DISSERTATION / DOCTORAL THESIS Titel der Dissertation /Title of the Doctoral Thesis „Phylogenetic community structure assessment of a mixed Dipterocarp forest using DNA barcoding and molecular phylogeny of the dominant tree family Dipterocarpaceae“ verfasst von / submitted by Jacqueline Heckenhauer, BSc, MSc angestrebter akademischer Grad / in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) Wien, 2017 / Vienna 2017 Studienkennzahl lt. Studienblatt / A 794 685 437 degree programme code as it appears on the student record sheet: Dissertationsgebiet lt. Studienblatt / Biologie/Biology field of study as it appears on the student record sheet: Betreut von / Supervisor: ao. Univ.-Prof. i. R. Dipl.-Biol. Mag. Dr. Mary Rosabelle Samuel ACKNOWLEDGEMENTS First of all I would like to express my deep gratitude to my supervisor Prof. Rosabelle Samuel for giving me the opportunity to write my PhD thesis about such an interesting topic, for her excellent supervision, and for supporting me to participate at conferences. She always had time when questions arose. I would also like to thank her for the helpful suggestions and the constructive criticism regarding the preparations of the manuscripts. I would like to thank all collaborators and co-authors for their great professional support, especially, Dr. Kamariah Abu Salim, for enabling trouble-free field work by dealing with the export permits and providing material; Ass. Prof. Ovidiu Paun, for being a brilliant advisor regarding the RADseq, during library preparation, as well as with analysis and interpretation of RADseq data; Prof. Mark W. Chase, for interesting debates during his visits to Vienna and his help in editing English texts; Charles Bullard Professor Peter S. Ashton, for sharing his knowledge and interesting ideas on Dipterocarpaceae that have contributed to this thesis; Dr. Michael H.J. Barfuss, who not only shared an office but also his expertise on DNA barcoding and sequencing with me; Prof. Toby Pennington and Dr. Kyle Dexter, for their advice during community structure analysis; and Prof. David Burslem for giving me the opportunity to spend time with his research group in Aberdeen. Thanks to Dr. Joseph Greimler, former deputy head and Prof. Christian Lexer, current deputy head of the Department of Botany and Biodiversity Research, Division of Systematic and Evolutionary Biology for providing infrastructural resources. Colleagues of our division, as well as PhD Lucia V. Castello are acknowledged for their support and for the great time we had during lunch breaks, seminars, or in the botanical garden when visiting the Sequoia tree. The friendships of MSc Marie K. Brandrud and PhD Jacky Hess was a great strength throughout my research and preparing the thesis. The following persons are acknowledged for technical support: Verena Klenja for contributing a lot to the lab work, especially during DNA extractions; Ing. Elfriede Grasserbauer for maintaining the lab; and MSc Juliane Baar for advice and help with the RADseq library preparations. Field assistants Fiona Willinathy Anak Amdani, Sawai Anak Amba and Teddy Chua of the KBFS, Brunei, are acknowledged for their support during field work. I would like to thank my friends, parents and grandmothers, especially Rosemarie, for supporting and motivating me since my bachelor studies and for their interest in the topic of my Ph thesis. Special thanks to my partner Benjamin for his strong emotional support and understanding over the duration of conducting this thesis. Last but not least, I would like to thank the two independent reviewers for reviewing the thesis. This project was funded by the Austrian Science Fund (FWF; project P26548-B22, grant given to Prof. Dr. Mary Rosabelle Samuel). CONTENT ABSTRACT ............................................................................................................................................... 7 ZUSAMMENFASSUNG ............................................................................................................................... 9 INTRODUCTION ..................................................................................................................................... 11 AIMS ..................................................................................................................................................... 18 REFERENCES ......................................................................................................................................... 19 PART 1: DNA BARCODES AND PHYLOGENETIC COMMUNITY STRUCTURE ....................................... 27 Chapter 1: Universal multiplexable matK primers for DNA barcoding of angiosperms .................. 27 Chapter 2: Plant DNA barcodes and assessment of phylogenetic community structure of a tropical mixed dipterocarp forest in Brunei Darussalam (Borneo) .................................. 37 PART 2: MOLECULAR PHYLOGENY AND PHYLOGENENOMICS OF DIPTEROCARPACEAE .............. 101 Chapter 3: Phylogenetic analyses of plastid DNA suggest a different interpretation of morphological evolution than those used as the basis for previous classifications of Dipterocarpacea (Malvales) .......................................................................................... 101 Chapter 4: Phylogenomics resolve evolutionary relationships and provide first insights into floral evolution in the tribe Shoreeae (Dipterocarpaceae) ..................................................... 141 CONCLUSION ....................................................................................................................................... 183 APPENDIX ............................................................................................................................................ 185 Conference contributions ................................................................................................................. 185 5 ABSTRACT For DNA barcoding, the two markers ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) and maturase K (matK) are widely used in plants. In contrast to the well conserved rbcL barcode region, the matk is rapidly evolving and therefore highly variable, approx. three times more variable than that of rbcL. PCR amplification of matK is often difficult especially when dealing with many different plant families. Earlier DNA barcoding studies have used different primer pair combinations for different plant families which is time consuming and costly. In this project, 14 universal matk primers were developed using complete matk sequences available from GenBank. These primers, when combined in a multiplex PCR, amplify the target region across a wide range of Angiosperms families. In this study, the matK, together with the rbcL barcode region, successfully identified trees and shrubs (3237 individuals) mostly at generic level, which helped to assess the phylogenetic community structure in several subplots of a 25 ha mixed Dipterocarp forest in Brunei Darussalam (Borneo, Southeast Asia). The combined matrix of rbcL + matK barcodes [555 haplotypes which belong to ≥154 genera, 68 families, 25 orders sensu Angiosperm Phylogeny Group (APG)] was used to reconstruct phylogenetic relationships, with and without constraining the topology of taxonomic orders to match that proposed by the Angiosperm Phylogeny Group (APG III). A third phylogenetic tree was reconstructed using the program Phylomatic which trims a reference tree to taxa occurring in the community. This program is traditionally used by many ecologists. The different phylogenetic trees obtained were used to calculate community metrics [net relatedness index (NRI) and nearest taxon index (NTI)]. The community indices detected the same patterns of community structure: in most cases it was either random community assembly or phylogenetically clustering, irrespective of the different phylogenetic trees used for calculations. Phylogenetic clustering indicates that habitat filtering plays a role in assembly processes. However, the Phylomatic tree produces greater variation across the plots for NRI and NTI, presumably due to noise introduced by using an unresolved phylogenetic tree. Dipterocarpaceae (Malvales) are the dominant trees in the study plot, which led to investigation of the molecular phylogeny of the whole family including all subfamilies. Dipterocarpaceae comprises three subfamilies: the largest Asian subfamily Dipterocarpoideae, Monotoideae from Africa, Madagascar and the Colombian Amazon, and Pakaraimaeoideae from Guaianan Highlands and Venezuela. Molecular clock analysis reveals that extant Dipterocarpoideae diverged approx. 55 Mya. Phylogenetic analysis of plastid regions including all three subfamilies as well as representatives of closely related families Sarcolaenaceae, Cistaceae, and Bixaceae, highlights differences from the previous morphological classifications. Pakaraimaea of the monotypic subfamily Pakaraimaeoideae is assigned to Cistaceae. Monotoideae is weakly supported as sister to Dipterocarpoideae. In the subfamily Dipterocarpoideae, Dipterocarpus is sister to Dryobalanops and tribe Shoreeae which contradicts the morphological concepts of the tribes Dipterocarpeae (Anisoptera, Cotylelobium, Dipterocarpus, 7 Stemonoporus, Vatica, Vateria, and Vateriopsis) and Shoreeae (Hopea, Parashorea, Neobalanocarpus und Shorea) sensu Ashton. Further, the genus Shorea (sensu Ashton) is not monophyletic. Genome sizes of the species examined are small (0.3264–0.6724 pg). New chromosome numbers are