Systematics and Evolution of New Caledonian Araucaria A thesis submitted to the University of Edinburgh for the degree of Doctor of Philosophy Mai-lan Kranitz PhD thesis The University of Edinburgh September 2005 ABSTRACT New Caledonia is a global biodiversity hotspot and contains more than 2300 endemic species including 7% of the world’s conifers. No other region in the world with such a small area possesses such a rich and distinctive conifer flora, and 13 of the world’s 19 Araucaria species are endemic to New Caledonia. This thesis has investigated the evolution and systematics of this group. A molecular phylogenetic study based on sequence data from two chloroplast regions resolved all 13 New Caledonian species as a monophyletic group, sister to the Norfolk Island Pine ( A. heterophylla ). The relationships between the New Caledonian species was not fully resolved as little sequence variability was detected, however, three main groups were defined. The species with bigger leaves occupied a basal polytomy, whereas the vast majority of species with smaller leaves were grouped together in a clade. Within this ‘small leaved’ clade, the three New Caledonian species with a coastal distribution formed another monophyletic group. The timing of the radiation of all these species was tested via a molecular clock approach using different calibration tools (fossil data, geological events, substitution rates). The precise dating of the New Caledonian radiation remains uncertain because different calibration methods give different dates. However, it seems likely to have occurred between 10 and 43 mya . What can be said is that the limited sequence divergence between these species (which in other groups would be typical of <3 million years divergence), does not tally with the fossil record and geological events. This suggests a reduction in evolutionary rates in Araucaria . A combination of molecular and morphological approaches was used to assess species limits and population identities. This resulted in re-determination of the identity of several populations and the distributions of some species. The current state of knowledge of the taxonomy of the New Caledonian species was summarised. Finally, the distribution of chloroplast haplotypes among 468 individuals from 49 populations representing all New Caledonian Araucaria revealed strong taxonomic signal, and high genetic diversity among the species with bigger leaves, and low diversity in the coastal species. The distribution of genetic variation is discussed in the context of the evolution and conservation of the New Caledonian Araucaria spp . a Declaration I hereby declare that this thesis is composed of work carried out by myself unless otherwise acknowledged and cited and that this thesis is of my own composition. Research towards this thesis was carried out between October 2001 and October 2004. No part of this dissertation has been previously submitted for any other degree. b Acknowledgements This work is the result of the collaboration between a great number of people and I hope that if I forgot some of them here, they will forgive me. My first thanks obviously goes to Dr Pete Hollingsworth, without whose help and support this thesis would have probably never reach completion. Thanks again for your hard work on this project and for cheering me up all the way. I am grateful to the Darwin Trust of Edinburgh for funding this project and giving me the opportunity to do such an interesting work. I would also like to thanks Dr Richard Ennos, for helping me in this work, giving me useful advice as to how to set my mind to think in the right direction and lessening the load of work from Pete. Mes remerciements vont aussi au Dr Tanguy Jaffré pour son aide et ses conseils tout au long de mes études et son intérêt sans faille pour la flore calédonienne. Many thanks to Alex Clark for her help in the lab and her support throughout these four years of hard life in Edinburgh. Thanks also for allowing me to be able to speak French from time to time. Thanks to Chris Kettle for sharing the same passion for New Caledonian’s “most charismatic Conifers” and avoiding me the trouble to try to climb them. Thanks to Martin Gardner, Phillip Tomas and Michelle Hollingsworth for sharing with me the joy of being in New Caledonia and doing some mining site explorations. Many thanks to all the people from the Royal Botanic Garden of Edinburgh for their support and kindness. Merci à tout le personnel de l’IRD de Nouvelle-Calédonie pour leur aide et leur conseils au cours de mes récoltes sur le terrain. Merci à toute les personnes rencontrées en Nouvelles-Calédonie dont l’aide à permis de faire en sorte que ce projet puisse aboutir. Et finalement, merci à ma famille et mes proches pour leur soutiens moral pendant ces quatres années de stress et de solitude loin d’eux. Table of contents c Page Chapter 1 – Introduction 1 1.1 Araucaria diversity on New Caledonia 1 1.1.1 New Caledonia: a biodiversity hotspot. 1 1.1.2 Araucariaceae (Gymnosperma, Coniferales) 2 1.1.3 Araucaria (Monkey-puzzle trees) 3 1.1.3.1 Extant species. 3 1.1.3.2 Extinct species. 5 1.1.4 New Caledonian Araucaria 6 1.2 Hypotheses on the origin of Araucaria diversity in New Caledonia 8 1.2.1 Dispersal attributes 9 1.2.2 Vicariance 9 1.2.3 Diversification of the genus in New Caledonia 10 1.3 Problems of species delimitation 12 1.4 Thesis Objectives 13 1.5 Summary of issues raised 14 1.6 Approaches: an overview of the different methods available 15 1.6.1 Sources of molecular data for plant taxonomy and phylogeny 15 1.6.2 Choice of the appropriate methods and regions for taxonomic and phylogenetic studies 18 1.6.2.1 Different tools for different questions 18 1.6.2.2 Polymerase chain of reaction (PCR) 19 1.6.2.3 PCR-Sequencing 19 1.6.2.4 PCR-Restriction fragment length polymorphism (RFLP) 20 1.6.2.5 Amplification fragment length polymorphism (AFLP) 20 1.6.2.6 Simple sequence repeats (SSRs) = microsatellites 21 1.6.3 Approach for each chapter 23 Chapter 2 – Reconstructing the phylogenetic history of Araucaria .24 d 2.1 Introduction: 24 2.1.1 New Caledonia: a hot spot of Araucaria evolution. 25 2.1.2 Previous work on New Caledonian Araucaria 25 2.2 Material and methods 29 2.2.1 Materials: field sampling 29 2.2.2 Approaches 29 2.2.2.1 DNA extraction 33 2.2.2.2 Choice of cpDNA primers 33 2.2.2.3 DNA Sequencing 35 2.2.2.4 Data analysis 36 2.3 Results 37 2.3.1 Sequence characteristics 37 2.3.2 Parsimony 38 2.3.2.1 trnS-trnFm 38 2.3.2.2 psbA-trnH 40 2.3.2.3 Combined analysis 40 2.3.3 Maximum Likelihood 44 2.3.3.1 Separate analyses of chloroplast regions 44 2.3.3.2 Combined analysis 48 2.4 Discussion 48 2.4.1 Relationships within the Araucariaceae 48 2.4.2 Relationships within Araucaria 49 2.4.3 Relationships within section Eutacta 50 2.4.3.1 Clade 1: A. rulei and A. laubenfelsii (supported by a G->A change at bp 412 and a G->A change at bp 560 of the psbA-trnH region) 51 2.4.3.2 Clade 2: A. columnaris, A. nemorosa, A. luxurians, A. biramulata, A. schmidii, A. subulata, A. scopulorum, A. bernieri (supported by A->G change at bp 412 and a A->G change at bp 560 of the psbA-trnH region) 51 2.4.3.3 Clade 3 (nested within clade 2): A. columnaris, A. nemorosa, A. luxurians (supported by a C->A change at bp 3 e and a C->T change at bp 185 of the trnS-trnFm region) 52 2.4.3.4 Comparison of the New Caledonian species with the phylogeny of Setoguchi et al. (1998) 53 2.4.4 Overview of the phylogeny of New Caledonian Araucaria 53 Chapter 3 – Dating the radiation of Araucaria on New Caledonia 59 3.1 Introduction 59 3.1.1 The debated origin of Araucaria species 59 3.1.2 The theory behind the molecular clock 63 3.1.2.1 The neutral theory 63 3.1.2.2 Dating the phylogeny 64 3.1.2.3 Time constraint and calibration point: the tools for dating the phylogeny 65 3.1.3 Substitution rates in plants 66 3.2 The geological and climatic history of New Caledonia (Table 3.2) 68 3.2.1 Geodynamics of the southwest Pacific (Kroenke, 1996; Pintaud, 1999; Picard, 1999; McLoughlin, 2001) 68 3.2.1.1 Precambrian to Early Cretaceous (700-85 mya) 68 3.2.1.2 Early Cretaceous to Lower Eocene (85-55 mya) 72 3.2.1.3 Eocene to present (55-0 mya) 73 3.2.2 Paleoclimate of the southwest Pacific (Jurassic to present) 74 3.2.3 Possible land bridges to New Caledonia 76 3.3 Material and methods: 77 3.3.1 Materials: field sampling 77 3.3.2 Methods 78 3.3.2.1 The phylogeny 78 3.3.2.2 Molecular Clock 78 3.3.2.3 Estimation of rates using time estimations from NPRS 79 3.4 Results 80 3.4.1 Molecular clock hypothesis rejected by modeltest 80 3.4.2 Divergence time estimations 80 f 3.4.2.1 Estimation using fossils as calibration points 82 3.4.2.2 Estimation using a volcanic island as a calibration point 83 3.4.2.3 Estimation using geological events as calibration points 84 3.4.3 Estimations of rates using the time estimate obtained in 4.2 and 4.3 as time estimation for the nodes 85 3.5 Discussion: 87 3.5.1 Variations in time and rate estimates from different partitions 87 3.5.1.1 Time estimations from the fossil record 87 3.5.1.2 Time estimations from a volcanic island dating 87 3.5.1.3 Time estimations from the geological events 88 3.5.1.4 Rate variation in the context of different calibration points 88 3.5.2 Interpretation: Time estimate and Biogeography 89 Chapter 4 – Taxonomic problems and conservations issues 92 4.1 Introduction 92 4.1.1 Weakness of the Flora.