Systematics and polyploid evolution in Potentilleae ( R o s a c e a e ) Magnus Lundberg Systematics and polyploid evolution in Potentilleae (Rosaceae) Magnus Lundberg ©Magnus Lundberg, Stockholm 2011 Cover illustration: Fragaria virginiana, Bergianska trädgården (top) Fragaria chiloensis, Oregon, USA (bottom) Phylogeny of Fragariinae (right) Photo: Magnus Lundberg ISBN 978-91-7447-227-1 Printed in Sweden by Universitetsservice US-AB, Stockholm 2011 Distributor: Department of Botany, Stockholm University Academic dissertation for the degree of Doctor of Philosophy in Plant Systematics presented at Stockholm University 2011 Abstract Lundberg, M., 2011. Systematics and polyploid evolution in Potentilleae (Rosaceae). This thesis comprises studies of the phylogenetic relationships in the flowering plant clade Potentilleae in Rosaceae. The relationships were elucidated by using DNA sequence data from the nuclear genome as well as from the plastid genome. In particular, the focus of the studies was the investigation of allopolyploidy, i.e. speciation as a result of hybridization and subsequent chromosome doubling. A phylogenetic method was used for identifying allopolyploidy through comparison of trees resulting from the analyses of different DNA sequences. Five sub-clades were investigated. First, both the sister clades that together contain all of Potentilleae: Fragariinae and Potentilla. Secondly, three subclades of Fragariinae, namely Alchemilla in wide sense, Sibbaldia and relatives, and Fragaria. The aim was to unravel the phylogenetic relationships, including instances of allopolyploidy. Classification issues were discussed in relation to the phylogenetic results. The split between Potentilla (=Potentillinae) and Fragariinae received better support than in previous studies. The phylogeny of Fragariinae was found to be consistent with classifying ten genera: Alchemilla in wide sense (incl. Aphanes and Lachemilla), Comarum, Sibbaldia, Sibbaldianthe, Sibbaldiopsis, Chamaerhodos, Drymocallis, Dasiphora, Potaninia, Fragaria, and also including a few orphan Potentilla species. The segregated genera Ivesia, Horkelia, Horkeliella and Duchesnea were found to be nested within Potentilla, corroborating earlier studies, while the segregated genus Argentina (P. anserina and close relatives) showed an ambiguous position. Plastid and nuclear (ribosomal) phylogenies were compared and incongruences were detected as potential instances of allopolyploid speciation. Five strongly supported incongruences were detected in Fragariinae and four of them were considered to be potentially caused by allopolyploidy. In addition, five supported incongruences were found in Potentilla. Alchemilla in the wide sense was found to contain four major clades, African Alchemilla, Eurasian Alchemilla, Lachemilla and Aphanes. Both Lachemilla and Aphanes were nested within Alchemilla and it was suggested that the name Alchemilla should be used in the wide sense, i.e. including both the genera Lachemilla and Aphanes. The genus Sibbaldia as commonly classified was shown to be polyphyletic in five different places in Potentilleae. Three Sibbaldia clades ended up in Fragariinae and two in Potentilla. A phylogeny of Fragaria, based on a nuclear low/single copy DNA region was estimated. The gene copy phylogeny was used to construct a reticulate tree hypothesizing allopolyploid speciation events. The evolution of Fragaria was shown to have been shaped by polyploidy. Keywords: Potentilleae, Fragariinae, Potentilla, Sibbaldia, Fragaria, Alchemilla, systematics, phylogeny, polyploidy, autopolyploidy, allopolyploidy, reticulate evolution. Preface This thesis is based on the following papers, referred to in the text by their Roman numerals. I Lundberg, M., Töpel, M., Eriksen, B., Nylander, J. A. A., Eriksson, T. 2009. Allopolyploidy in Fragariinae (Rosaceae): Comparing four DNA sequence regions, with comments on classification. Mol. Phylogenet. Evol. 51, 269-280. II Töpel, M., Lundberg, M., Eriksson, T., Eriksen, B. Molecular data and ploidal levels indicate several putative allopolyploidization events in the genus Potentilla (Rosaceae). Manuscript. III Gehrke, B., Bräuchler, C., Romoleroux, K., Lundberg, M., Heubl, G., Eriksson, T., 2008. Molecular phylogenetics of Alchemilla, Aphanes and Lachemilla (Rosaceae) inferred from plastid and nuclear intron and spacer DNA sequences, with comments on generic classification. Mol. Phylogenet. Evol. 47, 1030–1044. IV Eriksson, T., Lundberg, M., Töpel, M., Östensson, P. Sibbaldia – a molecular phylogenetic study of a polyphyletic genus in Rosaceae. Manuscript. V Lundberg, M., Eriksson, T., Zhang, Q., Davis, T. M. New insights of polyploid evolution in Fragaria (Rosaceae) based on the single/low copy nuclear intergenic region of RGA1- Subtilase. Manuscript. Paper I and Paper III is reprinted with the permission from the copyright holder, Elsevier. Lundberg's contribution to papers included in the thesis: I - Lundberg is first author and has produced all the sequences new to this study. Conducted phylogenetic analyses and has written the manuscript with comments and suggestions from co-authors. Convergence diagnostics was performed and interpreted with help from co-authors. II - Lundberg is co-author and has contributed by producing sequences for included outgroups, taken part of writing the manuscript with additional comments and suggestions from co-authors. III - Lundberg is co-author and has contributed by producing sequences for included outgroups, written part of the method section and given comments of the manuscript. IV - Lundberg is co-author and has produced a large set of the included sequences, taken part in analyzing the data, as well as the writing of the manuscript. V - Lundberg is first author and has contributed by producing the vast majority of the sequences, conducted phylogenetic and reticulate analyses and written the manuscript with comments and suggestions from co-authors. Contents Introduction.........................................................................................10 Aims....................................................................................................14 Material and Methods..........................................................................15 Results and Discussion........................................................................17 Conclusions.........................................................................................20 Svensk sammanfattning.......................................................................21 Tack/Thanks........................................................................................24 References...........................................................................................25 Abbreviations bp basepairs c.f. confer, compare DNA deoxyribonucleic acid et al. et alii, and others ETS external transcribed spacers ITS internal transcribed spacers pp. posterior probability rDNA ribosomal deoxyribonucleic acid RGA1 resistance gene analogue 1 s.l. sensu lato, in a broad sense s.s. sensu stricto, in a strict sense ssp. subspecies Subt subtilase trn transfer ribonucleic acid var. variety Introduction The research discipline of Systematic Biology involves studying various groups of organisms and their relationships. Systematists are often interested in natural groups (monophyletic clades), i.e. species groups that share a common ancestor. Conclusions of what natural groups are, and the relationships among organisms constituting the natural groups were traditionally based on macromorphological characters, i.e. the appearance of the organisms. As microscopes were improved, new possibilities emerged to study micromorphological characters such as flower development and pollen grains. As new techniques developed, information were also obtained from e.g. chemical compounds and chromosome numbers. In the beginning of the 1980's, molecular methods were developed and DNA sequences from either nuclear, plastid or mitochondrial genomes were a new source of information. Along with this new source of data, phylogenetic methods were also significantly developed. The results from DNA sequence analysis are usually depicted as a phylogeny (phylogenetic tree with nodes and branches; e.g. see Fig. 1) and used to make hypotheses about relationships among organisms, based on synapomorphies (presence of shared derived character states) to define natural groups. Information obtained from phylogenies can also be used to make hypotheses about speciation processes, biogeography, character evolution, co-evolution, biodiversity, gene evolution, nature conservation and ecology. In this thesis, the organisms that have been studied belong in the tribe Potentilleae (Potentillineae [= Potentilla] + Fragariinae; see also Fig. 1), belong in Rosoideae (Rosaceae; [Morgan et al., 1994; Eriksson et al., 2003; Potter et al., 2002; Potter et al., 2007]). Potentilleae includes at least 1000 species and most of them occur in northern temperate, arctic and alpine regions but a few are found in the southern hemisphere. The classification and circumscription of Potentilleae and closely related genera has been of much debate for the last century and no taxonomic consensus has been reached until recently with the aid of molecular sequence data. The various taxonomic hypotheses are due to that many morphological characters have been shown to be homoplasious, i.e. arisen or lost at several independent occasions in the evolution of this clade. Most Potentilla species are perennial