Are there any trees in the Arctic? Reconstruction of evolutionary histories in a young biome Tor Carlsen Dissertation presented for the degree of Philosophiae Doctor Department of Biology & National Centre for Biosystematics, Natural History Museum Faculty of Mathematics and Natural Sciences University of Oslo 2007 Guard: Found them? In Mercia? The coconut's tropical! King Arthur: What do you mean? G: Well, this is a temperate zone KA: The swallow may fly south with the sun or the house martin or the plover may seek warmer climes in winter, yet these are not strangers to our land? G: Are you suggesting coconuts migrate? KA: Not at all. They could be carried. G: What? A swallow carrying a coconut? KA: It could grip it by the husk! Monty Python on plant dispersal Table of contents Summary................................................................................................................................2 Sammendrag...........................................................................................................................4 List of papers..........................................................................................................................6 Introduction............................................................................................................................7 The evolution of a PhD project..................................................................................7 The genus Smelowskia................................................................................................8 The genus Cardamine.................................................................................................9 The genus Cassiope..................................................................................................11 Polyploidy.................................................................................................................12 Treebuilding methods...............................................................................................12 The main objectives of this PhD study.....................................................................13 Methods................................................................................................................................14 Laboratory methods..................................................................................................14 Treebuilding methods...............................................................................................15 Results & Discussion............................................................................................................16 Main results from the tree genera.............................................................................16 Origins of the Arctic flora........................................................................................17 Are there any trees in the Arctic? ............................................................................18 Dispersal...................................................................................................................20 Acknowledgements..............................................................................................................21 References............................................................................................................................23 Papers...................................................................................................................................28 1 Summary Reconstructing molecular phylogenies and unraveling biogeographic histories of arctic plants are needed to obtain better insights into the processes of evolution, dispersal and colonization in this young biome. Studies of dispersal into, and speciation within, the Arctic are important to obtain better knowledge of the source areas for arctic biodiversity. Unraveling the history of recently diverged lineages such as those typical for the young arctic biome is challenging, because it is difficult to find molecular markers with sufficient variation and to handle the problem of incomplete lineage sorting and hybridization. Thus several different molecular marker systems for many potentially suitable model groups were tested and developed. Three genera which are represented in Beringia (the Asian and American land masses surrounding the Bering Strait from Lena River to Mackenzie River) and which have assumed phylogenetic connections to plants living in East Asia and North America were selected. Two of these genera are also good candidates for studying polyploidization as a mode of speciation in the Arctic, as they show large variation in chromosome number within and among the species. This study was also intended to contribute to the PanArctic Flora project by providing data to help resolving the taxonomy of some challenging species complexes. - The history of the genus Smelowskia was reconstructed based on microsatellite loci combined with sequences of nuclear and plastid regions. An Asian origin of the genus and two independent dispersal events into the Beringian and North American regions were inferred. We also found evidence for merging the Beringian S. porsildii, S. spathulatifolia, and S. jurtzevii into one species; S. porsildii. - The biogeography and phylogeny of the large genus Cardamine were inferred based on nuclear and plastid sequences. The phylogenetic trees showed limited resolutions, supporting a hypothesis of recent and rapid speciation in the genus. We found evidence for several extremely long-distant dispersal events. Dispersal into the Southern Hemisphere and the Arctic has occurred repeatedly, and we identified at least three phylogenetically distinct arctic lineages. Polyploidization has occurred independently many times during the evolution of Cardamine. Rapid divergence combined with widespread polyploidization offer an explanation for the complex evolutionary history of the genus. Two species complexes within this genus were selected for more detailed studies. 2 - Six microsatellite loci originally developed for the Arabidopsis genome, were used to identify evolutionary and taxonomic units within the Beringian Cardamine digitata aggregate. Molecular groups corresponding to morphological differences suggested recognition of four species in this complex; C. blaisdellii, C. digitata, C. microphylla, and C. purpurea. Each species included at least two ploidy levels, indicating recurrent polyploidizations. - As a first step towards addressing the origin of the circumarctic Cardamine bellidifolia, we conducted a study with main focus on its two putatively most closely related European alpine species (C. alpina and C. resedifolia) using AFLPs. Surprisingly, the arctic species C. bellidifolia was distinctly differentiated from its putative alpine ancestral lineages. Contrasting phylogeographies were inferred between the two alpine species C. alpina and C. resedifolia. A high degree of genetic distinction was found between the Alpine and Pyreneean populations of C. alpina. In addition, a high level of diversity was found within Pyreneean populations compared to Alpine populations. In contrast, C. resedifolia showed more genetic variation among populations in the Alps than between the Alps and distant areas such as Corsica, the Carpatians and the Pyrenees. These results show that the two species have very different histories of glacial survival and recolonization. - To facilitate these and future studies of recently diverged taxa, we developed 72 new microsatellite loci and tested 15 previously published loci for the Brassicaceae. We found them to provide variation among and within three distantly related genera: Cardamine, Smelowskia, and Draba. Of these 87 loci, 18 were variable within Cardamine, while ten were variable within Smelowskia. Seventy-one of these primers were variable within Draba, and 50 were variable within Draba nivalis. The markers amplifying across these genera are potentially suitable for studying other genera in Brassicaceae as well. - A phylogeographic analysis of Cassiope tetragona including both Central Asian and Beringian relatives revealed that the circumpolar ssp. tetragona was well separated from the North American ssp. saximontana. A Beringian origin of C. tetragona ssp. tetragona was inferred, and the levels and geographical patterns of differentiation and gene diversity suggested that the latest expansion from Beringia into the Circumarctic was recent, possibly during the current interglacial. The results were in accordance with a recent leading-edge mode of colonization, particularly towards the east throughout Canada/Greenland and across the North Atlantic into Scandinavia and Svalbard. 3 Sammendrag Få detaljerte studier av spredning til og artsdannelse innen arktis er gjort. Slektskap og biogeografiske mønstre må analyseres for å forstå hvordan spredning og evolusjon foregår innen det arktiske området, og for å finne opphavet til den arktiske biodiversiteten. Å finne de evolusjonære historiene i arktis er utfordrende siden det ofte er lite variasjon i vanlige molekylære markører, og hybridiseringer og ufullstendig lineage sorting skaper ytterligere problemer. Flere ulike molekylære markørsystemer for mange potensielt passende modellgrupper ble derfor testet og utviklet. I dette studiet er det valgt ut tre planteslekter som alle har utbredelse i Beringia i tillegg til andre deler av Øst-Asia og Nord-Amerika. To av de valgte slektene har stor variasjon i kromosomtall innen og mellom arter og