THE SIGNIFICANCE OF FRUIT AND SEED ANATOMY IN THE EVOLUTION OF SABIACEAE Ludovica Santilli 2016 Thesis submitted in partial fulfillment for the MSc in Biodiversity and Taxonomy of Plants 1 Abstract Fruit and seed morphology and anatomy of selected species of Ophiocaryon and Meliosma were investigated to gain insight into the evolution of fruit and seed related characters in the Sabiaceae and to find possible distinctive characters for the family in relation to their position at the base of Proteales. The investigation implied the experimentation of three different embedding techniques, the optimisation of the protocols with regards to the material object of this study, and the use of microtomy and of light microscopy. Carpological characters are often indicators of taxonomic affinities and used to distinguish taxa at different level. Similarly, seed morphology and anatomy, especially when the seed is investigated in the context of its development -from its early embryonal phase to its maturity- are considered important source of phylogenetic relationships. The fruit of Sabiaceae has developed into a drupe in which the pericarp becomes hard and takes over the seed protection, while the seed coat is reduced and partly disintegrated or disappears during development. Some species of Sabiaceae have ovules that have become unitegmic probably by loss of the outer integument and are hemitropous to orthotropous. These two derived conditions from an ancestral bitegmic and anatropous ovule probably go together as the outer integument, is thought to be responsible for curvature of ovules in angiosperms. The ovules develop into orthotropous exalbuminous seeds with a collapsed and undifferentiated seed coat. The orthotropous and pachychalazal seed of Ophiocaryon might be derived from the orthotropous seed of Meliosma which does not show pachychalazaly. As for the possible characters shared with other early diverging eudicots, this study could not find unequivocal evidences. Nevertheless, the most interesting features to be further investigated in order to address the question have been identified. Additional, anatomical investigation on ovules and seeds of Sabiaceae would confirm the process through which unitegmy has been achieved and allow direct comparison with those members of Proteales that show orthotropy and reduction of the outer integument. 2 Acknowledgements I wish to thank my supervisors Wolfgang Stuppy and Louis Ronse de Craene for their support throughout the project. Lab work was a delicate, time consuming part of the project. Sincere thanks go to Frieda Christie for her assistance and precious advices during the lab work, which allowed me to improve the protocol and to get beautiful and clear LM images. I would also like to thank Michael Muller for finding the time to answer my questions on histochemical tests and staining procedure and Greg Kenicer for his help in the moment of need. Most of all, I want to express my gratitude to my friends. My classmates for being such a good, unforgettable team, particularly Camila for cycling together to the garden, Jia my darling for your sweetness, Marita for letting me complain the all year and Nicolas for all the time together and all the little things. Roberta, for spending the nights writing in the library of the MSB and make it incredibly fun. Chiara and Fruszi, for understanding everything I say and even more. Finally, to my family. My mother who made this possible, my sister who will be a great doctor and my little brother who has inspired me with his courage, I am very proud of you both. 3 Table of contents ABSTRACT ........................................................................................................................................................ 1 ACKNOWLEDGEMENTS .................................................................................................................................... 2 TABLE OF CONTENTS ........................................................................................................................................ 3 TABLE OF FIGURES ........................................................................................................................................... 4 1 INTRODUCTION ....................................................................................................................................... 5 1.1 GENERA OF SABIACEAE ................................................................................................................................ 5 1.2 TAXONOMY............................................................................................................................................... 6 1.2.1 Phylogenetic position of Sabiaceae .................................................................................................. 6 1.2.2 Infrageneric classification ................................................................................................................. 8 1.3 MORPHOLOGY ......................................................................................................................................... 10 1.3.1 Floral morphology ........................................................................................................................... 10 1.3.2 Gynoecium ...................................................................................................................................... 11 1.3.3 Fruit and seed morphology ............................................................................................................. 12 1.4 AIMS OF THE INVESTIGATION ...................................................................................................................... 13 2 MATERIALS AND METHODS ................................................................................................................... 14 2.1 PLANT MATERIAL ...................................................................................................................................... 14 2.2 EMBEDDING WITH TECHNOVIT .................................................................................................................... 14 2.3 EMBEDDING WITH LR WHITE RESIN .............................................................................................................. 16 2.4 CRYOSECTIONING ..................................................................................................................................... 17 2.5 HISTOCHEMICAL TESTS .............................................................................................................................. 18 2.6 LIGHT MICROSCOPY .................................................................................................................................. 18 2.7 TERMINOLOGY USED ................................................................................................................................. 18 3 RESULTS ................................................................................................................................................ 19 3.1 FRUIT DEVELOPMENT ................................................................................................................................ 19 3.1.1 Ovary wall ....................................................................................................................................... 20 3.1.2 Pericarp ........................................................................................................................................... 22 3.2 SEED DEVELOPMENT ................................................................................................................................. 27 4 DISCUSSION ........................................................................................................................................... 31 4.1 DIVERSIFICATION WITHIN SABIACEAE............................................................................................................ 31 4.2 EVOLUTIONARY TRENDS IN SABIACEAE .......................................................................................................... 32 4.2.1 Fruit development and evolution .................................................................................................... 32 4.2.2 Seed form and integuments ............................................................................................................ 34 4.3 COMPARISON WITH CLOSEST FAMILIES.......................................................................................................... 36 CONCLUSIONS ............................................................................................................................................... 40 REFERENCES ................................................................................................................................................... 42 APPENDIX ...................................................................................................................................................... 48 4 Table of figures Cover page Meliosma bogotana Steyerm. Photo by John Bernal, Jardin Botanico de Bogotà. Figure 1 Phylogeny of early diverging Eudicots .................................................................... 7 Figure 2 Phylogeny of Sabiaceae. .......................................................................................... 9 Figure 3 A-D= M. cuneifolia gynoecium at anthesis E-F= O. heterophyllum young fruits. A= LS along the frontal plane B=