Küpfer, Philippe

2010, Espíndola, María Anahí, Alvarez, Nadir, Rahier, Martine, Carstens, Bryan C., Després, Laurence, Gibernau, Marc, Gugerli, Felix, Küpfer, Philippe

Coevolution is defined as reciprocal evolutionary changes that might arise at any spatiotemporal scale. Despite every organism on Earth undergoes coevolutionary interactions, cases of one-to-one specific relationships are generally rare. However, because of the reduced number of interacting species they concern, these species-specific associations are interesting to evolutionary biologists because they allow testing hypotheses in simple frameworks. Despite the history and evolution of coevolutionary interactions have been studied in several cases in the last decade, this topic remains difficult to fully circumscribe because of the multiplicity of factors that affect one or the other species concerned. Moreover, a lot is known about coevolution at a small scale, but little has been done at larger and more integrative scales spanning wider spatiotemporal ranges. Phylogeography is a young area of biology that allows understanding the distribution of lineages in space and time. Despite that the idea of parallely studying the history of species involved in specific interactions appears simple, this has rarely been done until now probably because of the technical efforts this would represent. From a theoretical point of view, we could propose that in specific interactions, because of the dependence between the partners involved, we should observe some phylogeographic pattern associated to the type of interaction studied. In this way, while partners of mutualistic interactions should present similar postglacial histories, this should not be true for those associated by antagonistic relationships. In this thesis, we exploit different techniques and approaches to test this general hypothesis. The final aim of this study is thus to understand if it is possible to identify a pattern of comparative phylogeography in relation to the type of interaction, using as case-studies two specific and obligate European interactions: the antagonistic relationship established between Arum maculatum L. (Araceae) and its Psychodid (Diptera) pollinating flies, and the nursery pollination mutualism involving Trollius europaeus L. (Ranunculaceae) and the Chiastocheta (Diptera: Anthomyiidae) species complex. Before testing our comparative phylogeographic hypotheses and because studying the phylogeography of interactions requires a wide knowledge of the environmental, taxonomic and historical frameworks in which these ecological relationships arose, it was first needed to clearly delimitate the identity of species, their distribution and the environmental factors influencing their survival to finally understand their comparative history. We thus took advantage of the potentialities that interdisciplinary approaches provide, applying molecular taxonomy, biological and evolutionary methods, biogeographic inferences, ecological niche models and hindcasting techniques, as well as classical and recently-developed phylogeographic analyses. Our results indicate that the phylogeographic patterns of these specific and obligate antagonistic and mutualistic relationships appear to be related to the type of interaction. Antagonistic partners presented incongruent phylogeographic patterns, what can be notably explained by differences in their life-history traits. Species involved in mutualistic interactions partly showed congruent phylogeographic patterns (particularly in the cases of T. europaeus and C. dentifera). Flies interacting with T. europaeus appear moreover to present different histories, regardless of their important ecological similarities. These results demonstrate that the systems studied appear to be far more complex than initially supposed, with crossed effects of environmental and historical features on the dynamics of the interaction. Because of the high complexity and interdependency of factors affecting one or the other partner, performing investigations in an interdisciplinary framework appears indispensable to disentangle the dynamics of interactions.

2007, Clot, Bernard, Küpfer, Philippe

L'augmentation spectaculaire de la prévalence des allergies au pollen dans les dernières décennies a stimulé le développement de l’aérobiologie. Le but de ce travail consistait à mieux circonscrire les paramètres climatiques et météorologiques qui influencent la saison pollinique, dans une perspective prédictive, à évaluer l’influence du changement climatique sur la saison pollinique et à déterminer quels pollens pourraient exposer la population à de nouveaux risques dans le domaine des allergies. Une méthode de prévision du début de la saison pollinique a été proposée pour chacun des deux taxons les plus allergisants dans notre pays, les graminées et le bouleau. La maturité des fleurs est modélisée par un cumul de températures et donc prévisible à moyen terme (quelques semaines). La libération effective du pollen dépend des conditions météorologiques à court terme (de quelques heures à deux ou trois jours) et est à considérer comme un phénomène « intra saison ». L’influence du réchauffement climatique sur la végétation et sur la saison pollinique a été mise en évidence. L’intérêt de l’aéropalynologie et de la phénologie comme indicateurs de la réaction de la végétation au changement climatique a été souligné. Les hivers plus doux ont eu pour conséquence un développement de la végétation précoce au printemps, une avance moins marquée des phases estivales et automnales et un allongement de la saison de végétation dans son ensemble de l’ordre de deux à trois semaines. Les tendances concernant le pollen de l’air ont été étudiées pour un grand nombre de taxons, offrant ainsi une vision d'ensemble de la saison pollinique. L’avance du début de la saison est particulièrement remarquable, alors que l’augmentation des quantités de pollen n’est observée que pour certains taxons. Pour les personnes allergiques, la période de risque s’allonge et les seuils de concentrations qui font apparaître les symptômes sont plus fréquemment dépassés. L’intérêt de porter attention à des pollens autres que ceux considérés comme des allergènes majeurs a été souligné. En particulier, l'importance quantitative et qualitative du pollen de charme a été démontrée. En raison de l’importance croissante des allergies dans notre société, il est fort possible que du pollen d’autres plantes indigènes joue à l’avenir un rôle plus important comme cause d’allergies. De plus, que ce soit par l’introduction de variétés ornementales, la migration naturelle d’espèces ou l’installation de plantes envahissantes, l’arrivée de nouveaux allergènes dans l’air est relativement fréquente et vraisemblablement amenée à se poursuivre. La reconnaissance précoce des situations à risque particulier pour la santé humaine, comme les plantes dont l’allergénicité est connue ailleurs, est importante pour permettre de mener à bien des actions de prévention à un moindre coût. L’aéropalynologie s’est avérée très performante pour la prévention, puisqu’elle a permis la découverte de l’arrivée de l’ambroisie dans la région de Genève. Un contrôle des populations d'ambroisie a ainsi pu être mis en place en Suisse à un stade précoce de la phase d'envahissement. Le pollen d’ambroisie a été utilisé comme modèle pour la compréhension des phénomènes de transport à moyenne et longue distance et pour l'évaluation de l'importance relative des sources locales dans l’exposition de la population. Les changements qui interviennent dans notre environnement, provoqués directement ou indirectement par les activités humaines, favorisent la présence et l’augmentation d'allergènes "traditionnels" et celle d’allergènes nouveaux. Une exposition accrue au pollen conduit à un risque d’allergies plus important. Pourtant, dans la mesure où l'être humain est à l'origine de la plupart de ces changements, et que ses activités les maintiennent et les renforcent, on peut espérer que cette évolution n'ait pas un caractère inéluctable et que la tendance pourra être renversée.

2005, Schoenenberger, Nicola, Küpfer, Philippe

Genetic and ecological aspects of gene flow from wheat (Triticum aestivum L.) to Aegilops L. species Hybridisation and introgression from crops to wild relatives is a key issue in risk assessment. In the present study, hybridisation and introgression dynamics from hexaploid wheat (2n=42) to tetraploid Aegilops species (2n=28) were investigated by experiments in natural conditions or in the greenhouse, and by genetic analyses. In order to study crop-weed hybridisation as a function of distance, a field trial was set up where Ae. cylindrica was planted in plots at 0, 1, 5, 10 and 25m from a wheat field. In the progeny (14045 seeds sown) we detected hybrids up to 1m from the wheat field. Wheat-specific RAPD fragments were found in Ae. cylindrica x T. aestivum hybrids and BC1 plants. Using a set of Chinese Spring nulli-tetrasomic wheat lines, we were able to assign DNA fragments to wheat chromosomes. Introgressed wheat-specific markers were localised on the three genomes (A, B and D). Some of these markers were transformed into easy-to-use Sequence Characterised Amplified Regions (SCARs), and used to characterise an introgressive series. Ae. cylindrica x GM-wheat hybrids, BC1 and BC1S1 (self-fertilised first backcrosses) were manually produced, in order to study inheritance of transgenes. Female fertility of the hybrids was 0.03-0.6%, BC1 plants had 30-84 chromosomes and displayed highly irregular meioses, their self fertility ranged from 0 to 5.21 %. BC1S1 plants had 28-43 chromosomes and some of them recovered full fertility. One BC1S1 individual contained the bar gene issued from its transgenic wheat progenitor and survived herbicide treatment. A RAPD-based population genetics study was carried out in natural Ae. cylindrica populations, most of them from adventive locations in Switzerland, Italy and the USA. Genetic diversity was low and most of the variance resided among populations. Italian populations from the Aosta valley and a Swiss population were similar or identical to northern American populations indicating that the species may have crossed the Atlantic Ocean several times. Using 52 Ae. geniculata individuals planted in a winter wheat field, we obtained an overall Ae. geniculata x T. aestivum hybridisation rate of 0.94%, hybrid fertility, i.e. BC1 production rate, was 2.2%. Specific wheat SCAR markers were detected in the hybrids and backcrosses. Gene flow is thus possible between wheat and Aegilops, at a limited rate. Because of the large cultivation of wheat, trans-gene flow might occur significantly. Moreover, insertion of the transgene on A and B genome does not prevent introgression.

2009, Bürki, Sven, Küpfer, Philippe, Alvarez, Nadir

La famille des Sapindaceae est importante d’un point de vue économique et comprend plus de 1900 espèces (distribuées dans approximativement 140 genres; par exemple Litchi, Paullinia), majoritairement distribuées en zone tropicale. Cependant, certains genres peuvent coloniser les zones tempérées. Depuis plus d’un siècle, la définition de cette famille (plus particulièrement la possible inclusion des Aceraceae et Hippocastanacae au sein des Sapindaceae) ainsi que les relations entre les sous-familles, ont été largement débattues (voir chapitre 1 pour un résumé). Dans ce travail, les relations au sein des Sapindaceae, ainsi que celles entre les Aceraceae et Hippocastanaceae, sont étudiées sur la base de huit marqueurs moléculaires (nucléaire et chloroplastiques) en utilisant une approche complémentaire de types supermatrice (chapitre 1) et supertree (chapitre 6). Les deux approches supportent la monophylie des Sapindaceae lorsque les Aceraceae et Hippocastanaceae sont incluses, et montrent un haut taux de paraphylie et polyphylie au niveau des sous-familles et tribus. De plus, les résultats contestent la monophylie de plusieurs genres (par exemple, Cupaniopsis, Haplocoelum, Matayba). Afin de maintenir le critère de monophylie, une nouvelle classification informelle des Sapindaceae est proposée sur la base de caractères moléculaire et morphologique. La famille des Sapindaceae est donc subdivisée en quatre sous-familles et dix groupes comme suit (les sous-familles sont triées par ordre phylogénétique): Xanthoceroideae (comprend uniquement Xanthoceras sorbifolium), Hippocastanoideae (deux groupes; comprend les Aceraceae et Hippocastanaceae), Dodonaeoideae (deux groupes) et Sapindoideae (dix groupes). De plus, des analyses moléculaires et morphologiques complémentaires ont permis de reconnaître un nouveau genre endémique de Madagascar, Gereaua, ségrégé d’Haplocoelum (chapitre 4). Une révision taxonomique du genre Lepsianthes à Madagascar est également présentée, dans laquelle une espèce nouvelle est décrite, L. sambiranensis (chapitre 5). Finalement, l’arbre phylogénétique ainsi que les données sur les fossiles et la distribution des taxa ont été utilisés pour investiguer l’histoire évolutive des Sapindaceae. Cela a été rendu possible par l’application et la comparaison des toutes dernières méthodes développées en biogéographie. Une contribution au développement des analyses biogéographiques est également proposée par la présentation d’un modèle biogéographique basé sur les relations paléogéographiques (chapitre 2). De plus, l’incertitude sur l’estimation de l’âge des clades a été considérée lors de l’interprétation des scénarios biogéographiques (chapitre 2). Finalement, l’impact des facteurs abiotiques (par exemple, les intenses activités tectoniques ou les forces orbitales) et biotiques (par exemple, la co-évolution plantes/animaux) sur la diversification des Sapindaceae ont été étudiés. Ces analyses supportent une origine des Sapindaceae en Asie tempérée au début du Crétacé suivie par une colonisation des zones tropicales depuis la fin du Paléocène (chapitre 3). Cette étude montre, pour la première fois, que le changement climatique abrupt ayant eu lieu entre l’Eocène et l’Oligocène, a accéléré le taux de diversification des Sapindaceae. Ce résultat, qui s’oppose à la majorité des paradigmes (voir chapitre 3 pour plus d’information), est principalement dû aux propriétés géologiques et climatiques rencontrées en Asie du Sud Est. En effet, cette région a favorisé de multiples contacts entre les lignées de Sapindaceae et de successives spéciations ont eu lieu sur les continents Laurasien et Gondwanien. Cette étude montre l’importance jouée par l’Asie du Sud Est dans l’évolution des Sapindaceae (ainsi que probablement d’autres familles d’angiospermes) et souligne l’importance de préserver cette région qui subit de fortes pressions humaines., The economically important soapberry family (Sapindaceae; Sapindales) comprises about 1900 species (distributed into ca. 140 genera; e.g., Litchi, Paullinia) mainly found in tropical regions, with only a few genera being restricted to temperate areas. For more than a century, the circumscription of the family (especially the potential inclusion of Aceraceae and Hippocastanaceae within the Sapindaceae) as well as the relationships among subfamilial entities have been widely challenged (chapter 1 for a review). In this study, infrafamilial relationships within the Sapindaceae and its relationships to the closely related Aceraceae and Hippocastanaceae are investigated based on eight nuclear and plastid markers and inferred from the complementary supermatrix (chapter 1) and supertree (chapter 6) approaches. Both approaches support the monophyly of Sapindaceae when Aceraceae and Hippocastanaceae are included and highlight a high level of paraphyly and polyphyly at the subfamilial and tribal levels. The monophyletic status of several genera is even contested (e.g., Cupaniopsis, Haplocoelum, Matayba). In order to maintain monophyly, a new informal classification is proposed based on molecular and morphological evidence. The soapberry family is thus subdivided into four subfamilies and 14 groups as follows (sorted according to phylogenetic relationships): Xanthoceroideae (only composed by Xanthoceras sorbifolium), Hippocastanoideae (two groups; including the previous Aceraceae and Hippocastanaceae), Dodonaeoideae (two groups) and Sapindoideae (ten groups). In addition, further molecular and morphological investigations allow the recognition of a new Malagasy genus, Gereaua, segregated from Haplocoelum (chapter 4). A taxonomic revision of Lepisanthes in Madagascar is also proposed with the description of a new species, L. sambiranensis (chapter 5). Phylogenetic framework, fossils data and taxa distributions are used to infer the evolutionary history of the soapberry family. This is achieved by applying and comparing state-of-the-art biogeographic methods. Moreover, additional contributions to the biogeographic framework are proposed, for instance the implementation of a biogeographic model based on paleogeographic connections (chapter 2). The influence of divergence time uncertainty on biogeographic scenario is also considered (chapter 2). Finally, the impact of abiotic (e.g., intense tectonic activities, orbital forces) and biotic (e.g., co-evolution plants/animals) factors on the diversification of the Sapindaceae is investigated based on biogeographic inference and divergence time estimations (chapter 3). Results strongly suggest an origin of Sapindaceae in temperate Asia sometime in the Early Cretaceous with a subsequent spread all over the tropics since the Late Paleocene (chapter 3). In this study, it is show, for the first time, that abrupt climatic change in the Eocene-Oligocene boundary triggered the diversification rates of the Sapindaceae. This paradigm-breaking result is mainly due to the geological and climatic properties of South East Asia that favoured multiple contacts between lineages and further speciation across Laurasian and Gondwanian continents. This study highlights the importance of South East Asia in the evolution of the soapberry family (as well as that of additional angiosperms families) and underlines the importance to preserve this highly endangered area.

2007, Haemmerli, Marc, Küpfer, Philippe

Based on results from six different molecular studies and cytology, a taxonomic revision of Gentiana Sect. Calathianae Froel. is proposed. The revised section comprises two subsections: Subsect. Calathianae with three taxa having relatively low chromosome numbers, G. nivalis L. and G. utriculosa L. (annuals), and G. pumila Jacq. (perennial); and Subsect. Vernae (Tzevelev) Haemmerli stat. nov. with nine species and seven subspecies including G. verna L. (with two subspecies, G. verna subsp. verna, and G. verna subsp. delphinensis Kunz), G. tergestina Beck (with three subspecies, G. tergestina subsp. tergestina, G. tergestina subsp. balcanica Haemmerli, and G. tergestina subsp. pontica Haemmerli), G. sierrae Briquet, G. schleicheri Kunz, G. rostanii Reuter, G. bavarica L. (with two subspecies, G. bavarica subsp. bavarica , and G. bavarica L. subsp. subacaulis Müller), G. terglouensis Hacq., G. brachyphylla Vill., and G. orbicularis Schur . These results provide indications into the biogeography of the section. While several taxa such as G. pumila Hacq., G. bavarica L. subsp. subacaulis Müller, G. rostanii Reuter or G. verna L. subsp. delphinensis Kunz, G. terglouensis Hacq. or G. schleicheri Kunz are restricted to the Alpine chain, others occur on different European Mountain ranges (G. utriculosa L., G. sierrae Briq. and G. brachyphylla Vill.), but only few taxa also grow outside Europe (G. nivalis L., G. verna L, G. tergestina Beck and G. penetii Romo). Two species are not represented at all within the Alps: G. penetii Romo from Atlas Mts. and G. tergestina Beck including its subspecies from Dinaric Alps eastward, throughout minor Asia to the Altai Mts. Most taxa are limited to only a limited distribution area or to only one or few mountain ranges. Widespread taxa are the annual G. nivalis L. and the perennials G. verna L. s.str. and G. tergestina Beck s.l.

2004, Nessi, Luca, Rahier, Martine, Tabacchi, Raffaele, Küpfer, Philippe, Städler, Erich

La chrysomèle alpine Oreina gloriosa (Coleoptera: Chrysomelidae) est hautement spécialisée sur l'ombellifère Peucedanum ostruthium (Apiaceae), l'impératoire. Dans la nature, adultes et larves sont très sélectifs: les uns et les autres se nourrissent exclusivement de feuilles, mais surtout, ils choisissent certaines plantes et en ignorent complètement d'autres. En outre, des études préliminaires ont suggéré que les larves soient actives la nuit. Comme de nombreuses autres apiacées, P. ostruthium synthétise plusieurs furanocoumarines linéaires, métabolites secondaires décrits pour leur activité antiappétante et/ou toxique à l'égard d'insectes phytophages généralistes. Par ailleurs, les furanocoumarines linéaires sont connues pour leur action phototoxique: le rayonnement ultraviolet proche du spectre visible induit chez ces substances une excitation électronique qui peut déterminer différents processus toxiques au niveau cellulaire. Le but de cette étude a été de vérifier l'hypothèse selon laquelle la chimie secondaire de P. ostruthium constituerait un facteur explicatif de l'utilisation de la plante hôte par la strictement monophage O. gloriosa. Toute la recherche a été menée au sein d'une seule population de la plante et de la population du coléoptère qui lui est inféodée.

Dans un premier temps, nous avons étudié le rythme d'activité de la chrysomèle sur les 24 heures dans son mileu. Ce travail a révélé que les larves se nourrissent exclusivement pendant la nuit et s'enfoncent dans le sol pendant le reste de la journée. Certaines jeunes larves se retirent dans les gaines ou les bourgeons. Au contraire, les adultes ont été observés sur les plantes pendant les 24 heures. Nous avons ensuite vérifié si la croissance larvaire était affectée par la photoactivation des furanocoumarines. Pour cela, les larves ont été placées dans deux environnements lumineux, avec et sans rayonnement ultraviolet. La mortalité a été plus grande dans le deuxième traitement: nous n'avons néanmoins pas pu tirer de conclusions définitives sur l'effet de la photoactivation des furanocoumarines. Dans un travail de terrain, nous avons montré que les invertébrés antagonistes de O. gloriosa sont essentiellement actifs pendant la nuit. Au vu du chevauchement entre les périodes d'activité de la chrysomèle et de ses prédateurs, nous suggérons que les ennemis naturels ne constituent pas la raison d'être du comportement des larves. Nous proposons que l'activité nocturne représente une contre-adaptation à la photoactivité des furanocoumarines. Il s'agirait alors d'un rare exemple d'adaptation comportementale chez un insecte spécialisé sur une plante hôte avec furanocoumarines linéaires. En effet, les insectes avec un régime alimentaire strictement lié à ces métabolites secondaires, disposent en général de moyens de détoxification métaboliques. La différence comportementale entre larves et adultes est probablement liée à l'épaisseur et à la pigmentation de la cuticule imaginale, vraisemblablement opaque à la lumière ultraviolette.

Nous avons également vérifié l'hypothèse selon laquelle O. gloriosa dans la nature sélectionnerait les plantes en fonction de leur attributs chimiques. Nous avons échantillonné des plantes fortement consommées et des plantes libres de toute attaque d'O. gloriosa. Les feuilles ainsi récoltées ont été extraites et analysées par chromatographie liquide pour déterminer la composition qualitative et quantitative en furanocoumarines linéaires. La chimie secondaire a permis de bien discriminer plantes consommées et non consommées. O. gloriosa est probablement à même de reconnaître certaines de ces substances comme signaux ou elle est en mesure de mieux en tolérer certaines plutôt que d'autres dans leur action toxique. De toute façon, la chimie secondaire explique, du moins en partie, l'utilisation de la plante hôte par ce coléoptère strictement monophage au sein d'une population de sa plante nourricière. Cet exemple révèle la nécessité d'approfondir l'investigation, trop souvent négligée, du rôle de la variation chimique intrapopulationnelle dans les études concernant la convenance de l'hôte pour des insectes spécialistes., The alpine leaf beetle Oreina gloriosa (Coleoptera: Chrysomelidae) is highly specialized on the umbelliferous plant Peucedanum ostruthium (Apiaceae). Adults and larvae feed on leaves and clearly discriminate between individual plants in nature. Preliminary studies suggested that the larvae are active at night. Like many other apiaceous plants, P. ostruthium produces several linear furanocoumarins, secondary metabolites shown to be deterrent and/or toxic to generalist herbivorous insects. Moreover, linear furanocoumarins are well known as phototoxins: when irradiated with long-wave ultraviolet light, these compounds enter an excited state which can mediate different within cell toxic processes. The aim of the present thesis was to verify the hypothesis that the secondary chemistry of P. ostruthium is a factor explaining the actual host-plant use by the strictly monophagous O. gloriosa. All of our work concentrated on a single plant population and its affiliated beetle population.

We firstly investigated the daily activity rhythm of the beetles in the field. Larvae have been shown to feed exclusively at night and to burrow into the soil for the remainder of the day. Some young larvae hid within sheaths or buds during daylight. In contrast, adults were found on the plants over the 24 hours. We further verified if the performance of the larvae was affected by the photoactivation of the linear furanocoumarins. Larvae were forced to remain in two different light environments, with and without ultraviolet radiation. O. gloriosa was shown to suffer higher mortality when forced under light with ultraviolet radiation. Nonetheless, we were not able to draw definitive conclusions on the effect of the furanocoumarin photoactivation. In a field study, we showed that invertebrate natural enemies of O. gloriosa are mainly active at night. Because of the congruence in time between the beetle and the predators activities we suggest that natural enemies should not be considered as a driving force for the larval night behaviour. We propose that night activity counteracts the photoactive furanocoumarins and should be considered a rare example of behavioural adaptation in a linear furanocoumarin-specialized insect. In fact, specialists are reported to rely on metabolic detoxification. The difference in the behaviour between larvae and adults may be related to the thick and pigmented cuticle of the latter, probably opaque to ultraviolet light.

Finally we tested the hypothesis that individual plants remain free from O. gloriosa attack due to their chemical attributes. Samples of plants hosting actively feeding O. gloriosa (strongly consumed leaves) and plants exhibiting no damage (unattacked leaves) were collected in the field and examined by HPLC for their qualitative and quantitative linear furanocoumarin composition. The secondary chemistry allowed us to clearly segregate attacked and unattacked plants. We suggest that plant chemistry, relative to the ability of O. gloriosa to tolerate or recognise plant chemicals, can to some extent explain the host use of this monophagous leaf beetle within a population of its host plant. This shows the need for further work on the neglected impact, for specialists, of intrapopulation variation in secondary chemistry on host suitability., Il crisomelide alpino Oreina gloriosa (Coleoptera: Chrysomelidae) è altamente specializzato sull'ombellifera Peucedanum ostruthium (Apiaceae), l'imperatoria vera o erba rena. Adulti e larve in natura sono estremamente selettivi: gli uni e le altre si nutrono solo di foglie, ma distinguono tra le singole piante cibandosi di alcune e trascurandone completamente altre. Inoltre indagini preliminari hanno suggerito che le larve siano attive di notte. Analogamente ad altre ombellifere, P. ostruthium contiene varie furanocumarine lineari, metaboliti secondari con effetto deterrente e/o tossico per insetti fitofagi generalisti. Le furanocumarine lineari sono pure note per la loro fototossicità: la luce ultravioletta di lunghezza d'onda vicina allo spettro visibile induce in queste sostanze un'eccitazione elettronica che può provocare vari processi tossici a livello cellulare. La nostra tesi aveva lo scopo di verificare l'ipotesi secondo cui la chimica secondaria di P. ostruthium spiegherebbe l'utilizzazione della pianta ospite - così come l'osserviamo oggi - da parte dello specialista O. gloriosa. Per l'insieme della ricerca ci siamo concentrati su una singola popolazione della pianta e sulla popolazione dell'insetto ad essa legata.

Per prima cosa abbiamo studiato il ritmo di attività dei coleotteri in natura. Abbiamo mostrato che le larve si nutrono esclusivamente di notte e passano il resto della giornata nel suolo. Alcune giovani larve si ritirano dentro le guaine o i germogli. Gli adulti invece sono presenti sulle piante durante tutte le 24 ore. In una ricerca successiva abbiamo verificato se la crescita larvale sia compromessa dalla fotoattivazione delle furanocumarine lineari. Abbiamo tenuto le larve in due distinti ambienti luminosi, con e senza radiazione ultravioletta. Quest'ultima ha determinato una maggiore mortalità di O. gloriosa, tuttavia non siamo stati in grado di formulare conclusioni definitive sull'effetto della fotoattivazione. Un lavoro sul campo ha mostrato che gli invertebrati antagonisti del crisomelide sono essenzialmente attivi di notte. Dato che i periodi di attività di O. gloriosa e dei suoi predatori si sovrappongono, suggeriamo che i nemici naturali non costituiscano il fattore selettivo all'origine del comportamento notturno delle larve. Proponiamo che questa limitazione notturna nell'attività trofica rappresenti un'acquisizione adattativa contro la fotoattività delle furanocumarine. Si tratterebbe allora di un raro esempio di adattamento comportamentale in un insetto specializzato su una pianta contenente furanocumarine lineari. In effetti, in generale gli insetti con un regime alimentare strettamente legato a queste sostanze dispongono di strumenti di detossificazione metabolici. La differenza comportamentale tra larve e adulti potrebbe essere ricondotta al tegumento spesso, colorato e probabilmente opaco alla luce ultravioletta di questi ultimi.

Abbiamo infine verificato l'ipotesi secondo cui la chimica secondaria determinerebbe l'idoneità delle singole piante per la crescita di O. gloriosa. Abbiamo proceduto ad un campionamento sul campo di piante fortemente consumate e di piante prive di qualsiasi segno d'attacco del crisomelide. Le foglie così raccolte sono state estratte e analizzate con la cromatografia liquida per determinare la composizione qualitativa e quantitativa delle furanocumarine. La chimica secondaria ha permesso di ben caratterizzare le piante consumate rispetto a quelle non utilizzate. O. gloriosa è probabilmente in grado di riconoscere talune di queste sostanze quali segnali o di tollerarne alcune piuttosto di altre nella loro azione di tossine. In ogni caso, la chimica secondaria permette di spiegare perlomeno in parte l'utilizzazione della pianta in seno ad una popolazione da parte di questo coleottero altamente specializzato. Questo risultato rivela l'interesse di ulteriori ricerche su un aspetto spesso ignorato quale è l'impatto, per artropodi specialisti, della variazione intrapopolazionale nella chimica secondaria sull'idoneità della pianta ospite.

2007, Kissling, Jonathan, Küpfer, Philippe, Linder, Peter

In an attempt to understand the evolutionary history of the poorly studied genus Sebaea and its relationship to other genera of tribe Exaceae (Gentianaceae), intensive morphological and karyological character optimization based on robust molecular phylogeny was performed. Phylogenetic reconstructions support the monophyly of Exaceae, and further reveal a polyphyletic Sebaea, including four well-supported clades, hereafter treated as separate genera, based on non-molecular synapomorphies. The first clade contains the single species Lagenias pusillus, characterized by its medifix anthers, inserted at the base of the corolla tube and its seed testa cells (polygonal). The second clade, Sebaea s. str., contains most of the South African species having secondary stigmas and bilateral seeds, with rectangular testa cell. The third clade, Exochaenium, contains exclusively tropical African species, characterized by a stylar polymorphism and a papillose clavate stigma (versus smooth and bilobed). Finally the fourth clade, Klackenbergia, contains two species characterized by inflorescences with axillary subsessile flowers. Based on these results, the taxonomic reinstatement of Exochaenium (23 species) and Lagenias (1 species), along with the establishment of a new genus Klackenbergia (2 species), are proposed. In the light of the new phylogenetic relationships found within the Exaceae, new views on the evolution of (1) karyological and (2) morphological characters are proposed. Finally, the historical biogeography of the tribe is reevaluated (3). 1. Intensive chromosome counts based on material collected and fixed in the field (157 population and c. 60 species), and exhaustive literature survey, reveal a broad set of chromosome numbers (2n = 18, 28, 32, 34, 36, 42, 52, 54, 56, 60, 62, 64, 68), and the occurrence of polyploid systems within Exacum and Sebaea. These results allow us to postulate x = 7, 8, or 9 as the possible base chromosome numbers for the Exaceae. Karyological reconstruction, based on the molecular phylogeny, suggest a basic number of x=7 for the Exaceae, followed by dysploidy event leading to secondary base number of x=8 and x=9, and several polyploidization events. 2. Optimization of morphological characters suggests that the most recent common ancestor of Exaceae (MRCA) was similar to Lagenias pusillus by having pentamerous yellow actinomorphic flowers, with anthers included in the corolla tube and dehiscing by longitudinal slits, a bilobed stigma, and the absence of secondary stigmas, and cubical seeds with polygonal testa cells. This MRCA might have then developed particular floral syndromes as indicated by long corolla tubes or presence of enantiostyly in the tribe. 3. Dating analyses and dispersal-vicariance reconstructions suggest that the Exaceae evolved c. 32 million years ago in Africa and subsequently spread to Madagascar. The colonization of Australia, New-Zealand, and Asia involved at least three long-distance dispersals. Early diversification of Exaceae in Africa might be the consequence of the development of a temperate with dry summer climate, in the Cape region (South Africa), while the Quaternary climatic variation might explain most of the species diversity of Sebaea and Exochaenium. At the generic level, molecular phylogenies of Sebaea, based on chloroplastic and nuclear DNA markers, reveals five well-supported clades. Sebaea sulphurea seems to have evolved early, and is distinct from all the remaining extant species. Each clade is supported by several characters (morphological, vegetative, phenological or geographical), and a preliminary infrageneric classification is proposed.

2006, Song, Yi, Küpfer, Philippe

Balsaminaceae has two genera: Hydrocera with only one species and Impatiens, with over 1000 species. Hydrocera is found in the Indo-Malaysia areas, and Impatiens is highly clustered in tropical Africa, Madagascar, southern India, the eastern Himalayas, Southeast Asia. Only a few Impatiens species are found in the temperate areas of the northern hemisphere. In order to have a better understanding of the morphological and karyological evolution, an seedcoat micromorphology investigation with 38 species of Impatiens and a karyological investigation with 45 species of Impatiens was carried out. A high diversity were found in seedcoat micromorphology of Impatiens. Four morphological types were distinguished. Taxonomic and phylogenetic implications of the seedcoat micromorphology are also discussed. Karyological investigations revealed the somatic chromosome numbers vary greatly from 2n=6 to 2n=66 in Impatiens. Considering all the available chromosomal data, x=7, 8, 9, 10 are the most frequent basic numbers of the family. Geographic distributions of the most frequent basic numbers show interesting patters in hotspots of Impatiens. The DNA molecular phylogenetics of this family were then explored. One nuclear DNA region, internal transcribed spacers (ITS) and two non-coding chloroplast DNA regions, trnL-trnF and aptB-rbcL have been chosen as DNA mark. 182 species of this family coming from different distribution center and three species of Marcgraviaceae and two species of Tetrameristaceae were amplified by PCR, sequenced and phylogenetically analyzed applying distance estimates and Bayesian method. Five phylogenetic topologies were received. They are congruent with each other, but not identical. Only combined Bayesian tree is well supported in most of the lineages. Molecular phylogeny results confirmed the monophyly of Balsaminaceae and Impatiens and it showed that the phylogenetic relationships in Impatiens are associated with geographic distribution, less defined by gross morphology. The parsimonious optimization of the centers of endemism onto the molecular phylogenies revealed that extant Impatiens species are of mainland Southeast Asian origin, from where dispersal to boreal Eurasia and North America, to central Asia and Eastern Europe via the Himalayas, and to India and Africa have occurred. The Madagascan Impatiens show an African origin. The character-state optimization suggested that spurred flower, narrowly-fusiform capsule, 4 lateral sepals, 3-colpate pollen and reticulate seedcoat represent the plesiomorphic states within Impatiens. Both x=8 and 10 might be the ancestral basic chromosome number in Balsaminaceae. The diverse and complicate specialization of floral structures of Balsaminaceae is pollinator-mediated, and therefore highly homoplasious.