COPYRIGHT AND CITATION CONSIDERATIONS FOR THIS THESIS/ DISSERTATION o Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. o NonCommercial — You may not use the material for commercial purposes. o ShareAlike — If you remix, transform, or build upon the material, you must distribute your contributions under the same license as the original. How to cite this thesis Surname, Initial(s). (2012) Title of the thesis or dissertation. PhD. (Chemistry)/ M.Sc. (Physics)/ M.A. (Philosophy)/M.Com. (Finance) etc. [Unpublished]: University of Johannesburg. Retrieved from: https://ujdigispace.uj.ac.za (Accessed: Date). Combretaceae: Phylogeny, Biogeography and DNA Barcoding by JEPHRIS GERE THESIS Submitted in fulfilment of the requirements for the degree PHILOSOPHIAE DOCTOR in BOTANY in the Faculty of Science at the University of Johannesburg December 2013 Supervisor: Prof Michelle van der Bank Co-supervisor: Dr Olivier Maurin Declaration I declare that this thesis has been composed by me and the work contained within, unless otherwise stated, is my own. _____________________ J. Gere (December 2013) Table of contents Table of contents i Abstract v Foreword vii Index to figures ix Index to tables xv Acknowledgements xviii List of abbreviations xxi Chapter 1: General introduction and objectives 1.1 General introduction 1 1.2 Vegetative morphology 2 1.2.1 Leaf morphology and anatomy 2 1.2.2. Inflorescence 3 1.2.3 Fruit morphology 4 1.3 DNA barcoding 5 1.4 Cytology 6 1.5 Fossil record 7 1.6 Distribution and habitat 7 1.7 Economic Importance 8 1.8 Taxonomic history 9 1.9 Aims and objectives of the study 11 i Table of contents Chapter 2: Molecular phylogeny of Combretaceae with implications for infrageneric classification within subtribe Terminaliinae. 2.1 Introduction 24 2.2 Materials and methods 26 2.2.1 Taxon sampling, outgroups and loci selection 26 2.2.2 DNA extraction, amplification, sequencing and alignment 27 2.2.3 Phylogenetic analyses of molecular data 29 2.3 Results 31 2.3.1 Characteristics of genes sequences 31 2.3.2 Congruence tests 32 2.3.3 Combined plastid analysis 32 2.3.4 ITS analysis 33 2.3.5 Combined data set analysis Bayesian Inference analysis 34 2.4 Discussion 36 2.5 Taxonomic treatment 42 Chapter 3: Reconstructing the biogeographical history of the family Combretaceae (Myrtales) using DNA sequences 3.1 Introduction 81 3.2 Materials and methods 83 3.2.1. Taxon sampling and outgroups selection 83 3.2.2. DNA Extraction, amplification, sequencing and alignment 84 3.2.3. Tree reconstruction and divergence time estimation 84 3.2.4. Biogeographic analysis 86 ii Table of contents 3.3 Results 88 3.3.1 Beast analysis and lineage dating 88 3.3.2 Tribe Laguncularieae 88 3.3.3 Subtribe Terminaliinae 88 3.3.4 Subtribe Combretineae 89 3.3.5 Ancestral area reconstructions 90 3.4 Discussion 91 3.4.1 Tribe Laguncularieae 93 3.4.2 Subtribe Combretineae 95 3.4.3 Subtribe Terminaliinae 97 3.4.4 Ancestral area reconstructions 98 3.5 Conclusion 101 Chapter 4: Incorporating trnH-psbA to the core DNA barcodes improves discrimination of species within southern African Combretaceae 4.1 Introduction 112 4.2 Materials and methods 114 4.2.1 Taxon sampling 114 4.2.2 DNA extraction, amplification and alignment 115 4.2.3 Data analysis 115 4.3 Results 118 4.4 Discussion 122 iii Table of contents Chapter 5: Conclusions 5.1 General conclusions 158 5.2 Future research 161 Chapter 6: References 162 iv Abstract Abstract Several studies have focussed on Combretaceae, however, phylogenetic relationships still remain unresolved within certain groups in the family. This study represents the most comprehensive phylogenetic inference using molecular data from both plastid (rbcL, matK, trnH-psbA, and psaA-ycf3) and nuclear nrITS regions. The phylogeny of Combretaceae was used to examine relationships, test biogeographic hypotheses, infer divergence age estimates for major lineages and explore the influence of dispersal, vicariance and extinction in shaping the current intercontinental disjunct distribution. The sister position of Combretaceae to the rest of the order Myrtales is supported with two distinct subfamilies, Combretoideae and Strephonematoideae. Monophyly of tribe Laguncularieae was observed with Macropteranthes, a genus endemic to Australia, included. Within tribe Combreteae, two subtribes are distinguished, Combretineae and Terminaliinae, with Gueira and Calycopteris sister to subtribe Combretineae. Two clades, within genus Combretum were noted, subgenus Combretum and Cacoucia with Meiostemon sister to subgenus Combretum. Within subtribe Terminaliinae, Terminalia is observed to be paraphylectic with two main clades distinguished. Clade I comprised of Old World and a few New World species, while clade II is mainly comprised of New World species and a few Old World species. Within clade II, genera Buchenavia, Anogeissus, Bucida, and Pteleopsis are embedded within Terminalia. Conocarpus is sister to clade II. This result allowed the formal transfer of the genera Anogeissus, Buchenavia, and Pteleopsis into Terminalia. v Abstract Divergence times were estimated using the Bayesian MCMC approach implemented in BEAST, suggesting a crown date for Combretaceae of ca, 110 mya, corresponding to the split of the two subfamilies during the Late Cretaceous period. Ancestral area reconstructions inferred using RASP and DEC model (Lagrange), suggested a Gondwanan origin for Combretaceae, with long - distance dispersal as the major event to account for the current intercontinental disjunct distribution pattern. Finally, the use of DNA barcoding is proposed as a complementary tool for species discrimination and identification, in addition to traditional morphological approaches. Core barcodes (rbcLa + matK) with the addition of trnH-psbA were identified as the best barcodes for the family Combretaceae. vi Foreword Foreword My thesis is presented in six chapters with research chapter’s 2 to 4 preceded by the introduction, motivation and general objectives of the study in chapter 1. Chapter 2 is the first research chapter dealing with taxonomy of Combretaceae with special emphasis on subtribe Terminaliinae. My results reinforce the amalgamation of the genera Anogeissus, Buchenavia, and Pteleopsis with Terminalia. These suggestions are formalised and have been submitted to Botanical Journal of the Linnean Society for publication. The title of the paper is as follows: “The inclusion of Anogeissus, Buchenavia and Pteleopsis in Terminalia (Terminaliinae, Combretaceae)”. Chapter 3 deals with the biogeographic history of Combretaceae focussing on divergence times and ancestral area reconstructions. The manuscript is in preparation and will be submitted for publication in January 2014. The title of the paper is as follows: “Reconstructing the biogeographical history of the family Combretaceae (Myrtales) using DNA sequences”. Chapter 4 is the last research chapter dealing with the assessment of the efficacy of the DNA barcodes to discriminate species within Combretaceae of southern Africa. This study has been published in Zookeys, and the format presented in this thesis has been standardised to provide uniformity in the thesis. The title of the paper is as follows: “Incorporating trnH-psbA to the core DNA barcodes improves significantly species discrimination within southern African Combretaceae”, Zookeys, Accepted 13 September 2013. The research vii Foreword chapters are followed by general conclusions presented in chapter 5 and lastly references in chapter 6. The research outputs from this study have been presented at national conferences (37th – 39th annual conference for South African Association of Botanists) and abstracts were published in the South African Journal of Botany. viii Index to figures Index to figures Chapter 1 Figure 1.1 Map showing the distribution of Combretaceae 12 Worldwide (APG III 2009) Figure 1.2 Unique multicellular structures of Combretaceae. a. Leaf 13 scale of Combretum hereroense Schinz. observed under electron microscope. b. Combretaceous hairs of the lower leaf surface. c. Scanning electron micrograph of leaf glandular hair of Quisqualis indica Linn. Photographs: (a) O, Maurin, (b-c) P.M. Tilney. Figure 1.3 Extra floral nectaries of Terminalia arjuna. Photograph: 14 O Maurin. Figure 1.4 Tomentose leaf of Buchenavia tetraphylla (Aubl.) R. A. 14 Howard. Photograph: A.Popovkin Figure 1.5 Glands of petioles and leaf lower surfaces. a. Arrows (A 15 and B) pointing to glands on petiole (leaf stalk) and on surface of leaf blade of Laguncularia racemosa (L.) C.F. Gaertn. b. Arrow pointing to paired glands near the base of the petiole (leaf stalk) of Conocarpus erectus DC. Photographs: B. Holst Figure 1.6 Variation in Combretaceae inflorescences. a. Cone- 16 shaped inflorescence of Anogeissus leiocarpa (DC.) Guill. and Perr. b. Axillary and simple Inflorescence of ix Index to figures Terminalia chebula Retz. c. Inflorescence of Combretum kraussii Hochst. d. Terminal and ramified inflorescence of Combretum collinum Engl. and Diels. e. Inflorescence of Combretum mossambicense (Klotzsch) Engl. f. Branch showing the position of leaves and the inflorescence of Quisqualis Indica. Photographs: (a-e) O. Maurin, (f) G.D. Carr. Figure 1.7 Different fruits of Combretaceae species: a. Round fruit 17 of Combretum bracteosum (Hochst.) Engl. and Diels. b. Ovoid fruit of Buchenavia tetraphylla (Aubl.) R. A. Howard. c. 2-winged fruit of Terminalia trichopoda Diels. d. 4-winged fruit of Combretum microphyllum Klotzsch. Photographs: (a) O. Maurin; (b) A. Popvkin; (c,d) G.D.Carr. Chapter 2 Figure 2.1 One of the most parsimonious trees (2 764 steps, 64 CI=0.45, RI=0.79) from the combined plastid data set. Numbers above the branches are bootstrap percentages above 50%. Current generic and tribal classification (Maurin et al. 2010, Stace 2010) is indicated on the right. Taxa marked with asterisk (*) are now considered part of Terminalia x Index to figures Figure 2.2 One of the most parsimonious trees (2 115 steps, 66 CI=0.37, RI=0.65) from the nuclear ITS data set.