (T. Sarto) EF680573 EF680626 EF680681 EF680520 Glyphidoceridae Pseudodoxia achlyphans JAP: Hokkaido, Sapporo (K. Sugisima) EF680571 EF680624 EF680679 EF680518 Gelechiidae Chionodes stefaniae EC: Galápagos, Pinzón (L. Roque) Schmitz and Landry (2007b) EF680620 EF680675 EF680514 Ingroup: Autostichidae: Symmocinae Galagete cinerea (1) EC: Galápagos, Fernandina EF680591 EF680645 EF680700 EF680539 G. cinerea (2) EC: Galápagos, Isabela, Puerto Villamil EF680580 EF680634 EF680689 EF680528 G. consimilis (1) EC: Galápagos, Floreana EF680605 EF680659 EF680714 EF680553 G. consimilis (2) EC: Galápagos, Sta Cruz EF680614 EF680669 EF680724 EF680563 G. consimilis (3) EC: Galápagos, Pinta EF680616 EF680671 EF680726 EF680565 G. consimilis (4) EC: Galápagos, Plaza Sur EF680617 EF680672 EF680727 EF680566 G. cristobalensis* EC: Galápagos, San Cristóbal EF680582 EF680636 EF680691 EF680530 G. darwini (1) EC: Galápagos, Baltra EF680612 EF680666 EF680721 EF680560 G. darwini (2) EC: Galápagos, Española EF680602 EF680656 EF680711 EF680550 G. darwini (3) EC: Galápagos, Fernandina EF680600 EF680654 EF680709 EF680548 G. darwini (4) EC: Galápagos, Floreana EF680615 EF680670 EF680725 EF680564 G. darwini (5) EC: Galápagos, Isabela EF680606 EF680660 EF680715 EF680554 G. darwini (6) EC: Galápagos, Pinta EF680607 EF680661 EF680716 EF680555 G. darwini (7) EC: Galápagos, Pinzón EF680609 EF680663 EF680718 EF680557 G. darwini (8) EC: Galápagos, San Cristóbal EF680590 EF680644 EF680699 EF680538 G. darwini (9) EC: Galápagos, Santa Cruz EF680618 EF680673 EF680728 EF680567 G. darwini (10) EC: Galápagos, Santiago EF680588 EF680642 EF680697 EF680536 G. darwini (11) EC: Galápagos, Seymour Norte EF680589 EF680643 EF680698 EF680537 G. espanolaensis* EC: Galápagos, Española EF680604 EF680658 EF680713 EF680552 G. gnathodoxa (1) EC: Galápagos, Fernandina EF680587 EF680641 EF680696 EF680535 G. gnathodoxa (2) EC: Galápagos, Floreana EF680576 EF680629 EF680684 EF680523 G. gnathodoxa (3) EC: Galápagos, Isabela, Puerto Villamil EF680585 EF680639 EF680694 EF680533 G. griseonana* EC: Galápagos, Santa Cruz EF680583 EF680637 EF680692 EF680531 G. levequei (1) EC: Galápagos, Fernandina EF680592 EF680646 EF680701 EF680540 G. levequei (2) EC: Galápagos, Isabela, Volcano Alcedo EF680595 EF680649 EF680704 EF680543 G. levequei (3) EC: Galápagos, Santiago EF680594 EF680648 EF680703 EF680542 G. levequei (4) EC: Galápagos, Santa Cruz (L. Roque) EF680574 EF680627 EF680682 EF680521 G. levequei (5) EC: Galápagos, Pinzón EF680610 EF680664 EF680719 EF680558 G. pecki (1) EC: Galápagos, Isabela, Volcano Alcedo EF680579 EF680633 EF680688 EF680527 G. pecki (2) EC: Galápagos, Santa Cruz EF680581 EF680635 EF680690 EF680529 G. protozona (1) EC: Galápagos, Baltra Schmitz and Landry (2007a) EF680668 EF680723 EF680562 G. protozona (2) EC: Galápagos, Fernandina EF680586 EF680640 EF680695 EF680534 G. protozona (3) EC: Galápagos, Isabela, Volcano Alcedo EF680584 EF680638 EF680693 EF680532 G. protozona (4) EC: Galápagos, Seymour Norte Schmitz and Landry (2007a) EF680630 EF680685 EF680524 G. protozona (5) EC: Galápagos, Santa Fé (L. Roque) EF680575 EF680628 EF680683 EF680522 G. seymourensis (1) EC: Galápagos, Fernandina EF680599 EF680653 EF680708 EF680547 G. seymourensis (2) EC: Galápagos, Seymour Norte EF680577 EF680631 EF680686 EF680525 G. turritella (1) EC: Galápagos, Baltra EF680613 EF680667 EF680722 EF680561 G. turritella (2) EC: Galápagos, Fernandina EF680603 EF680657 EF680712 EF680551 G. turritella (3) EC: Galápagos, Floreana EF680598 EF680652 EF680707 EF680546 G. turritella (4) EC: Galápagos, Isabela, Volcano Alcedo EF680578 EF680632 EF680687 EF680526 G. turritella (5) EC: Galápagos, Pinta EF680608 EF680662 EF680717 EF680556 G. turritella (6) EC: Galápagos, Plaza Sur EF680611 EF680665 EF680720 EF680559 G. turritella (7) EC: Galápagos, Santiago EF680593 EF680647 EF680702 EF680541 G. turritella (8) EC: Galápagos, Seymour Norte EF680597 EF680651 EF680706 EF680545 G. turritella (9) EC: Galápagos, Santa Cruz EF680596 EF680650 EF680705 EF680544 G. turritella (10) EC: Galápagos, San Cristóbal EF680601 EF680655 EF680710 EF680549 *single island endemic

TABLE 1. Species of Galagete and outgroups included in the phylogenetic analyses. Samples not collected by the first or third author are indicated by collector's name in parentheses.

41 CHAPTER 4

2.2. DNA extraction, PCR amplification, and sequencing Whole genomic DNA was extracted from the adult thorax and head using the Nucleospin kit (Macherey-Nagel), except for the specimen of G. protozona collected on Baltra, a larva found in Land Iguana droppings (Schmitz and Landry, 2007a). Abdomens and wings were conserved in gelatin capsules as vouchers and for confirmation of specimen identification by dissection of the genitalia. The vouchers are deposited in the Muséum d'histoire naturelle, Geneva, Switzerland (MHNG). Detailed data on collection localities and DNA GenBank accession numbers of all sequences included in our analysis are given in Table 1. Fragments of cytochrome oxidase I and II (COI and COII), elongation factor-1α (EF1α), and wingless (WG) were amplified using polymerase chain reaction (PCR) with the primer combinations listed in Table 2. The rationale for the choice of these genes is that they have proven to be informative at different levels in several phylogenetic studies of Lepidoptera (Caterino et al., 2000). The length of COI is ~1500 bp but we focused on its second half following the variability survey presented by Lunt et al. (1996) in other Insecta. Furthermore, because females are the heterogametic sex in Lepidoptera, the inviability of interspecific hybrid females expected under Haldane’s Rule makes mtDNA less vulnerable to introgression (Sperling, 2003) that might lead to a disagreement between species trees and gene trees (Maddison, 1997; Pamilo and Nei, 1988; but see Funk and Omland (2003) and Rubinoff and Holland (2005) for different inheritance of mtDNA). As a contrast to the faster evolving sequence in COI and COII, we also sequenced part of the nuclear gene EF1α and WG which are usually considered useful at higher taxonomic levels (Sperling, 2003).

Gene Primer (forward or reverse) Sequence of primer (5'-3') Reference COI k698 (f) TAC AAT TTA TCG CCT AAA CTT CAG CC Caterino and Sperling (1999) Pat2 (r) TCC ATT ACA TAT AAT CTG CCA TAT TAG Caterino and Sperling (1999) Jerry (f) CAA CAT TTA TTT TGA TTT TTT GG Caterino and Sperling (1999) COII Stefi (f) ATA CCT CGT CGT TAT TCT GAT TAT CC present study Eva (r) GAG ACC ATT ACT TGC TTT CAG TCA TCT Caterino and Sperling (1999) EF1α EF44 (f) GCY GAR CGY GAR CGT GGT ATY AC Monteiro and Pierce (2000) EFrcM4 (r) ACA GCV ACK GTY TGY CTC ATR TC Monteiro and Pierce (2000) WG LepWG1 (f) GAR TGY AAR TGY CAY GGY ATG TCT GG Brower and DeSalle (1998) LepWG2a (r) ACT NCG CAR CAC CAR TGG AAT GTR CA Brower and DeSalle (1998)

TABLE 2. Primers used in the amplification of COI, COII, EF1α, and WG.

42 CHAPTER 4

The amplifications were performed in a total volume of 50 µl. Thermal profiles for COI and COII (94°C for 30 s; 47°C for 30 s; 72°C for 2 min), EF1α (95°C for 1 min; 55°C for 1 min; 72°C for 2 min), and WG (95°C for 1 min; 50°C for 1 min; 72°C for 2 min), started with 5 min of denaturation at 95°C, were repeated for 35 cycles, and were followed by a final step for 10 min at 72°C.

FIGURE 1. Distribution of species of Galagete occurring in the Galapagos Islands. Species names in parentheses refer to populations for which samples could not be obtained for sequencing, but which are known from specimens in collections. Asterisks indicate the species considered as single-island endemics. Numbers in parentheses refer to the proposed maximum geological ages for each island (Bailey, 1976; Cox, 1983; Geist et al., 1994; White et al., 1993), estimated in millions of years. The star on Fernandina indicates the position of the archipelago's hotspot. Inset on the right is the geographic location of the Galapagos Islands relative to the equator and the South American continent.

43 CHAPTER 4

The amplified PCR products were purified using the PCR Purification kit (Qiagen) and run on an ABI Prism 377 automated DNA sequencer, or an ABI Prism 3130xl genetic analyzer. The amplification primers were used for direct sequencing of the PCR fragments, and an additional internal primer, "Jerry", was used to replace "k698" to sequence the terminal region of COI. The same individual was sequenced for each gene, and sequences were translated into amino acids to check for reading frame. To avoid contamination, two or more individuals for some populations were sequenced days apart for the end of COI only to test for concordance between them (not shown). The resulting sequences encompass a 555 bp fragment in COI, 453 bp in COII, 711 bp in EF1α, and 351 bp in WG, representing 2070 bp altogether.

2.3. Phylogenetic analysis Sequences were easily assembled manually, edited and aligned with the software BIOEDIT 7.0.5 (Hall, 1999). Alignments were unambiguous. Homogeneity tests of base frequencies were conducted on all alignments by using the base frequencies Chi-square test as implemented in PAUP* 4.0b10 (Swofford, 2003) to estimate if the sequences have evolved with the same pattern of nucleotide substitution. A partition homogeneity test, using a heuristic search of 1,000 replicates, was applied on the alignments of mtDNA and nDNA to verify if the two partitions could be analyzed in concatenation. Both data sets were first analyzed independently to look for potential conflicts. The concatenated data sets were used then for further phylogenetic analyses. Hierarchical-likelihood ratio tests performed with MODELTEST 3.04 (Posada and Crandall, 1998) determined that a General Time Reversible model with rate variation among sites and a proportion of invariable sites (GTR+Γ8+I) represent the best fit model of nucleotide substitution for the combined datasets. The Maximum Likelihood (ML) method was then performed with a heuristic search and random addition of sequences as implemented in PAUP* 4.0b10, with the starting tree obtained via stepwise addition of taxa, and then swapped using the tree-bisection-reconnection (TBR) algorithm. Outgroup taxa (Table 1) used were chosen from each subfamily inside the Autostichid assemblage (Kaila, 2004). The reliability of internal branches was assessed using the bootstrap method (Felsenstein, 1985) with 1000 replicates by using the program PHYML 2.4.4 using the same model (Guindon and Gascuel, 2003). Bayesian posterior probabilities were calculated using a Metropolis-coupled, Markov Chain, Monte Carlo (MCMCMC) sampling approach, as implemented in MRBAYES

3.1.2 (Huelsenbeck and Ronquist, 2003), using a GTR+Γ8+I model. Four simultaneous

44 CHAPTER 4

Markov chains were run in parallel twice for 1 million generations with trees sampled every 10 generations. Stationarity was evaluated graphically and the first 250,000 trees were discarded as burn-in. MRBAYES was set to estimate model parameters independently and simultaneously for each gene partition. All above-mentioned analyses were run through the Bioportal web-based service platform for phylogenomic analysis at the University of Oslo (www.bioportal.uio.no). The combined data provided robust phylogenetic reconstructions that we considered the departure point for further examination of the Galagete radiation.

2.4. Molecular dating The Bayesian relaxed molecular clock approach was applied using the program package MULTIDIVTIME (Thorne et al., 1998; Kishino et al., 2001; Thorne and Kishino, 2002). This program allows the incorporation of multiple time constraints, and takes into account both molecular and geological uncertainties to estimate the variance of divergence times. Calculations were performed on the ML tree obtained from the combined dataset. The module ESTBRANCHES was first used to estimate branch lengths of the constrained topology and the corresponding variance-covariance matrices. Taygete sphecophila was used as the outgroup because it was found to be the closest Galapagos relative based on morphological characters (Landry, 2002; Landry et al., 2006). The F84+Γ8 model (model complexity is limited by the dating program) was used with maximum likelihood parameters previously estimated by PAML 3.14 (Yang et al., 1997). The output from ESTBRANCHES was then used as the input file for the module MULTIDIVTIME to run a Markov Chain Monte Carlo (MCMC) for estimating the mean posterior divergence times on nodes, with associated standard deviation, and 95% credibility interval. MCMC was sampled every 100 generation over 1 million generations, after a burn-in period of 100,000 cycles. The following priors were used for the data set: 9.1 million years ago (MYA) for the expected time units between root and tips, corresponding to the age of the oldest drowned island east of the Galapagos hotspot (Christie et al., 1992), and 17 MYA (Werner and Hoernle, 2003) for the highest possible number of time units between root and tips. Other priors for gamma distribution of the rate at root node and the Brownian motion constant describing the rate variation (i.e. the degree of rate autocorrelation along the descending branches of the tree), were derived from the median branch length for the data set as advised by Thorne et al. (1998). Two internal calibration dates corresponding to the upper estimates for the age of emergence of two islands were used as constraints in the analysis under the assumption that the Galagete taxa colonized the islands shortly after their emergence: these dates correspond

45 CHAPTER 4 to the oldest volcan on Isabela (Alcedo, 0.5 MYA; Geist et al., 1994), and the emergence of Floreana (1.52 MYA; White et al., 1993). These dates reflect our choice to use lower internal calibrations points scattered through the topology to avoid discarding potentially useful information at the base of the tree. To estimate rates of speciation, we used the Yule estimator as implemented in Mendelson and Shaw (2005). The speciation rate (SR) for young monophyletic clade is estimated as SRln = ([lnN]/t) where N is the number of extant species and t is the divergence time.

3. RESULTS

3.1. Sequence data The final concatenated alignment, excluding outgroups, was 2070 bp long, including 1008 mtDNA (COI and COII) characters, of which 317 were variable (31.4%), 62 of which were first codon position, 12 second codon position, and 243 third codon position, and 1062 nDNA (EF1α and WG) characters of which 197 were variable (18.5%), 21 of which were first codon position, 7 second codon position, and 169 third codon position. The data showed an A/T ratio of 74% for mtDNA, and 44% for nDNA. The Chi-square homogeneity test of base frequencies indicated no significant deviations between taxa for both sets (mtDNA: χ2 = 12; df = 138; P = 1.00, and nDNA: χ2 = 15; df = 138; P = 1.00). The partition homogeneity test on the concatenated alignments was performed and showed no significant incongruence between the two data sets (P = 0.13). Therefore the combined mitochondrial and nuclear dataset was used in further phylogenetic analyses.

The ingroup genetic distance (corrected for multiple hits with GTR+Γ8+I) ranged up to 18.5% for the mtDNA, and 6.6% for the nDNA. The combined alignment was unambiguous for both sets and without indels, as expected for protein-coding genes, except for a 3 bp insertion observed in COII for the whole ingroup. Several facts indicated that the PCR mtDNA products were mitochondrial, rather than nuclear pseudogenes (Bensasson et al., 2001; Zhang and Hewitt, 1996). First, no unexpected stop codons or frameshift mutations were present in the coding sequences. Second, several long PCR were performed in one specimen for each species. Third, low guanine content (26%) suggests a mitochondrial origin.

46 CHAPTER 4

3.2. Phylogenetic analyses The ML tree of the combined mitochondrial and nuclear dataset is shown in Fig. 2, with the supports of nodes estimated with the two methods (MRBAYES, ML). The concatenated dataset shows increasing Bayesian posterior probabilities (PP) and ML bootstrap supports (BS) than found for the separate genes alone and no incongruence was observed between the resulting reconstructions of both data sets (not shown). The tree topologies obtained with MRBAYES and ML for the combined analysis were identical. All sequenced species of Galagete are grouped in a strong monophyletic clade in both reconstructions (1.00 PP; 100% BS). This clade is further subdivided in 11 fully resolved clades (all with 1.00 PP; 100% BS; except the G. darwini clade with 1.00 PP and 85% BS) which represent all the known species of Galagete including nearly all extant populations from the various islands, with two exceptions: G. espanolaensis which is nested inside the G. turritella clade (1.00 PP; 100% BS) and the population of G. protozona from Santa Fe which is nested inside the G. gnathodoxa clade (1.00 PP; 100% BS). Other interesting features appearing in the combined data tree is the monophyletic grouping of the three larger species, G. gnathodoxa, G. protozona, and G. seymourensis, (1.00 PP; 99% BS) recovered from the cladistic analysis based on morphology (Landry, 2002), and the basal placement of G. cinerea and G. consimilis (0.91 PP; 83% BS).

3.3. Molecular dating The first radiation event on the archipelago within the Galagete lineage obtained with the Bayesian relaxed molecular clock on the combined dataset and ML tree (Fig. 3) occurred around 3.3 ± 0.4 MYA. The genus radiated relatively quickly in about 1.8 million years, and the analysis gives an estimated speciation rate of 0.8 species per million years. The mini- radiation of the three larger species happened at 2.0 ± 0.3 MYA. G. cristobalensis and G. griseonana, two single-island endemics, split off at 2.7 ± 0.4 MYA and 2.5 ± 0.4 MYA respectively, and represent the oldest extant lineages of the radiation. The first split in the radiation coincides with the emergence of the oldest islands of Espanola and San Cristobal, the second with the emergence of Santa Fe. The radiation of 11 species of Galagete took place on the islands of Espanola, San Cristobal, Santa Fe, Santa Cruz, and Floreana, and was completed at 1.5 ± 0.3 MYA before the emergence of the other islands (dates indicated in Fig. 3).

47 CHAPTER 4

FIGURE 2. ML phylogram of Galagete species and population relationships based on a combined molecular analysis of 1008 bp mtDNA and 1062 bp nDNA sequence data, with nodal support indicated (Bayesian analysis, ML). A node is shown as fully resolved (*) if its posterior probability is ≥ 95 % and its bootstrap value is ≥ 75, respectively. Nodal support is indicated if its posterior probability is ≥ 75 % or its bootstrap value is ≥ 55. The▲ symbol indicates the G. protozona population on Santa Fe.

48 CHAPTER 4

4. DISCUSSION

4.1. Outgroups It is worth emphasizing three interesting features observed within the relationships to the outgroup taxa. First, the Autostichid assemblage sensu Kaila (2004) is strongly supported by both methods (MRBAYES, ML), excluding the Lecithoceridae Odites leucostola and Rhizosthenes falciformis. Second, the Bayesian analysis supports the paraphyly of the Autostichidae sensu Hodges (1998) and the basal position of Holcopogon bubulcellus (Holcopogonidae: Holcopogoninae), which is consistent with the results obtained by Kaila (2004). Third, regarding the Symmocinae sensu Hodges (1998), in which Galagete and Taygete were placed (Landry, 2002) with Oegoconia, the posterior probabilities support the close relationship of Taygete sphecophila with Galagete, but also the paraphyly of the subfamily because of the position of Oegoconia novimundi. Taygete sphecophila was originally described from specimens reared from a nest of Polistes wasps on Trinidad and was probably introduced to the Galapagos by anthropological means, jointly with Polistes nests (Causton et al., 2006; Landry et al., 2006) It is now identified as the closest relative of Galagete in morphological (Landry, 2002; Landry et al., 2006) and molecular analyses (this study). Interestingly, Darwin’s finches diverged also from a likely common ancestor occurring within the Caribbean islands (Burns et al., 2002). However, Central and South America are basically terra incognita for Gelechioidea and Taygete sphecophila may be more widespread than currently known.

4.2. Galagete phylogenetic relationships Our analysis of mtDNA and nDNA sequence data strongly supports the monophyly of Galagete and thus, the hypothesis of a single colonization event. Although some clades have unstable positions probably because of the very short internal branches in the likelihood analyses, all main lineages are supported. The lack of support in the internal structure of the trees could reflect the signature of the isolation and divergence of many populations in a short time frame, and thus, the tree topology probably is an accurate representation of historical relationships between taxa. The large number of genes used in this study and the extensive sampling reinforce this point of view. Therefore it should be considered as a valid phylogenetic hypothesis because it might represent a recent simultaneous speciation event (Hoelzer and Melnick, 1993). Galagete does not yet represent an adaptive radiation according to Schluter (2000) because it does not satisfy the evolution of ecological diversity within a

49 CHAPTER 4 rapidly multiplying lineage, but only the evolution of its phenotypic diversity. However, the life history of most Galagete species is poorly known and further studies will be necessary to assess the ecological diversity of the genus. The rapid speciation mechanism within Galagete may have happened twice in the Galagete colonization scenario, resulting in two major polytomies in the phylogeny (Fig. 3). The first event occurred at the base of the radiation just after the early colonizers arrived on the archipelago at around 3.3 MYA, and the second event is situated inside the radiation, with the simultaneous appearance of the three larger Galagete species between 1.5 and 2.0 MYA. The occurrence of more than one species within a clade could be explained by problems in recognizing species boundaries due to morphological variation, interspecific introgressive hybridization, or incomplete lineage sorting of ancestral polymorphism (Funk and Omland, 2003). Two examples can be found in the phylogeny of Galagete. First, Galagete espanolaensis, nested inside the G. turritella clade, may represent a geographical variant of the second species present on the oldest island of the archipelago, but it may also represent a valid taxon distinct from G. turritella, occurring on Espanola and San Cristobal. Interestingly, the three superficially indistinguishable species, G. consimilis, G. darwini, and G. espanolaensis, are genetically distinct and evolved separately. Two other cases of cryptic speciation were observed in the giant Galapagos tortoise population on Santa Cruz (Russello et al., 2005) and the Galapagos warbler finch in the archipelago (Petren et al., 1999; Tonnis et al., 2005), with one species occurring on the larger central islands while the other species are dispersed on smaller islands. Second, the peripheral population of G. protozona on Santa Fe, nested inside the G. gnathodoxa clade, is also of interest. Hybridization cannot be ruled out between the morphologically similar G. gnathodoxa and G. protozona as they often occur in sympatry or are present on adjacent islands. The peripheral population on Santa Fe may have been differentially and profoundly affected by past introgression. On the other hand, the isolation of this population may be more easily explained by incomplete lineage sorting. This hypothesis is supported by the budding speciation concept which produces patterns of paraphyly in both nuclear and mitochondrial loci (Funk and Omland, 2003), as shown with this G. protozona peripheral isolate. But since distinguishing between introgression and incomplete sorting is often difficult (Funk and Omland, 2003), it is tentative yet to decide between the two causes for this paraphyletic relationship. One factor that can accelerate lineage sorting is the relatively smaller population size due to bottlenecks presumably occurring on numerous occasions during the colonization of

50 CHAPTER 4 new islands in the Galapagos archipelago: this theory, first developed by Mayr (1954), is based on the influence of the founder effect in reducing levels of heterozygosity and leads, through random sampling error, to rapid differentiation between source and founder populations (Chakraborty and Nei, 1976; Hartl and Clark, 1989). During this study we found two examples of intra-archipelago diversification, G. levequei and G. pecki, which show both remarkable variation of the wing pattern between different island populations and well defined intraspecific relationships (see Fig.2). Specimens of G. levequei encountered on Fernandina are almost completely dark-brown compared to those found on the other islands, which are predominantly white, but there are intermediate forms. This great phenotypic variation encountered within G. levequei illustrates an interesting case of linear morphological gradation through the archipelago, with distinct genetic differentiation. The same may apply to the G. pecki populations in which variation is so striking that the population on Santa Cruz was thought to be a new species until it was described as a new subspecies based on morphological and molecular characters (Schmitz and Landry, 2005). Further studies will be necessary to estimate the role of founder effect in the differentiation of these taxa. Ricklefs and Bermingham (1998) have analyzed phylogenies of the West Indian avifauna and suggested that as populations get older, they become more restricted in their distribution and suffer an increased probability of extinction. Subsequent extinction among local populations may lead to global island extinction, or a new phase of range expansion leading to a renewal of the cycle (the “taxon cycle” theory, for a review see Emerson, 2002; Ricklefs and Bermingham, 2002). Within the Galapagos archipelago, according to this theory one can assume that the ranges of two species, G. cristobalensis and G. griseonana, which occur now only on older islands, may be due to local extinctions of once widespread ancestors. No clear pattern emerges from the G. darwini and G. turritella lineages. Both are common and present on each island and islet in the archipelago, often found close to the shore. Although some island populations are obviously closely related, the lack of branch support inside these clades suggests that gene flow has occurred recently between the different populations.

51 CHAPTER 4

FIGURE 3. Chronogram obtained from the combined mtDNA and nDNA data sets for the Galagete ingroup. Ages are inferred with the Bayesian rate autocorrelation method using five nodes under geological constraints (black dots). Horizontal boxes stand for ± one standard deviation around divergence ages. Vertical dashed lines indicate the estimated age of emergence of the islands. The▲ symbol indicates the G. protozona population on Santa Fe.

4.3. Origin, dating, and biogeography Changes in ocean currents were affected since 4.6 MYA due to the closure of the Isthmus of Panama, which was completed 3.6 MYA (Haug and Tiedemann, 1998), and were probably accompanied by alterations in the direction of the prevailing winds. The source of the founder of the Galagete group is probably coastal South America, which lies 1000 kilometers from the archipelago. Maybe one of the southeast trade winds carried the early colonizers of Galagete from the continent across the open waters of the Pacific Ocean to the Galapagos Islands by passive aerial transport, a mode of dispersal which is thought to account

52 CHAPTER 4 for the presence of most of the small-winged insects in the Galapagos (Peck, 2001). The oldest split within the Galagete lineage is estimated in this study at 3.3 ± 0.4 MYA, a result that seems reasonable compared to the earliest emergence date of the archipelago (3 MYA for the islands currently emerged but 9 MYA for the drowned seamounts). The rate of speciation of 0.8 species per million years corresponds to that calculated for the entire radiation of the Hawaiian Laupala criquets (Mendelson and Shaw, 2005). Compared to other native elements of the Galapagos fauna (see Table 3 for references), the Galagete colonization event happened after the arrival of the endemic iguanas (10.5-19.5 MYA) and Galapaganus weevils (10.7-12.1 MYA), which have diverged on the actual drowned islands, and before the giant Galapagos tortoises (1.5-2.0 MYA), the Darwin finches (1.2-2.3 MYA), and the Galapagos hawk (0.05-0.25 MYA), which represent recent arrivals on the extant islands. Although molecular estimates for Galapagos taxa are often plagued with problematic calibrations and emphasize previous use in the literature as justification for models (Grehan, 2001), new analytical methods, such as used here, enable the possibility to challenge recurrent questions on the chronological colonization scenario of oceanic islands. The common ancestor of all Galagete species, which may be phenotypically similar to G. cinerea and G. consimilis, could have arrived on the extant island of Espanola, or even before on the currently submerged islands like iguanids and weevils (Table 3). During initial colonization, opportunities for allopatric differentiation were probably limited. The radiation may have accelerated later as the archipelago increased in number of islands, but was accomplished before the last extant islands emerged. The apparent lack of any simple evolutionary pattern from older to younger islands distinguishes the Galapagos from more linear examples of animal radiation on hotspot archipelagos such as Hawaii (Roderick and Gillespie, 1998) and the Marquesas (Cibois et al., 2004). Although the simple stepping-stone colonization model is predominant in the Canary Islands, deviations from such a pattern have also been observed on this archipelago (Juan et al., 2000). The complex geological history and settings of the Galapagos Islands whose volcanoes are not aligned in a chain (White et al., 1993) may be reflected by the complexity of the colonization patterns. In the Galapagos, the historical biogeographic pattern expected if speciation followed in tandem with island formation, which is recovered in reptile and Land snail phylogenies (Caccone et al., 2002; Kizirian et al., 2004; Parent and Crespi, 2006), is in contrast with that found for birds (Bollmer et al., 2006; Tonnis et al., 2005) and our findings for Lepidoptera. The main cause is certainly the high vagility of the winged species which obscures the evolutionary pathways within the archipelago with probable back-colonizations underlining a stochastic dispersal

53 CHAPTER 4 pattern (Cowie and Holland, 2006). Although the nature of colonization in Galagete is stochastic, the arrival of the ancestor and the diversification of the radiation coincide with the chronological emergence of the major islands. It would be of interest to add microsatellites to the dataset as they have demonstrated use for reconstructing phylogenies of closely related taxa like Darwin's finches (Petren et al., 1999; Petren et al., 2005) or giant Galapagos tortoise populations (Ciofi et al., 2002; Ciofi et al., 2006; Russello et al., 2005). Such a detailed level of information would help to refine our understanding of this endemic radiation. A more comprehensive study on the ecology of the various Galagete species would also be worthwhile to confirm its adaptive nature and to elucidate the environmental causes underlying this insular radiation. Critical knowledge on Galagete is still missing and necessary for further inference regarding the conservation status of its species, ecological role, and importance. All these would be useful and contribute to strategies of arthropod conservation on the archipelago.

5. ACKNOWLEDGEMENTS

We would like to thank the authorities of the Galapagos National Park and the Charles Darwin Research Station, especially Lazaro Roque-Albelo, for allowing fieldwork and providing logistical support. We are also thankful for the assistance and companionship provided to B. Landry and P. Schmitz in the field by Stefania Bertoli, Novarino Castillo, Bill Conner, Valentina Cruz, Sarah Garrett, Henri Herrera, Jill Key, Jose Loaiza, Ana Maria Ortega, Stewart Peck, Lazaro Roque-Albelo, and Eduardo Vilema. We are grateful to Jackie Guiard and Benoît Stadelmann for technical assistance and to José Fahrni for help with sequencing. We are also deeply indebted to G. Baldizzone, R. Fritschi, L. Roque, T. Sarto, and K. Sugisima for their generous donations of specimens. Felix Sperling kindly suggested advices in the early stage of the molecular work and Daniel Rubinoff provided helpful comments on a previous draft of the manuscript. This work was supported by the Fondation Lombard, the Fondation Schmidheiny, the Fondation Claraz, the Swiss Academy of Sciences, the Baslerstiftung für biologische Forschung, and the Département des Affaires culturelles of the city of Geneva.

54 CHAPTER 4

TABLE 3. Molecular estimates (millions of years) determining the initial split within the lineage and the temporal window of divergence from the ancestor in different organism lineages of the Galapagos. et al. 1999, 2002 1999, al. et NANA 8.9 10.2 data electrophoretic for estimations Sarich and Nei with calibrated data allozyme data electrophoretic for estimations Sarich and Nei with calibrated data allozyme 1983 Wright 1983 Wright NANA 33.0 - 24.0 data rbc-L encoded plastid for on comparison based divergence sequence cpDNA 6.2 - 1.9 1992 Albert & Wendel data rbc-L encoded plastid for on comparison based divergence sequence cpDNA 1994 al. et Schilling 2.93.7 - NA Bayesianrelaxed molecular clockapproach andnDNA for mtDNA present study 3.0 - 34.0 - 3.0 48.0 - 33.0 clock molecular albumin on based distance immunological 1992 al. et Lopez 10.7 - 12.1 - 10.7 NA ratesarthropods with calibrated COI for clock molecular mtDNA 2000 al. et Sequeira within lineagewithin ancestor with weevils microlepidoptera Gossypium Scalesia lizards lava lizards lava lizards Galagete Galapaganus Darwin's finchesDarwin's Galapagos hawk Galapagos NA NA 2.3 - 1.2 0.051 - 0.254molecular clock andCOI, mtDNA forND2 calibrated Cytb, with geese rates ratesgeese with calibrated Cytb for clock molecular mtDNA et Bollmer al. 2006 2001 al. et Sato Tropidurus iguanidsGalapagos Tropidurus 20.0 - 15.0 NA clock molecular albumin on based distance immunological 1983 Sarich & Wyles Species split Initial Divergence data and Method Reference Phyllodactylus cotton Galapagos Flightless Galapagos daisy daisy Galapagos iguanidsGalapagos GiantGalapagos tortoise 1.52.0 - 19.5 - 10.5 6.0 - 12.0 NAmolecular clock mtDNA for and Cytb calibrated 16S rDNA with ectotherms rates Caccone rates ungulate with calibrated rDNA 16S and 12S for clock molecular mtDNA 1997 Rassmann

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CHAPTER 5

EVIDENCE FOR CRYPTIC SPECIATION ON GALAPAGOS VOLCANOES IN THE MICROMOTH GALAGETE DARWINI (LEPIDOPTERA, AUTOSTICHIDAE).

P. SCHMITZ a, b, A. CIBOIS a & B. LANDRY a

a Muséum d’histoire naturelle, C. P. 6434, CH-1211 Geneva 6, Switzerland. b Département de Zoologie et Biologie Animale, Molecular Systematic Group, Université de Genève, 30 quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland

Manuscript submitted.

57 CHAPTER 5

ABSTRACT

To get insight into the early stages of speciation, we reconstructed a DNA-based phylogeny, using combined mitochondrial and nuclear markers, of the extant populations of a moth species (Galagete darwini) endemic to the Galapagos that belongs to an insular radiation similar in size to that of Darwin’s finches. We present the first case in the Archipelago of speciation correlated with elevation and to some extent with vegetation zones on the Galapagos volcanoes. Cryptic speciation of the micromoth G. darwini may be driven by reproductive mechanisms or habitat preferences, rather than geographic barriers to dispersal alone, which are generally accepted to play the key roles in species formation and maintenance.

58 CHAPTER 5

1. INTRODUCTION

Volcanic oceanic islands represent an ideal microcosm for studying evolution in terrestrial arthropods (Roderick & Gillespie 1998, 2002). They show considerable habitat diversity as a result of gradients in topology and humidity, which, combined with their isolation, result in lower competition and empty ecological niches (Whittaker 2002). In the Galapagos, as observed in plants (Porter 1979), the greatest insect diversity and endemism is in the arid lowlands (Peck 2001), the largest ecological zone of the archipelago. This zone is predominant on the low islands and on the larger islands it extends inland to an elevation of over 120 meters on the southern slopes, and up to 300 meters or higher on the northern sides, where it is replaced by more humid vegetation zones at higher elevations (Wiggins & Porter 1971). In insect genera that have undergone extensive speciation in the Galapagos there is a general pattern in which the ancestral species were lowland colonists and evolutionary radiation within lineages took place in the arid regions (Peck 2001). Such a hypothesis is consistent with the fact that the Galapagos are known to have been drier than they are currently, with the vegetated areas of the wetter upland forests diminished or even absent (Colinvaux 1972). The micromoth Galagete darwini belongs to the largest endemic Galapagos radiation of Lepidoptera, being comparable in species numbers to that of Darwin's finches (Landry 2002; Schmitz & Landry 2005; Schmitz et al. in press). Although their feeding habits are known imprecisely, larvae appear to be scavengers (Landry 2002; Schmitz & Landry 2007). Nested inside the Galagete insular radiation, G. darwini is the most widespread species of the genus, occurring on each major island at almost all elevations (Landry 2002). In this paper we present a detailed phylogenetic analysis of Galagete darwini, based on several individuals per islands collected at different altitudes. This reveals the occurrence of cryptic taxa and suggests a speciation event linked to altitudinal zones.

2. MATERIAL AND METHODS

2.1 Samples and DNA methods Adults of G. darwini were attracted to light and collected in arid lowlands on eleven islands of the Galapagos Archipelago and in humid highlands on a subset of five of them (Fig. 1A). Accurate elevations and slope orientation of collecting localities were measured with a GPS (Garmin, GekoTM 301). Methods for DNA extraction, PCR amplification and sequencing of

59 CHAPTER 5

mitochondrial cytochrome oxidase I and II (COI and COII), and nuclear elongation factor-1α (EF1α), and wingless (WG) are available in Schmitz et al. (in press). The resulting sequences encompass a 555 bp fragment of COI, 453 bp of COII, 711 bp of EF1α, and 351 bp of WG, representing 2070 bp altogether. The new sequences obtained for the G. darwini specimens are deposited in GenBank (Table 1; accession numbers XXX) and were integrated in the global data set (Schmitz et al. in press).

Species Sample localities Altitude GenBank COI GenBank COII GenBank EF1α GenBank Wingless Galagete darwini (1) EC: Galápagos, Baltra 52m EF680612 EF680666 EF680721 EF680560 G. darwini (2) EC: Galápagos, Española 5m EF680602 EF680656 EF680711 EF680550 G. darwini (3) EC: Galápagos, Fernandina 353m EF680600 EF680654 EF680709 EF680548 G. darwini (4) EC: Galápagos, Fernandina 815m XXX XXX XXX XXX G. darwini (5) EC: Galápagos, Floreana 6m EF680615 EF680670 EF680725 EF680564 G. darwini (6) EC: Galápagos, Floreana 329m XXX XXX XXX XXX G. darwini (7) EC: Galápagos, Isabela 9m EF680606 EF680660 EF680715 EF680554 G. darwini (8) EC: Galápagos, Isabela, Volcano Alcedo 892m XXX XXX XXX XXX G. darwini (9) EC: Galápagos, Pinta 8m EF680607 EF680661 EF680716 EF680555 G. darwini (10) EC: Galápagos, Pinzón 14m EF680609 EF680663 EF680718 EF680557 G. darwini (11) EC: Galápagos, San Cristóbal 75m EF680590 EF680644 EF680699 EF680538 G. darwini (12) EC: Galápagos, Santa Cruz 22m EF680618 EF680673 EF680728 EF680567 G. darwini (13) EC: Galápagos, Santa Cruz 135m XXX XXX XXX XXX G. darwini (14) EC: Galápagos, Santa Cruz 225m XXX XXX XXX XXX G. darwini (15) EC: Galápagos, Santiago 6m EF680588 EF680642 EF680697 EF680536 G. darwini (16) EC: Galápagos, Santiago 527m XXX XXX XXX XXX G. darwini (17) EC: Galápagos, Seymour Norte 16m EF680589 EF680643 EF680698 EF680537

TABLE 1. List of Galagete darwini specimens used in the analysis and GenBank accession numbers of DNA sequences.

2.2. Data analysis Sequences were easily assembled manually, edited and aligned with BIOEDIT 7.0.5 (Hall 1999) and alignments were unambiguous. The concatenated alignments were used in phylogenetic analyses. The presence of stop codons or indels, which could reveal pseudogene sequences, were checked using MEGA 3.1 (Kumar et al. 2004). Hierarchical-likelihood ratio tests performed with MODELTEST 3.04 (Posada & Crandall 1998) determined that a General Time Reversible model with rate variation among sites and a proportion of invariable sites

(GTR+Γ8+I) represent the best-fit model of nucleotide substitution for the combined datasets. The Maximum Likelihood (ML) method was then performed with a heuristic search and random addition of sequences as implemented in PAUP* 4.0b10 (Swofford 2003), with the starting tree obtained via stepwise addition of taxa, and then swapped using the tree-bisection- reconnection (TBR) algorithm. The reliability of internal branches was assessed using the bootstrap method (Felsenstein 1985) with 1000 replicates by using the program PHYML 2.4.4 (Guindon & Gascuel 2003). Bayesian posterior probabilities were calculated using a Metropolis-coupled, Markov chain, Monte Carlo (MCMCMC) sampling approach, as implemented in MRBAYES 3.1.2 (Huelsenbeck & Ronquist 2003), using the same

GTR+Γ8+I model. Four simultaneous Markov chains were run in parallel twice for 1 million

60 CHAPTER 5 generations with trees sampled every 10 generations. Stationarity was evaluated graphically and the first 250,000 initial trees were discarded as burn-in. MRBAYES was set to estimate model parameters independently and simultaneously for each gene partition. In the maximum likelihood tree, when posterior probabilities and bootstrap support did not reach ≥95% and ≥75% of significant support values, respectively, the nodes were collapsed. All above- mentioned analyses were run through the Bioportal web-based service platform for phylogenomic analysis at the University of Oslo, Norway.

3. DISCUSSION

The combined mtDNA and nDNA phylogeographic analysis surprisingly revealed that G. darwini populations at higher elevation on the western large islands (Fernandina, Isabela, and Santiago) represent a distinct lineage from the one found in the low arid zones of these same islands (Fig. 1B). This result suggests the presence of at least two cryptic taxa, which inhabit different elevations on these islands. The "arid lowland" lineage includes all populations of G. darwini present on the small islands as well as the populations occurring in the arid zones of the western large islands. On the opposite, no differences appear between highland and lowland moths on the islands of Floreana and Santa Cruz. On Fernandina, Isabela, and Santiago, the presence of at least two cryptic taxa suggests a geographical isolation process that is correlated with elevation and to some extent with vegetation zones. This pattern suggests that speciation probably occurred first on Floreana or Santa Cruz by allopatric isolation of an arid lowland ancestor, followed by a niche shift into humid highland habitat on these two original islands and subsequent colonization of the higher zones of Fernandina, Isabela, and Santiago. This scenario is consistent with the phylogenetic reconstruction in that the basal taxa are found in coastal arid habitats whereas the more derived taxa occur in the higher and more humid zones. A trend observed also in other insects inhabiting the Archipelago like the flightless Galapaganus weevils (Sequeira et al. 2000). The colonization of the summit of the volcanoes by the "humid highlands" lineage was followed by secondary contact between the resultant species of the "arid lowlands" lineage on the western islands. The strong intraspecific genetic homogeneity in the "humid highlands" lineage suggest that gene flow or incomplete lineage sorting has been maintained between the populations of Santa Cruz and the highland populations on Fernandina, Isabela, and Santiago. The recent and historical volcanic activity of the young western islands (White et al. 1993) could also have influenced the colonization and evolutionary diversification of the

61 CHAPTER 5 summit by the "humid highlands" lineage, as observed for Galapagos tortoises (Beheregaray et al. 2003). Although cryptic species in the Galapagos occur also in other taxa (Russello et al. 2005; Tonnis et al. 2005), our results highlight the first case in the archipelago of cryptic speciation correlated with elevation. Our data support the idea that these small-winged insects are vagile taxa whose speciation may be driven by reproductive mechanisms or habitat preferences, rather than geographic barriers to dispersal alone, which are generally accepted to play the key roles in species formation and maintenance (Lack 1947).

4. ACKNOWLEDGEMENTS

We thank the Galapagos National Park Service for permission to conduct this research, the Charles Darwin Research Station and the Charles Darwin Foundation for logistical support. We are also thankful for the assistance and companionship provided to B. Landry and P. Schmitz in the field by Stefania Bertoli, Novarino Castillo, Bill Conner, Valentina Cruz, Sarah Garrett, Henri Herrera, Jill Key, Jose Loaiza, Ana Maria Ortega, and Lazaro Roque-Albelo. We thank Ken Petren and Dan Rubinoff for comments on early drafts of the manuscript. This project was supported by Foundations Claraz, Lombard, Schmidheiny, the Swiss Academy of Sciences, the Baslerstiftung für biologische Forschung, and the Département des Affaires culturelles of the City of Geneva.

62 CHAPTER 5

FIGURE 1. Galagete darwini. (A) Geographical locations within the Galapagos Archipelago (map modified from (7)), and (B) combined mtDNA and nDNA-based phylogenetic relationships of populations. Distributions of G. darwini (yellow), with analyzed "arid lowlands" populations (red), and "humid highlands" populations (green) in the archipelago are indicated. The map shows the 300 and 600 m elevation contours and abbreviated island names in parentheses. The maximum likelihood tree is shown with elevation and slope orientation of collecting localities identified with abbreviation of island names (a = arid zone; h = humid zone). When bootstrap support and posterior probabilities did not reach ≥80% and ≥95% of significant support values, respectively, the nodes were collapsed.

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64

GENERAL CONCLUSIONS AND PERSPECTIVES

65 GENERAL CONCLUSIONS AND PERSPECTIVES

Our investigations of the molecular phylogeny and biogeography of an insular endemic radiation have considerably broadened our knowledge on the diversity, distribution, ecology, evolutionary history, and speciation processes of the microlepidoptera genus Galagete. First, our morphologic studies lead us to describe several new taxa used afterwards in our phylogenetic reconstructions (Chapter 1 and 3). Second, we discovered the biology of one species of Galagete and described and illustrated the immature stages in details (Chapter 2). Third, our molecular and biogeographic analyses allowed us to obtain a solid phylogeny of the species of Galagete and the various insular populations. They provided also crucial information regarding the evolutionary history and chronological colonization scenario of the Galapagos by this endemic radiation (Chapter 4). Moreover, our results identified inside the radiation an event of cryptic speciation observed between two distinct populations of G. darwini subdivided according to elevation on the western islands (Chapter 5).

However, several aspects await further development to open new insights in our understanding of this insular endemic radiation:

1) It would be of interest to add microsatellites to the dataset. Such a detailed level of information would help to refine the resolution of the interspecific relationships inside this endemic radiation.

2) Carrying on the collecting efforts on the South American coast for which the inventory of the microlepidoptera fauna is still incomplete will certainly lead to discovering new species closely related to Galagete or a potential common ancestor.

3) A more comprehensive study of the ecology of the various Galagete species would also be worthwhile to confirm its adaptive nature and to elucidate the environmental causes underlying this insular radiation.

4) Critical knowledge on Galagete is still missing and necessary for further inference regarding the conservation status of its species, ecological role, and importance. All these would be useful and contribute to strategies of arthropod conservation on the archipelago.

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APPENDIX

81 REVUE SUISSE DE ZOOLOGIE 113 (2): 307-323; juin 2006

Taygete sphecophila (Meyrick) (Lepidoptera; Autostichidae): redescription of the adult, description of the larva and pupa, and impact on Polistes wasps (Hymenoptera; Vespidae) nests in the Galapagos Islands

Bernard LANDRY (BL)1, David ADAMSKI (DA)2, Patrick SCHMITZ (PS)3, Christine E. PARENT (CEP)4 & Lazaro ROQUE-ALBELO (LR)5 1 Muséum d'histoire naturelle, C.P. 6434, 1211 Genève 6, Switzerland. E-mail: [email protected] 2 Department of Entomology, National Museum of Natural History, P.O. Box 37012, NHB - E523, Smithsonian Institution, Washington, DC, 20013-7012, USA. E-mail: [email protected] 3 Muséum d'histoire naturelle, C.P. 6434, 1211 Genève 6, Switzerland. E-mail: [email protected] 4 Behavioural Ecology Research Group, Department of Biological Sciences, Simon Fraser University, Burnaby, British-Colombia V5A 1S6, Canada. E-mail: [email protected] 5 Department of Entomology, Charles Darwin Research Station, Casilla 17-01-3891, Quito, Ecuador; Biodiversity and Ecological Processes Research Group, Cardiff School of Biosciences, Cardiff University, PO Box 915, Cardiff CF10 3TL, United Kingdom. E-mail: [email protected]

Taygete sphecophila (Meyrick) (Lepidoptera; Autostichidae): redescrip- tion of the adult, description of the larva and pupa, and impact on Polistes wasps (Hymenoptera; Vespidae) nests in the Galapagos Islands. - Taygete sphecophila (Meyrick) (Lepidoptera; Autostichidae) is reported on the Galapagos Islands. The morphology of the moth, larva, and pupa are described and illustrated in details. Part of the mitochondrial DNA was sequenced and made available on GenBank. The incidence of predation by T. sphecophila on nests of Polistes versicolor Olivier (Hymenoptera; Vespidae) was measured in four different vegetation zones of Floreana and Santa Cruz Islands. The percentages of infested nests varied greatly (from 13.9% to 66.7% on Floreana and from 20.0 to 100% on Santa Cruz) and no clear ecological trends could be ascertained. Keywords: Micro moths - Autostichidae - Taygete - Polistes - Galapagos Islands - mitochondrial DNA - larval predation - morphology - ecology.

INTRODUCTION Taygete was described by Chambers (1873) to accommodate Evagora diffi- cilisella Chambers, 1872 (Nye & Fletcher, 1991). The latter name proved to be a synonym of T. attributella (Walker, 1864). The genus appears to be restricted to the

Manuscript accepted 06.12.2005 308 B. LANDRY ET AL.

New World. Becker (1984) lists 13 names in this genus for the Neotropical fauna while Hodges (1983) lists six species for the North American fauna, including five that are stated to be misplaced in this genus. BL's examination of the type specimens of the Neotropical species at the Natural History Museum, London, points to the possibility that only T. sphecophila (Meyrick, 1936) is congeneric with T. attributella in this re- gion. However, the types of Epithectis consociata Meyrick, E. notospila Meyrick, and E. altivola Meyrick have lost their abdomen and cannot be assigned to genus, and the type of E. lasciva Walsingham, deposited in the USNM, Washington, could not be found. Taygete Chambers was considered to belong to the Gelechiidae until Landry (2002) moved it to the Autostichidae, Symmocinae sensu Hodges (1998). Taygete sphecophila was described from three specimens bred in Trinidad from "bottom of cells of the Hymenopteron Polistes canadensis" (Meyrick, 1936). The moth and male genitalia were later illustrated with black and white photography by Clarke (1969). On the Galapagos Islands moths of T. sphecophila were first collected in 1989 by BL, but the species probably arrived earlier within nests of Polistes versicolor Olivier (Vespidae). The purposes of this paper are to redescribe and illustrate the moth of T. sphe- cophila, to describe and illustrate the larva and pupa, to present part of its mito- chondrial DNA, and to report on a few aspects of its biology, particularly with regard to the incidence of damage to P. versicolor nests by larvae.

MATERIAL AND METHODS Moths of T. sphecophila were first collected at night with a mercury vapor light set in front of a white sheet and powered by a small generator, and with an ultra-violet lamp powered by a battery. Other adult specimens were reared from contained nests of Polistes versicolor. Immature stages were found by dissecting Polistes nests and by exposing them to the sun, which causes larvae to exit nests and run away from them (Fig. 2). Specimens are deposited in the Charles Darwin Research Station (CDRS), Santa Cruz, Galapagos, Ecuador; the Canadian national Collection of Insects (CNC), Ottawa, Ontario, Canada; the United States National Museum of Natural History, Washington, D.C., U.S.A. (USNM), and the Muséum d'histoire naturelle (MHNG), Geneva, Switzerland. For the study of specimens using electron microscopy, larvae and pupae were first rinsed several times in water, cleaned in 10% EtOH with a camel hairbrush, and then dehydrated in EtOH as follows: 10% EtOH for 15 minutes, 20% for 15 minutes, 40% for 15 minutes, 70% for 1/2 hour, 90% for 1/2 hour, and 100% for 1/2 hour each in two separate baths. After dehydration, specimens were critical-point dried using a Tousimis critical point dryer, mounted on stubs, and coated with gold-palladium (40- 60%) using a Cressington sputter coater. The ultrastructure of the larvae and pupa was studied with an Amray scanning electron microscope. Gross morphological observations and measurements of the larvae and pupae were made using a dissecting microscope (reflected light) with a calibrated micrometer. TAYGETE SPHECOPHILA IN THE GALÁPAGOS ISLANDS 309

FIGS 1-2 1, Taygete sphecophila, female; 2, part of an abandoned nest of Polistes versicolor exposed to the sun with at least 8 larvae of Taygete sphecophila exiting from it. Maps of the larval chaetotaxy were initially drawn using a WILD dissecting micro- scope with a camera lucida attachment. Terminology for chaetotaxy follows Stehr (1987). 310 B. LANDRY ET AL.

In order to certify that the larvae corresponded to the adults found we sequenced a fragment of the mitochondrial gene Cytochrome oxidase I (COI) of both. Whole genomic DNA was extracted using the Nucleospin kit (Macherey-Nagel). The COI gene was amplified by PCR with two primers: k698 (5’-TACAATTTATCGCC- TAAACTTCAGCC-3’), and Pat2 (5’-TCCATTACATATAATCTGCCATATTAG-3’). The thermal profile started with an initial denaturation at 95°C for 5 min, followed by 35 cycles at 94°C for 30 s, 47°C for 30 s, and 72°C for 1 min 30 s, and a final step at 72°C for 10 min. The purified PCR product was sequenced in both directions using fluorescent dye terminators in an ABI 377 automated sequencer. The sequence is available from GenBank (Accession No. DQ309437). In order to determine the distribution and the density of Taygete sphecophila as predator on Polistes versicolor nests, several study sites were selected in four of the vegetation zones of Santa Cruz and Floreana Islands. In each vegetation zone a series of quadrats of 10 m x 10 m were made at random, and the number of active and inactive nests of Polistes versicolor were counted. The delimitation of vegetation zones was based on vegetation composition (Wiggins & Porter, 1971). Nests were found by visually searching the study sites. In addition, nests found in and near Puerto Ayora, a small town located on the littoral and arid zones on the south coast of Santa Cruz Island, were included in the study. The presence of T. sphecophila in Polistes nests was determined by the presence of little holes on the back of the nests (Fig. 2) and distinc- tive breaches on the capped cells normally occupied by wasp pupae. In 1999, nests of Polistes versicolor were monitored weekly in the area of Puerto Ayora, and nests that were abandoned after being infested by T. sphecophila were collected during that period of time. Some adults of T. sphecophila that emerged from these nests were preserved dry for taxonomic identification. The ecological observations were made between April and August 1999, February and April 2002 and 2003 on Santa Cruz Island, and between April and August 1999 on Floreana Island. To test for ecological or insular trends in the frequency of parasitism of P. versicolor nests by T. sphecophila, we performed a G-test for goodness of fit (Sokal & Rohlf, 1995) on each island dataset using the proportion of P. versicolor nests in a given zone to infer the expected frequency of parasitism by T. sphecophila.

TAXONOMIC TREATMENT Taygete sphecophila (Meyrick) Epithectis sphecophila Meyrick, 1936: 624; Gaede, 1937: 113; Clarke, 1955: 290; Clarke, 1969: 63, pl. 31 figs 4-4b; Makino, 1985: 25; Yamane, 1996: 85. Taygete sphecophila (Meyrick); Becker, 1984: 47; Landry, 1999: 68; Landry, 2002: 818-819. MATERIAL EXAMINED FOR MORPHOLOGICAL WORK: Moths (13 specimens from the Galapagos Islands, Ecuador): SANTA CRUZ: 1 &, C[harles] D[arwin] R[esearch] S[tation], arid zone, 19.i.1989, M[ercury] V[apor] L[amp] (B. Landry); 4 & (two dissected with genitalia on slides CNC MIC 4586 & BL 1196, the latter with right wings on slide BL 1313), CDRS, arid zone, 3.ii.1989, MVL (B. Landry); 2 ( (one dissected, slide BL 1126), Barranco, ex larva en nido Polistes versicolor, 8.ii.1999 (L. Roque, No. 99.20); 1 &, NNW Bella Vista, GPS: 225 m elev., S 00° 41.293’, W 090° 19.665’, 18.ii.2005, u[ltra] v[iolet] l[ight] (B. Landry, P. Schmitz); 1 & (dissected, slide BL 1195), 2 km W Bella Vista, 27.ii.1989, MVL (B. Landry); 1 &, casa L. Roque-Albelo & V. Cruz, GPS: 137 m elev., S 00° 42.595’, W 090° 19.196’, 27.ii.2005, uvl (B. TAYGETE SPHECOPHILA IN THE GALÁPAGOS ISLANDS 311

FIG.3 Taygete sphecophila, male genitalia (sizes not proportionate). 3a, dorsal view of valvae + vin- culum + juxta and ventral view of tegumen + uncus + gnathos detached on right side and spread on left side, phallus removed, setae shown on right side only; 3b, side view of phallus with vesica everted; 3c, dorsal view of phallus, vesica inverted, scale = 0.1 mm; 3d, lateral view of whole genitalia. 312 B. LANDRY ET AL.

Landry); 2 ( (dissected, slides BL 1208 & 1209), émergé d’un nid de Polistes, 1999 (C. Parent). SAN CRISTOBAL: 1 (, antiguo botadero, ca. 4 km SE P[uer]to Baquerizo, GPS: 169 m elev., S 00° 54.800’, W 089° 34.574’, 22.ii.2005, uvl (B. Landry). Larvae (166 specimens) and pupae (10 specimens) collected on Santa Cruz by P. Schmitz in 2004 and 2005.

DIAGNOSIS: The presence in males of this species of a corematal organ at the base of the abdomen (Fig. 4) and a trifurcated uncus (Fig. 3a) are excellent diagnostic features with regards to the rest of the Galapagos fauna. Males of Galagete Landry are the only other Galapagos moths to share a corematal organ, but their uncus is made of a single projection. In females the shape of segment VIII (Fig. 5), especially dorsally, will separate T. sphecophila from any other species in the Galapagos and probably the rest of its range. On the archipelago, some species of Galagete Landry (2002) or Gelechiidae may appear superficially similar, especially because they share a similarly shaped hindwing, a similar wingspan, upturned labial palpi, and scales on the proboscis basally, but the forewing markings of T. sphecophila (Fig. 1) are unique among these groups. REDESCRIPTION: General appearance of moth greyish brown with dark brown markings on forewing (Fig. 1); scales usually dark brown at their base and paler apically. Head scales longer laterally and directed medially and ventrally, except on occiput, directed medially and dorsally. Ocellus and chaetosema absent. Labial palpus gently curving upward, darker brown laterally than medially, with white rings of scales mostly at apex of segments; segments II and III shorter together than segment I. Antenna mostly greyish brown, darker brown toward base; flagellomeres in both sexes simple and with erect scales ventrally from about middle of flagellum. Thorax conco- lorous with head, sometimes darker brown at base. Foreleg mostly dark brown, with beige scales at apex of tarsomere I and on all of tarsomere V. Midleg mostly dark brown laterally, with paler scales at apex of tarsomeres I and II, and on all of tarsomere V, uniformly beige medially on femur and tibia, also with short tuft of dark brown scales dorsally on basal half of tibia. Hindleg paler than other legs, with some dark brown laterally on femur and tibia, mostly dark brown on tibial spines and at base of tarsomeres I-IV, also with tuft of long dirty white scales on dorsal margin of tibia. Wingspan: 7.5-9.0 mm. Forewing mostly greyish brown, with three dark brown trian- gular markings on costa, largest marking at base, reaching inner margin, smallest sub- medially situated, barely reaching cell, third marking large, reaching middle of wing; with dark brown scaling also at apex and as 1-3 small patches of 10 scales or less below postmedian costal marking; also with variable amounts of yellowish-orange to rusty- brown scales usually within basal dark brown marking, below postmedian marking, and toward apex; fringe dark brown at apex, more greyish brown elsewhere. Hindwing greyish brown without markings, with concolorous fringe. Wing venation (based on slide BL 1313, female) (Fig. 6): Forewing Sc to about 2/5 wing length; R1 from about middle of cell; R2 and R3 separate, both from before upper angle of cell; R4, R5, and M1 from upper angle of cell, connected, R4 and R5 directed toward costa before apex, M1 directed toward outer margin below apex; M2 and M3 separate, M2 from lower angle of cell, M3 from shortly before lower angle of cell; CuA1 and CuA2 separate, both from shortly before lower angle of cell; CuP absent; cell a little more than half TAYGETE SPHECOPHILA IN THE GALÁPAGOS ISLANDS 313

FIGS 4-5 Taygete sphecophila. 4, ventral view of first abdominal segment; 5, ventral view of female genitalia, setae shown on right side only. wing length; A1 and A2 joined at about 1/5 their lengths. Female forewing retinaculum consisting of anteriorly directed scales at base of cubital stem and posteriorly directed scales at base of Sc. Hindwing Sc closely following costa, reaching it at about 3/5 wing length; Rs connected with M1 after upper angle of cell, Rs reaching costa at about 4/5 314 B. LANDRY ET AL. wing length, M1 directed toward apex; M2 from slightly above lower angle of cell, reaching outer margin below middle; M3 and CuA1 connected for about 1/2 their lengths after lower angle of cell, M3 to tornus, CuA1 to inner margin shortly before tornus; CuA2 from about 2/3 cell to inner margin at 7/10; CuP and anal veins indis- tinct; apex distinctly produced; outer margin distinctly concave; female frenulum with 2 acanthae. Abdomen dorsally mostly dark greyish brown, with dirty white scales at apex of all segments except last; ventrally dark brown on each side of large dirty white band except for last segment, mostly concolorous, greyish brown; male first abdominal segment (Fig. 4) ventrally with an invaginated pouch containing a membranous structure bearing scales (see Note below). Male genitalia (Fig. 3). Uncus moderately long, with pair of fixed lateral, pointed and gently tapering glabrous projections; also with movable median projec- tion, slightly longer than lateral projections, enlarged at apex and bifid, with each end bulbous and setose, also slightly setose at base laterally. Gnathos a long curved rod pointing posteriorly, apically more heavily sclerotized, tapered, glabrous, and rounded. Tegumen broad medially, with moderately narrow pedunculi. Valva with unsclerotized setose cucullus, tapering, rounded apically, with slightly sclerotized setose ridge at base on inner side, also with medium sized apodemes directed anteriorly from base of costa; sacculus with pair of short, narrow, setose, and apically rounded projections, dorsal projection curved and directed dorsally, ventral one straight and directed posteriorly. Vinculum narrow, slightly projected anteriorly and upturned. Juxta poorly developed, small, better sclerotized at posterior edge around phallus. Phallus (= aede- agus of authors, but see Kristensen, 2003) narrow, with shaft flattened dorsoventrally beyond middle, better sclerotized on left side in narrow band, slightly upturned apically; coecum penis medium-sized with pair of very small peduncles laterally; vesica with minute scobination. Female genitalia (Fig. 5). Papillae anales large and long, moderately setose, sclerotized dorsally and laterally at base. Posterior apophyses slightly curved apically, slightly longer than papillae. Tergum VIII well sclerotized, with few long setae espe- cially on margin, with deep rounded concavity in middle apically; middle of concavity with posteriorly directed projection variable in length and bearing two setae. Anterior apophyses straight, slightly enlarged apically, about as long as papillae. Sternum VIII with apical margin bell shaped, well sclerotized, with few long setae mostly posteriorly along margin and midventrally. Intersegmental membrane between sternites VII and VIII slightly sclerotized on each side of midventral line and with pair of short projec- tions inside body at apical margin. Ostium bursae in middle of sternite VIII, ventrally protected by slightly protruding crescent of sclerotization. Ductus bursae short, gradually enlarging, basal half well sclerotized, distal half spiculose and with wrinkles patterned like brood cells in bee hive. Corpus bursae slightly longer than wide, spicu- lose, with one large, spiny, curved, and pointed cornutus; latter set in small sclerotized patch with pair of bumps on each side of its base.

DESCRIPTION OF THE LARVA AND PUPA: Larva. (Figs 7-17): Length 5.0-8.2 mm (n = 72), < 5.0 mm (n = 94). Body pale gray, textured with microconvolutions; head capsule amber; prothoracic shield amber, gradually darkening posteriorly; pinacula pale brown; anal plate pale amber; setae with widened, circular, and slightly raised TAYGETE SPHECOPHILA IN THE GALÁPAGOS ISLANDS 315

FIG.6 Wing venation of Taygete sphecophila. sockets. Head (Figs 7-10, 17): hypognathous, textured with slightly raised, confluent, polygonal ridges except on area between adfrontal sclerites (Figs 7-8); adfrontal scle- rites widened distally, frontal setae about equal in length, AF2 above apex of frons, AF1 below; F1 slightly closer to AF1 than to C1; C2 at least 2 1/2 times longer than C1; clypeus with 6 pairs of setae, 3 pairs on medial half, 3 on distal half; mandible angular (Fig. 17), shallowly notched subapically forming small apical dentition, bearing pair of subequal setae on outer surface near condyle, and with 1 large dentition on inner surface; sensilla types and arrangement on antenna (Fig. 9) and on maxillary palpi (Fig. 10) similar to those of other Gelechioidea studied by Adamski & Brown (1987), Adamski (1999), Adamski & Pellmyr (2003), Landry & Adamski (2004), and Wagner et al. (2004), and other Lepidoptera studied by Adamski & Brown (2001), Albert (1980), Avé (1981), Grimes & Neunzig (1986a, b), and Schoonhoven & Dethier (1966). Three stemmata in genal area, 1 approximate pair above antenna, and 1 stemma below antenna; substemmatal setae about equal in length, arranged as in Fig. 8; S3 and S1 elongate and about equal in length, S2 short; S3 lateroventral to S2, S2 approximate to stemma 3, and S1 approximate to stemma 5 (stemmata 1, 2, and 6 absent); A-group setae above gena, mesal to L1; P1 dorsolateral to AF2, P2 dorsomesal to P1. Thorax (Figs 11, 14): T1 with L-group trisetose, on large pinaculum extending beneath and posterior of spiracle; setae anterior to spiracle; L1 approximate and posteroventral to L2, about 2 1/2 times lengths of L2 and L3; SV-group setae on anterior part of elon- gate pinaculum; SV1 about 1/3 longer than SV2; coxae nearly touching, V1s very approximate (not shown); segments of leg textured with slightly elongate ridges, many produced distally into hairlike spines, claw single (Fig. 11); shield with SD1 slightly posterior to and about 1/3 longer than XD2 and XD1; XD2, XD1, D1, and SD2 about 316 B. LANDRY ET AL.

FIGS 7-13 Scanning electron micrographs of larva of Taygete sphecophila. 7, Frontolateral view of head capsule, scale = 100 µ; 8, Ventrolateral view of head capsule, scale = 100 µ; 9, Sensilla of antenna: 1 = sensilla basiconica, 2 = sensillum chaetica, 3 = sensillum styloconicum, 4 = sen- sillum trichodeum, scale = 10 µ; 10, Sensilla of maxillary palpus: A2 = sensillum styloconicum, A1, A3, M1-2, L1-3 = sensilla basiconica, SD = sensillum digitiform, scale = 10 µ; 11, Distal portion of left prothoracic leg showing claw, scale = 10 µ; 12, Left proleg on A4, scale = 100 µ; 13, Anal plate of A10, scale = 100 µ. TAYGETE SPHECOPHILA IN THE GALÁPAGOS ISLANDS 317 equal in lengths, XD2 about twice distance from XD1 than from SD1; D1 in straight line with XD1, slightly posterior to SD2 and D2; D2 about same length as SD1, in straight line with SD2. T2-T3 (Fig. 14): D2 about 2 times length of D1, both on small pinaculum; SD1 about 2 times length of SD2, both on small pinaculum; L1 about 1/3 longer than L2, both on small pinaculum, L3 slightly shorter than L2, posterior to or in vertical line with SV1; MV1 on anterior margin between T2-T3, slightly above SV1 (hard to see); V1s on T2-T3 about equal distance apart, at least 4 times distance between V1s on T1. Abdomen (Figs 12, 13, 15, 16): A1-A2 (Fig. 15): D2 and D1 equal in lengths or D2 slightly longer, MD1 on anterior part of segment anteroventral to D1; SD1 above spiracle, about 1/3 longer than D2, with minute SD2 (anterior part of pina- culum); small opening on ventroposterior margin of pinaculum bearing SD1 and SD2; spiracle on A1 slightly larger than those on A2-A7; L1 2 times length of L2, both on same pinaculum, slightly anterior of spiracle; L3 about same length as L2, anterior to, in vertical line with, or posterior to D2; SV-group bisetose on A1, trisetose on A2, on same pinaculum; V1s equal distance apart (not shown). A3-A10 (Figs 12, 13, 16): A3- A6 with 4 pairs of protuberant prolegs, crochets biordinal, in circle (Fig. 12); setae as above; A7 as above except, SV-group bisetose and on same pinaculum; A8 as above except with spiracle slightly larger than on previous segments and SV-group unisetose; A9 with D2 about 2-2 1/2 times longer than D1; D1 anterior to D2 and SD1, equi- distant to both setae; SD1 about same length as D1; L-group setae slightly anterior to D1; L1 about 3 times length of L2, on same pinaculum; L3 slightly longer than L2; SV1 slightly shorter than L1; V1s as previous segments; A10 (Figs 13, 16): anal plate with SD2 and SD1 equal in lengths, about twice length of D2; D1 slightly shorter than D2; crochets of proleg biordinal, in semicircle, gradually shortened mesally and laterally. Pupa. (Figs 18-21): Length 3.6-4.6 (n = 10): amber, smooth, spiracles protu- berant; all dorsal setae apically hooked except long seta associated with axillary tubercle (Figs 19-20). Sclerites of antennae annulated, widely separated anteriorly, gradually convergent from beyond basal 1/3 of sclerites of maxillae, fused for short distance beyond distal apices of sclerites of maxillae, gradually divergent posteriorly, exposing distal part of sclerites of hindlegs; sclerites of midleg not fused distally; paired nodular scars of prolegs on A5-A6 (Fig. 18); A6-A10 fused, rotating as unit; cremaster dorsolaterally flattened, trapezoidal basally, extending posterolaterally into 2 slightly divergent and elongate spine-like processes (Fig. 21).

DISTRIBUTION AND PHENOLOGY: The species was described from Trinidad (Meyrick, 1936) and never mentioned from anywhere else subsequently. In the Galapagos Islands it has been found on Floreana (from the littoral to the humid zones), San Cristobal (in the arid zone), and Santa Cruz (from the littoral to the humid zones). In the Galapagos we have collected live moths of this species in January, February, March, April, September, November, and December.

NOTES: Preliminary phylogenetic analyses, both morphological and molecular, support the placement of Taygete sphecophila within Autostichidae (PS, unpublished data). For example, Kaila’s (2004) matrix was reanalyzed with T. sphecophila data, and the species clusters in Kaila’s autostichid assemblage with Galagete Landry. 318 B. LANDRY ET AL.

FIGS 14-17 Larva of Taygete sphecophila. 14, Chaetotaxy of thorax; 15, Chaetotaxy of abdominal segments 1-2; 16, Chaetotaxy of abdominal segments 6-10; 17, Mandible. A comparison of a 1283 base pairs fragment (consisting of most of the COI gene except the first 254 base pairs) sequenced for a larva and an adult of Taygete spheco- phila showed no substitution, which clearly indicates conspecificity. The larva has only three stemmata, a condition that is highly unusual in Gelechioidea and that may be due to the unique host relationship. It proved impossible to evaginate the ventro-abdominal pouch (Fig. 4) in several male specimens of this species. However, BL was able to evaginate this core- matal organ from a specimen of Taygete attributella (Walker). The organ consists of a narrow membranous tube, almost as long as the abdomen, on which narrow scales are connected all around. The membrane of the tube is very thin and the tube collapsed as soon as specimens were transferred to lactic acid for temporary storage. An illustration of this structure for the closely related Galagete turritella Landry is provided by Landry (2002: Figs 17, 18). TAYGETE SPHECOPHILA IN THE GALÁPAGOS ISLANDS 319

FIGS 18-21 Pupa of Taygete sphecophila. 18, Ventral view; 19, Dorsal view; 20, Segments 8-10; 21, Lateral view.

ECOLOGICAL STUDY

PATTERNS OF PREDATION Although egg-laying was never observed, it is possible that the female moths lay their eggs within the pupal cells of P. versicolor through numerous small holes of 1-2 mm in diameter that we observed on the back of the nests. In a sample of 25 P. versicolor nests, the number of T. sphecophila moths found per nest varied between 3 and 13. However, 42 T. sphecophila larvae were recovered by PS from a rather small nest collected on Santa Cruz in 2004. The food source needed for the development of the moth’s larvae are the wasps’ pupae which are defenseless because of their isolation in their capped cells. When ready to emerge from the wasp's cell, the moth makes a distinctive breach through the cap covering the top of the cell.

DISTRIBUTION OF INFESTED NESTS The level of T. sphecophila infestation could only be assessed for nests of P. versicolor that were abandoned. A total of 103 such nests were found on the different study sites on Santa Cruz Island between 1999 and 2003, and 141 nests on Floreana Island in 1999. The percentages of nests that presented signs of predation by T. sphe- cophila are given in Table 1, along with the vegetation zones in which they were found. 320 B. LANDRY ET AL.

TABLE 1. Percentage of Polistes versicolor nests found in four different vegetation zones of Santa Cruz and Floreana Islands presenting signs of Taygete sphecophila predation. Number of nests per sample are in parentheses. Vegetation Zone % of nests with T. sphecophila predation Santa Cruz Island Floreana Island Littoral 35.3 (n=17) 40.0 (n=5) Arid 43.0 (n=79) 13.9 (n=101) Transition 20.0 (n=5) 66.7 (n=6) Humid 100.0 (n=2) 51.7 (n=29)

On Santa Cruz island the arid zone was the area of highest abundance of nests. This result is similar to that obtained by Roque-Albelo & Causton (1999) for abun- dance of adult foragers. The percentages of infestation varied between zones (Table 1). However, very few nests were collected in the littoral, transition, and humid zones. Nests of P. versicolor were again more common in the arid zone of Floreana. However, only 13.9% of them were infested by T. sphecophila in this zone. In contrast to Santa Cruz, on Floreana nests also were abundant in the humid zone, where 51.7 % of them were infested. The results of the G-test for goodness of fit allow us to test for ecological trend in nest infestation according to vegetation zonation. The proportion of parasitism in Polistes versicolor nests in the four vegetation zones on Santa Cruz Island does not show deviation from the expected (based on the proportion of P. versicolor nests found in each vegetation zone; G = 4.806, df = 3, P > 0.05). However, the situation on Floreana Island appears different as P. versicolor nests found in the arid zone are infested by T. sphecophila less than expected, and nests found in the transition and humid zones are infested more than expected (G = 15.482, df = 3, P < 0.01).

DISCUSSION Different factors, including climatic conditions, infestation by nest scavengers and parasitoids, and predation affect the wasp colony cycle (Yamane, 1996). Across its range of distribution, from Costa Rica to Southern Argentina, P. versicolor seems to prefer dry forest habitats (Richards, 1978). Data from previous studies suggest that in the Galapagos the wasps are more abundant in the arid zone of the islands (Roque- Albelo & Causton, 1999; Lasso, 1997). This preference in distribution could be asso- ciated with climatic conditions (Parent, 2000). In the Galapagos the higher zones of the islands are cooler and receive more rainfall than lower zones, particularly on the southern slopes, and this factor probably affects nest development. Collection data of T. sphecophila suggest a similar pattern of distribution. Most moth specimens were collected in the dryer zones of the islands suggesting a close correlation with nest abundance. On Santa Cruz Island the occurrence of T. sphecophila in different vegetation zones is a reflection of the frequency of P. versicolor nests. However, T. sphecophila seems to be more abundant than expected in the transition and humid zones of Floreana Island and less frequent in the arid zone. Therefore, T. sphecophila’s occurrence on Floreana Island is not strictly a reflection of the abundance of P. versicolor nests, TAYGETE SPHECOPHILA IN THE GALÁPAGOS ISLANDS 321 suggesting that other ecological or climatic factors might influence its distribution. It is not clear why there is such a difference between Floreana and Santa Cruz islands, but one possible hypothesis is that T. sphecophila has colonized these two islands at different points in time, so that populations on one of the island have had more time to adapt to the island’s ecological and climatic context. Polistes nests, as in many other social wasps, are scavenged and parasitized by various insects including more than 11 moth species from four families (Makino, 1985). Only Taygete sphecophila was found in the Galapagos, where the species apparently prefers to attack large nests, and all infested nests collected were large enough to presume that they were in an advanced stage of the reproductive phase. If predation by T. sphecophila is restricted to this stage of the wasp colonies the proba- bilities for this moth species to be an effective agent of biological control are reduced. These results support the idea of Miyano (1980) that parasitic and scavenging Lepidoptera reduce notably the colony’s productivity but are not thought to be a direct cause of colony failure. However, the possibility to use T. sphecophila as a biological control agent against P. versicolor needs to be evaluated. We believe that the first individuals of Taygete sphecophila probably arrived within a nest of P. versicolor built on some human-made structure that would have traveled by boat from the continent. It is actually quite possible that both animals arrived together on the Galapagos. The wasp was first detected in 1988 on Floreana, and is thought to have arrived with a shipment of bananas (Abedrabbo, 1991), but Eduardo Vilema, resident of Santa Cruz, says that he first saw a nest of Polistes versi- color near Bella Vista, on Santa Cruz, in 1984 or 1985 (pers. comm. to BL in 2004). And we think it unlikely that the wasps came on banana regimes as they are not known to build their nests there.

ACKNOWLEDGEMENTS BL would like to thank the authorities of the CDRS and Galapagos National Park Service (GNPS) for providing authorizations and logistical support for sampling micro moths in 1989, 1992, 2002, 2004, and 2005. The first two expeditions were organized by Stewart B. Peck, Carleton University, Ottawa, and benefited from financial support of the National Sciences and Engineering Council of Canada. Professor Peck, as well as Bradley J. Sinclair and several others were excellent field companions. BL is also grateful to Jean-François Landry, Agriculture & Agri-Food Canada, Ottawa, for providing lab space, equipment and technical support in 1999- 2000. We are thankful to curator Kevin Tuck of the Natural History Museum, London, for his kind help to BL in London and for providing information on references regarding T. sphecophila and on specimens of other species. Financial support for BL’s investigations in London in 2000 were provided by the Galapagos Conservation Trust, United Kingdom, and the Charles Darwin Foundation. PS would like to thank the CDRS and GNPS for allowing field work and providing logistical support in the Galapagos in 2004 and 2005. Research funds to PS were provided by the A. Lombard Foundation, the E. & L. Schmidheiny Foundation, the Swiss Academy of Sciences, and the Department of cultural affairs of the city of Geneva. 322 B. LANDRY ET AL.

CEP would like to thank the CDRS and GNPS for providing logistical support and required permits to conduct field work in Galapagos in 1999. Research funds to CEP were provided by CIDA, the Canadian International Development Agency. DA thanks Diana Marques, scientific illustrator, for the illustrations (Figs 14-21) of the larva and pupa and the digital production of the plates, and Scott D. Whittaker, Manager of the Scanning Electron Microscopy Laboratory, Smithsonian Institution, Washington, DC, for the help with the preparation of the larval specimens for study. LR thanks Henry Herrera of CDRS for his help in the field and in the lab. Finally, we are grateful to Lauri Kaila and Klaus Sattler for reviewing the manuscript.

REFERENCES

ABEDRABBO, S. 1991. Nueva avispa introducida en las islas. Carta Informativa 31: 4. ADAMSKI, D. 1999. Blastobasis graminea, new species (Lepidoptera: Gelechioidea: Coleo- phoridae: Blastobasinae): A stem borer of sugar cane in Colombia and Venezuela. Proceedings of the Entomological Society of Washington 101: 164-174. ADAMSKI, D. & BROWN, R. L. 1987. A new Nearctic Glyphidocera, with descriptions of all stages (Lepidoptera: Blastobasidae: Symmocinae). Proceedings of the Entomological Society of Washington 89: 329-343. ADAMSKI, D. & BROWN, J. W. 2001. Systematic review of the Ecdytolopha group of genera (Lepidoptera: Tortricidae: Grapholitini). Entomologica Scandinavica, Suppl. 58: 1-86. ADAMSKI, D. & PELLMYR, O. 2003. Immature stages and redescription of Blastobasis yuccae- colella Dietz (Lepidoptera: Gelechioidea: Coleophoridae: Blastobasini), with notes on its biology. Proceedings of the Entomological Society of Washington 105: 388-396. ALBERT, P. J. 1980. Morphology and innervation of mouthpart sensilla in larvae of the spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae). Canadian Journal of Zoology 58: 842-851. AVÉ, D. A. 1981. Induction of changes in the gustatory response by individual secondary plant compounds in larvae of Heliothis zea (Boddie) (Lepidoptera, Noctuidae). Ph.D. disser- tation, Department of Entomology, Mississippi State University, 89 pp., 4 tables, 37 figs. BECKER, V. O. 1984. Gelechiidae (pp. 44-53). In:HEPPNER, J. B. (ed.). Atlas of Neotropical Lepidoptera, Checklist: Part 1, Micropterigoidea - Immoidea. Dr. W. Junk Publishers, The Hague, xxvii + 112 pp. CHAMBERS, V. T. 1873. Micro-Lepidoptera. The Canadian Entomologist 5: 221-232. CLARKE, J. F. G. 1955. Catalogue of the Type Specimens of Microlepidoptera in the British Museum (Natural History) described by Edward Meyrick. Volume 1. Trustees of the British Museum, London, vii + 332 pp. CLARKE, J. F. G. 1969. Catalogue of the Type Specimens of Microlepidoptera in the British Museum (Natural History) described by Edward Meyrick. Volume 7. Trustees of the British Museum (Natural History), London, 531 pages. GAEDE, M. 1937. Familia: Gelechiidae (pp. 1-630). In:WAGNER, H., STRAND, E. & BRYK,F. (eds). Lepidopterorum Catalogus, part 79, Vol. IV. Dr. W. Junk, Berlin. GRIMES, L. R. & NEUNZIG, H. H. 1986a. Morphological survey of the maxillae in last-stage larvae of the suborder Ditrysia (Lepidoptera): Palpi. Annals of the Entomological Society of America 79: 491-509. GRIMES, L. R. & NEUNZIG, H. H. 1986b. Morphological survey of the maxillae in last-stage larvae of the suborder Ditrysia (Lepidoptera): Mesal lobes (Laciniogaleae). Annals of the Entomological Society of America 79: 510-526. TAYGETE SPHECOPHILA IN THE GALÁPAGOS ISLANDS 323

HODGES, R. W. 1983. Gelechiidae (pp. 19-25). In:HODGES, R. W. et al. (eds). Check List of the Lepidoptera of America North of Mexico. E. W. Classey Ltd. and the Wedge Entomo- logical Research Foundation, London, xxiv + 284 pp. HODGES, R. W. 1998. The Gelechioidea (pp. 131-158). In:KRISTENSEN, N. P. (ed.). Handbook of Zoology, Lepidoptera, Moths and Butterflies, Vol. 1: Evolution, Systematics, and Bio- geography. Walter de Gruyter, Berlin & New York, x + 491 pp. KAILA, L. 2004. Phylogeny of the superfamily Gelechioidea (Lepidoptera: Ditrysia): an exemplar approach. Cladistics 20: 303-340. KRISTENSEN, N. P. 2003. Skeleton and muscles: adults (pp. 39-131). In:KRISTENSEN, N. P. (ed.). Handbook of Zoology, Lepidoptera, Moths and Butterflies, Vol. 2: Morphology, Physio- logy, and Development. Walter de Gruyter, Berlin & New York, x + 564 pp. LANDRY, B. 1999. Quatre premières mentions de Lépidoptères pour la province de Québec (Opostegidae, Gelechiidae et Tortricidae). Fabreries 24: 66-72. LANDRY, B. 2002. Galagete, a new genus of Autostichidae representing the first case of an extensive radiation of endemic Lepidoptera in the Galápagos Islands. Revue suisse de Zoologie 109: 813-868. LANDRY, J.-F. & ADAMSKI, D. 2004. A new species of Nanodacna Clarke (Lepidoptera: Elachistidae: Agonoxeninae) feeding on the seeds of Austrocedrus chilensis (Cupres- saceae) in andean Argentina. Journal of the Lepidopterists’ Society 58: 100-113. LASSO, T. L. 1997. Ecologia e impacto de la avispa introducida (Polistes versicolor, Vespidae- Hymenoptera) en las Islas Floreana y Santa Cruz, Galapagos Ecuador. B.Sc. Thesis, Universidad Central del Ecuador, Quito, 211 pp. MAKINO, S. 1985. List of parasitoides [sic] of Polistine wasps. Sphecos 10: 19-25. MEYRICK, E. 1936. Exotic Microlepidoptera 4: 609-642. MIYANO, S. 1980 Life tables of colonies of workers in a paper wasp, Polistes chinensis anten- nalis, in central Japan (Hymenoptera: Vespidae). Research in Population Ecology 22: 69-88. NYE, I. W. B. & FLETCHER, D. S. 1991. The generic names of moths of the World. Volume 6. Microlepidoptera. Natural History Museum Publications, London, xxix + 368 pp. PARENT, C. 2000. Life cycle and ecological impact of Polistes versicolor versicolor (Olivier) (Hymenoptera: Vespidae), an introduced predatory wasp on the Galapagos Islands, Ecuador. M.Sc. Thesis, Carleton University, Ottawa, Canada, 139 pp. RICHARDS, O. W. 1978. The social wasps of the Americas excluding the Vespinae. Trustees of the British Museum (Natural History), London, Publication No. 785, vii + 580 pp. + 4 pls. ROQUE-ALBELO,L.&CAUSTON, C. 1999. Monitoreo de avispas introducidas en las Islas Galápagos. Charles Darwin Research Station report,12pp. SCHOONHOVEN, L. M. & DETHIER, V. G. 1966. Sensory aspects of host-plant discrimination of Lepidopterous larvae. Archives néerlandaises de zoologie 16: 497-530. SOKAL, R. R. & ROHLF, F. J. 1995. Biometry (3rd ed.). W. H. Freeman and Company, New York, 887 pp. STEHR, F. 1987. Cosmopterigidae (Gelechioidea) (pp. 391-392). In:STEHR, F. (ed.). Immature in- sects, Volume 1. Kendall/Hunt Publishing Company, Dubuque, Iowa, 754 pp. WAGNER, D. L., ADAMSKI, D. & BROWN, R. L. 2004. A new species of Mompha Hbn. from Mississippi (Lepidoptera: Gelechioidea). Proceedings of the Entomological Society of Washington 106: 1-18. WIGGINS, I. L. & PORTER, D. M. 1971. Flora of the Galápagos Islands. Stanford University Press, Stanford, xx + 998 pp. YAMANE S. 1996. Ecological factors influencing the colony cycle of Polistes wasps (pp. 75-97). In:TURILLAZZY, S. & WEST-EBERHARD, M. J. (eds). Natural History and evolution of paper-wasps. Oxford University Press, xiv + 400 pp. Ringing & Migration (2006) 23, 33-38

Autumn migration of Reed Buntings Emberiza schoeniclus in Switzerland

PATRICK SCHMITZ1* and FLORIAN STEINER2 1Department of Biology, Faculty of Science, University of Geneva, Switzerland 2Rue de Genève 60, 1225 Chêne-Bourg, Switzerland

To elucidate the migration strategies of Reed Buntings Emberiza schoeniclus migrating through Central Europe, we analysed data from 595 Reed Buntings ringed at La Touvière, Rhône River, Geneva, Switzerland, during the autumn migration from September to November 2004. These data were used to investigate age, sex, biometrics and body condition in relation to timing of migration. The overall sex ratio and the ratio of first-years to adult birds were 1:1 and 3:1, respectively, but there was a chronological sequence of young females, adult females, young males and adult males during the autumn. The mean bill depth varied during the study period with individuals migrating during October having deeper bills. The greater bill depth of males by comparison with other European studies suggests a more northeasterly origin of these birds, compatible with a leap-frog migration.

The Reed Bunting Emberiza schoeniclus has a wide (Villarán 1999, Villarán & Pascual-Parra 2003). In Palearctic distribution (Cramp & Perrins 1994) and is contrast, published studies from Switzerland refer only ringed in large numbers in Switzerland. In 2004, 4,243 to the spring migration (Pedroli & Gogel 1972, Christen individuals were ringed, representing 4.3% of the 1984). In this paper, we consider the autumn migration ringing totals (Wiprächtiger et al 2005). Most Swiss of Reed Buntings from September to November 2004 passage migrants breed in Germany, Poland, Austria at a study site in the western tip of Switzerland, situated and the former Czechoslovakia, with a smaller number at a junction between the breeding and the wintering originating from Scandinavia. The Swiss winter grounds in Europe. We have examined the sex ratio, recoveries show a large concentration in southeast age ratio, and biometric data in relation to timing of France, particularly in the Camargue, although this may migration. We have tested the hypothesis that females be exaggerated by the large-scale ringing in this region. depart earlier than males in order to reach their more Other birds ringed on passage or in the breeding season southerly wintering grounds, and consider the possibility in Switzerland have been recovered in southwest ^ that some of the birds migrating through Switzerland France, Spain, and northern Italy (Prys-Jones 1984). may belong to northeast European populations. In Switzerland, overwintering Reed Buntings are regular but scarce and occur mostly on the Swiss plateau near Lake Léman (Maumary et al in prep). The breeding METHODS population in Switzerland is estimated at 3,000–5,000 pairs (Schmid et al 1998). Because of the loss of Study area adequate breeding sites, particularly reedbeds, in the The study was carried out from September to November canton of Geneva, the Reed Bunting has declined there 2004 at La Touvière (46°10’N 5°59’E), in the Rhône by 63% between 1977 and 2001 (Lugrin et al 2003). In basin, about 13 km W of Geneva, Switzerland. The the area alongside the River Rhône used for this present site is a 1.4 ha, thin stretch of reedbed along the study, Reed Buntings occur only as sporadic breeders; southern bank of the River Rhône, dominated by however, during autumn migration large flocks converge Phragmites australis. The trapping area covered at dusk to roost in the reedbeds. approximately 0.2 ha (14%) of the reedbed. Mist nets The autumn migratory patterns of the Reed Bunting were always placed in the same positions during the have been described for Europe (Prys^ -Jones 1984) and study (n = 7 nets; 3 m high, total length 54 m). Trapping component countries such as France (Olioso 1987), was carried out from afternoon till dawn twice a week, Germany (Tauchnitz 2000, George 2002), and Spain giving a total of 16 days. Since few birds were present *Correspondence author in November we ringed on only one day per week during Email: [email protected] this month.

© 2006 British Trust for Ornithology 34 P. Schmitz and F. Steiner

Biometrics Table 1. Number of Reed Buntings, captured at La Touvière, The routine data recorded were sex, age, capture date, Switzerland, by sex and age-class. wing length (maximum chord, Svensson 1992) and Males Females Total body mass using a 100 g Pesola balance read to the nearest 0.5 g. We also recorded bill depth (at the limit First-year birds 197 250 447 of feathering on the upper and lower mandibles), width Adult birds 78 67 145 (at base) and length (from tip to skull), with a dial Total 275 317 592 calliper to the nearest 0.1 mm (Svensson 1992). Bill 2 = 3.332; P = 0.068 measurements are more reliable for comparisons of χ 1 populations than wing measurements because the latter during the start of October, when the number of vary due to feather abrasion and age-related differences captures generally increased, and were most abundant between juveniles and adults (Stewart 1963, Collette at the end of the study period (29 October – 13 1972). All birds were sexed and aged although some November). Capture totals reached a peak during the individuals were not measured during very busy periods second half of October (Fig 1) and during this period when large numbers of birds were caught at once. Wing the sex ratio was not significantly different from 1:1. and bill measurements were taken by the same ringer With respect to age, first-year birds were most (FS) to ensure consistency in measurements. Reed abundant at the beginning and at the end of the study Buntings were aged using flight-feather abrasion period, and adults were most abundant in the middle (Svensson 1992, De La Puente & Seoane 2001), moult (Fig 1). Over the whole of the study period, first-year limits of wing and tail (Jenni & Winkler 1994), and Reed Buntings (n = 447) outnumbered adults (n = 145) iris coloration (Karlsson et al 1985) as criteria. by 3:1; this age ratio was similar for both sexes (first- year males: n = 197 and first-year females: n = 250) and significantly different from 1:1 (χ2 = 153; P < 0.01). Statistical analysis 1 To compare the proportions of different sex and age- classes captured, chi-squared tests with Yates’ correction Wing length, body mass, body condition (2x2 contingency tables) were used. Body condition was and bill depth estimated from the residuals of a regression of body mass A summary of biometric data is presented in Table 2. on wing length. Biometrics and body condition with Mean wing lengths differed significantly with respect respect to date of capture, sex and age-classes were to sex (F1, 544 = 333.51; P < 0.001), age (F1, 544 = 15.89; P analysed using Analysis of Variance (ANOVA) and < 0.001) and date of capture (F7, 544 = 15.94; P < 0.001; Generalised Linear Models (GLM) using the statistical corrected for sex and age). The mean wing length of package S-PLUS (MathSoft, Inc). Since Reed Buntings adult males was 6.2 mm longer than adult females and were consistently caught from afternoon until dusk, 2.0 mm (2.5%) longer than first-year males; similarly, time of day was not included as a factor in ANOVA for females the mean wing length of adults was 1.1 mm and GLM analyses of body mass. 160

140 13.5

RESULTS 120 8.8 20.9 3.5 7.0 100 17.1 Sex and age ratio and timing of movements 16.4 35.1 29.5 During the study, 595 Reed Buntings were ringed. Two- 80 17.1 thirds of this total were captured at dusk and the rest 60 32.8 in the afternoon. All birds were sexed and the age was 33.4 Numbercaptures of determined for 592 individuals (99.5%) (Table 1). 40 60.0 42.6 Overall, with 319 females (54%) and 276 males (46%) 3.5 19.7 24.1 20 32.4 29.9 50.0 11.2 captured, the sex ratio was not significantly different 72.4 44.4 66.7 2 2 33.3 44.4 from 1:1 (χ 1 = 2.7; P > 0.05). However, sex (χ 7 = 28.7; 0 33.3 2 18/09 25/09 01/10 08/10 15/10 22/10 29/10 06/11 13/11 P < 0.001) and age (χ 7 = 45.7; P < 0.001) ratios varied significantly with date of capture. Reed Buntings were Date first caught in the middle of September, and all were Figure 1. Frequency of captures of adult male (dotted bars), adult female; females were also more abundant than males female (hatched bars), first-year male (filled bars), and first-year female (open bars) Reed Buntings captured at La Touvière, during the beginning of the study period (18 September Switzerland, in relation to date of capture. Percentages adjacent – 8 October). Conversely, the first males were captured to the bars are shown for each week.

© 2006 British Trust for Ornithology, Ringing & Migration, 23, 33-38 Reed Bunting migration 35

(1.5%) longer than in first-year birds (Fig 2). Body (F1, 470 = 3.74; P = 0.008), but not between sexes (F1, 470 mass also varied with sex (F1, 451 = 49.8; P < 0.001) and = 3.04; NS) or with age (F1, 470 = 2.05; NS; Fig 4). date of capture (F7, 451 = 9.4; P < 0.001; sex corrected), Bill depth values for the four age and sex categories but not with age (F1, 451 = 0.009; non-significant [NS]); were normally distributed (Fig 5) and differed males were heavier than females (Fig 3). Maximum significantly between sexes (F1, 451 = 13.38; P < 0.001) mean body mass was recorded in October during the and with date of capture (Fig 6; F7, 451 = 10.83; P = peak of migration, and minimum mean body mass 0.002; sex corrected), but not with age (F1, 451 = 2.87; occurred during the start and final stages of autumn NS); males had deeper bills than females (Fig 6). migration. Body condition, expressed as the magnitude Interactions between sex, age, and date of capture on of the residuals from a linear regression of body mass biometric data and body condition were investigated on wing length, varied significantly with date of capture using GLM, and were not significant (all P > 0.05), except for the effect of the interaction between sex and age on wing length (F = 6.56; P = 0.01). 84 1, 544

82 DISCUSSION 80 During migration, Reed Buntings of the schoeniclus subspecies are present at La Touvière between September 78 and November. The timing of autumn migration at 76 this site, in the western part of Switzerland, is consistent with other observations for Switzerland as a whole 74 (Winkler 1999). In spring, adult males depart from Wing length (mm) lengthWing 72 their wintering grounds in Spain (Villarán & Pascual- Parra 2003) and arrive in Switzerland earlier than adult 70 females (Pedroli & Gogel 1972, Christen 1984), 68 followed by young birds (Christen 1984). A similar sequence is observed during the prenuptial migration

25/09 01/10 08/10 15/10 22/10 29/10 06/11 13/11 along the North Sea coast in Belgium (Collette 1972). Date Although the autumn migration data from La Touvière reported here refer to one year only, the chronological Figure 2. Wing length (mm) of adult male (open circles), first-year sequence was the opposite to that during spring male (filled circles), adult female (open squares), and first-year female migration in Europe, with females migrating through (filled squares) Reed Buntings captured at La Touvière, Switzerland, in relation to date of capture. Mean and standard error are shown. the site approximately two weeks before males and a

3 23 2 22 1 21 t

eigh 0

20 w

19 -1 Residual 18 -2 Body mass (g)

17 -3 16 -4 15 25/09 01/10 08/10 15/10 22/10 29/10 06/11 13/11

25/09 01/10 08/10 15/10 22/10 29/10 06/11 13/11 Date Date Figure 3. Body mass (g) of adult male (open circles), first-year male Figure 4. Body condition of adult male (open circles), first-year (filled circles), adult female (open squares), and first-year female male (filled circles), adult female (open squares), and first-year female (filled squares) Reed Buntings captured at La Touvière, Switzerland, (filled squares) Reed Buntings captured at La Touvière, Switzerland. in relation to date of capture. Mean and standard error are shown. Mean and standard error are shown.

© 2006 British Trust for Ornithology, Ringing & Migration, 23, 33-38 36 P. Schmitz and F. Steiner

5.8 20 a) 15 5.6 10

5 5.4

0 5.2 20 b) Bill depth (mm) 15 5.0 10

5 4.8

0 25/09 01/10 08/10 15/10 22/10 29/10 06/11 13/11 40 Date c) 35 Figure 6. Bill depth (mm) of adult male (open circles), first-year male (filled circles), adult female (open squares), and first-year female 30 (filled squares) Reed Buntings captured at La Touvière, Switzerland,

Frequency in relation to date of capture. Mean and standard error are shown. 25

20 high proportion of first-year birds passing through earlier

15 than the adults, although there was considerable overlap (Fig 1). A similar pattern was observed for Chaffinches 10 Fringilla coelebs in Switzerland (Schifferli 1963) and

5 may be a general trend within passerines for short- distance migrants. 0 The phenology of the sex ratio observed on the study 40 site suggests that females leave their breeding grounds d) first, disperse earlier and possibly migrate longer 35 distances than males to reach their wintering grounds 30 in southern Europe. For males, a shorter spring migration distance would allow them to return to their 25 breeding sites earlier, where they can occupy better 20 territories and so increase their chances of obtaining a mate (George 2002). However, the relationship between 15 the Reed Buntings passing through the study site and 10 wintering populations further south is not clear. During the winter in Europe a skewed sex ratio according to 5 latitude is observed with three females to each male 0 present from October to February in Spain (Villarán 4.5 5.0 5.5 6.0 6.5 1999, Villarán & Pascual-Parra 2003), whereas a higher Bill depth (mm) percentage of males is present in Switzerland (Pedroli & Gogel 1972), and Germany (Tauchnitz 2000). In contrast, although slightly more females than males were caught at the study site in autumn, the sex ratio was not significantly different from 1:1. Furthermore, Figure 5. Frequency of bill depth (mm) of (a) adult male, (b) adult female, (c) first-year male, and (d) first-year female Reed Buntings the first-year:adult ratio of 1:1 observed in Spain during captured at La Touvière, Switzerland. winter (De La Puente & Seoane 2001) contrasts with the large age ratio difference of three first-year birds for each adult during migration through the Swiss study site. One interpretation of this is that young birds might disperse without migrating further southwest as females

© 2006 British Trust for Ornithology, Ringing & Migration, 23, 33-38 Reed Bunting migration 37

Table 2. Biometrics of Reed Buntings in migration from September to November 2004 at La Touvière, Geneva, Switzerland.

Wing length, mm Body mass, g Bill depth, mm Bill width, mm Bill length, mm mean ± SD (N); range mean ± SD (N); range mean ± SD (N); range mean ± SD (N); range mean ± SD (N); range

First-year males 78.3 ± 1.9 (181); 20.6 ± 1.8 (150); 5.5 ± 0.3 (150); 5.3 ± 0.3 (150); 12.0 ± 0.4 (150); 72.0 – 83.0 16.0 – 27.0 4.7 – 6.4 4.6 – 5.9 11.2 – 13.2 First-year females 73.0 ± 1.7 (226); 18.3 ± 1.8 (200); 5.3 ± 0.3 (189); 5.2 ± 0.3 (189); 1.7 ± 0.4 (189); 69.0 – 79.0 15.0 – 25.0 4.5 – 6.0 4.0 – 5.9 10.4 – 12.8 Adult males 80.3 ± 2.3 (74); 21.0 ± 2.0 (54); 5.5 ± 0.3 (61); 5.4 ± 0.3 (61); 12.1 ± 0.5 (61); 74.0 – 86.0 17.5 – 26.5 4.9 – 6.1 4.7 – 5.9 10.5 – 13.6 Adult females 74.1 ± 2.2 (60); 18.7 ± 2.0 (44); 5.4 ± 0.3 (50); 5.3 ± 0.2 (50); 11.8 ± 0.5 (50) 70.0 – 79.0 15.5 – 24.5 4.6 – 6.1 4.9 – 5.9 10.8 – 12.8 do, combined with a greater mortality rate of first-year due to fat accumulation and flight-muscle hypertrophy birds during migration. (Kaiser 1992). Body condition values were slightly One possible bias which might affect the proportion lower than those reported by Villarán & Pascual-Parra of the sex and age-classes is the turnover rate of the (2003) and did not vary between the sexes (Fig 4). birds captured at the study site. If for example, there is Mean bill depth (corrected for sex) increased a greater chance of females moving on to winter further progressively during the migration period until the southwest than males, then it may be that females spend middle of October when the highest bill depths were on average less time in the reedbed. The same applies recorded, and then decreased (Fig 6). A continuous to the high proportion of first-year birds caught, which cline of increasing bill depth exists towards the may due to the fact that they tend towards spending northeast of the Western Palearctic (Cramp & Perrins more time at the study site. This could affect our 1994). A comparison of the bill depth of males in interpretation of the relative numbers of different Geneva with those of birds from other European studies classes trapped. (Table 3) suggests that, at the peak of migration, the Our biometric data (Table 2) confirm other data on passage migrants included birds from large-billed sexual size dimorphism within Reed Buntings (Svensson populations with a more northeasterly origin, although 1992, Cramp & Perrins 1994, Villarán & Pascual-Parra these birds were not bigger overall on the basis of wing 2003) and show that adults are longer-winged than first- length. For example, an individual with a 6.4 mm bill year birds. At the study site, mean wing length depth is likely to have originated from northeastern increased progressively over the migration period (Fig Europe (Fig 6). Bill depth is a highly heritable trait 2). The lowest values for body mass during the autumn (Grant 1983) and, in contrast to wing length, does not migration were recorded at the beginning of October vary greatly with age or season (Boag 1984), and can be when the birds were starting to disperse, and at the therefore considered as a reliable and repeatable end of November when the birds started to remain on measurement for comparisons between distinct the study site as their wintering quarters. Maximum geographical populations. As proposed by Prys^ -Jones values for mean body mass corresponded to the peak of (1984) and Villarán (1999), the fact that birds with the migration in October (Fig 3) and may have been deeper bills are present during the peak of migration

Table 3. Bill depth of male Reed Buntings from European studies, according to Cramp & Perrins (1994).

Location Bill depth (mm) subspecies

S Sweden, Finland 5.3 ± 0.2 (17); 5.0 – 5.6 schoeniclus S Sweden, E Germany, Poland, SW Belarus 5.3 ± 0.18 (16); 5.1 – 5.6 schoeniclus Lapland 5.3 ± 0.2 (32); 4.7 – 5.8 schoeniclus Russia, St Petersburg 5.4 ± 0.3 (25); 5.1 – 6.0 schoeniclus Switzerland* 5.5 ± 0.3 (212); 4.7 – 6.4 schoeniclus + ? N Ukraine 5.7 ± 0.3 (37); 5.2 – 6.5 ukrainae E Austria, Hungary, N former Yugoslavia 6.0 ± 0.25 (23); 5.7 – 6.4 stresemanni NE Bulgaria, NE Romania, S Ukraine 6.9 ± 0.35 (8); 6.4 – 7.4 tschusii

© 2006 British Trust for Ornithology, Ringing & Migration, 23, 33-38 38 P. Schmitz and F. Steiner

may indicate leap-frog migration, with northeastern Kaiser, A. (1992) Fat deposition and theoretical flight range of small migratory populations tending to overwinter further to autumn migrants in southern Germany. Bird Study 39, 96-110. the southwest of Europe, overtaking the more sedentary Karlsson, L., Persson, K. & Walinder, G. (1985) Fotografisk dokumentation av alders- och konsskillnader hos faglar- malsattning, populations of Reed Buntings from Central Europe. arbetssatt och exempel pa resultat. Vår Fågelvärld 44, 465-478. Lugrin, B., Barbalat, A. & Albrecht, P. (2003) Atlas des oiseaux ACKNOWLEDGEMENTS nicheurs du canton de Genève. Editions Nicolas Junod, Genève. Maumary, L., Vallotton, L. & Knaus, P. (In prep) Les Oiseaux de We thank Corine Schopfer, Isabelle and Michel Poscia, and Suisse. Distribution, biologie et conservation des oiseaux de Suisse et rives limitrophes. Station Ornithologique Suisse et Nos Oiseaux, Brigitte and Jean-Marc Hayoz for assistance with ringing, Sempach. Elisabeth Wiprächtiger from the Swiss ringing station of Olioso, G. (1987) Migration et hivernage du Bruant des Roseaux Sempach for logistic support, Raphaël Covain for statistical Emberiza schoeniclus (L) en region Rhône-Alpes: Analyse des reprises assistance, and Alice Cibois and Raffael Winkler for de bagues. Bièvre 9, 1-8. improving an earlier version of the manuscript. Pedroli, J.C. & Gogel, R. (1972) Étude simultanée de la migration printanière dans 18 camps de baguement. Premiers résultats de l’opération Bruants 1972. Nos Oiseaux 31, 252-267. Prys-Jones,^ R.P. (1984) Migration patterns of the Reed Bunting, REFERENCES Emberiza schoeniclus schoeniclus, and the dependence of wintering distribution on environmental conditions. Le Gerfaut 74, 15-37. Boag, P.T. (1984) Growth and allometry of external morphology in Schifferli, A. (1963) Vom Zug der Buchfinken Fringilla coelebs in der Darwin’s finches (Geospiza) on Isla Daphne Major, Galápagos. Schweiz. Proceedings of the XIII International Ornithological Congress Journal of Zoology 204, 413-441. 13, 468-474. Christen, W. (1984) Durchzug, Geschlechtverhältnis und Flügelmasse Schmid, H., Luder, R., Naef-Daenzer, B., Graf, R. & Zbinden, der Rohrammer Emberiza schoeniclus im Frühjahr bei Rothrist AG. N. (1998). Atlas des oiseaux nicheurs de Suisse: Distribution des Der Ornithologische Beobachter 81, 227-231. oiseaux nicheurs en Suisse et au Lichtenstein en 1993 – 1996. Station Collette, P. (1972) Contribution à l’étude de la migration prénuptiale Ornithologique Suisse, Sempach. du Bruant des Roseaux (Emberiza schoeniclus). Aves 9, 226-240. Stewart, I.F. (1963) Variation of wing length with age. Bird Study 10, Cramp, S. & Perrins, C.M. (1994) Handbook of the Birds of Europe, 1-9. the Middle East and North Africa Vol. IX. Oxford University Press, Svensson, L. (1992) Identification Guide to European Passerines. Oxford. Svensson, Stockholm. De La Puente, J. & Seoane, J. (2001) The use of primary abrasion Tauchnitz, H. (2000) Zum Durchzug und Winteraufenthalt der for ageing Reed Buntings Emberiza schoeniclus. Ringing & Migration Rohrammer in Mitteldeutschland. Apus 10, 329-340. 20, 221-223. Villarán, A. (1999) Migracion e invernada del escribano palustre George, K. (2002) Winterquartiere und geschlechtsdifferenzierte (Emberiza schoeniclus) en España. Ardeola 46, 71-80. Zugstrategien in Thüringen beringter Rohrammern Emberiza schoeniclus. Villarán, A. & Pascual-Parra, J. (2003) Biometrics, sex ratio and Anzeiger des Vereins Thüringer Ornithologen 4, 337-340. migration periods of Reed Bunting Emberiza schoeniclus wintering Grant, P.R. (1983) Inheritance of size and shape in a population of in the Tajo Basin, Spain. Ringing & Migration 21, 222-226. Darwin’s finches, Geospiza conirostris. Proceedings of the Royal Winkler, R. (1999) Avifaune de Suisse. Nos Oiseaux, suppl. 3. Society of London B 220, 219-236. Wiprächtiger, E., Häfliger, G. & Kestenholz, M. (2005) Numbers Jenni, L. & Winkler, R. (1994) Moult and Ageing of European of birds ringed and recovered by the Sempach ringing centre. Station Passerines. Academic Press, London. Ornithologique Suisse, Sempach.

(MS received 29 June 2005; MS accepted 28 February 2006)

© 2006 British Trust for Ornithology, Ringing & Migration, 23, 33-38