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 or annual, rarely biannual herbs, or small shrubs. The leaves are pinnate or palmately compound. Most Potentilla are yellow flowered, almost consistently with 5 epicalyx segments, 5 sepals alternating with the epicalyx segments and 5 petals. Other common characters are usually 20 stamens, numerous free carpels and a basal chromosome number of x=7. 10 Figure 1. Phylogeny of Rosaceae. Phylogeny was redrawn based on Fig. 1 Potter et al. (2007). The clades of Fragariinae, Potentilleae and Rosoideae among others are marked to the right.

11 Potentilla was found to be polyphyletic, with its species intermixed with other genera based on nuclear ribosomal DNA data (Eriksson et al., 1998) and several genera grouped in a clade of their own as sister to Potentilla in the strict sense. This was the first support for the clade Fragariinae and further support was received after combining the nuclear sequences with plastid DNA sequences (Eriksson et al., 2003). The clade Fragariinae comprise 10 genera, Alchemilla in the wide sense, Chamaerhodos, Comarum, Dasiphora, Drymocallis, Fragaria, Potaninia, Sibbaldia, Sibbaldiopsis and Sibbaldianthe and a few orphan Potentilla species. Fragariinae share the morphological synapomorphy of anthers with confluent thecae at apex (cf. Potentilla with anthers where the thecae are separated at apex; Sojak, 2008), and possibly a second character based on lateral to basal position of styles on achenes (but see discussion in Paper IV). Many genera that belong in Fragariinae have according to earlier classifications been placed either inside Potentilla or considered to be closely related as separate genera, but Alchemilla and Potaninia have in many cases been placed elsewhere in Rosaceae (Hutchinson, 1964; Kalkman, 1988; Kalkman, 2004).

Recent studies have provided new insights that the mode of speciation known as allopolyploidy may be a major evolutionary force of speciation and today allopolyploidy is considered much more common than previously thought. For a review of hybridization and polyploidy see Soltis and Soltis (2009). In allopolyploid speciation two species hybridize followed by a chromosome doubling. If this hybrid is functional and survives, a new species may have been formed. Another similar process involving chromosome doubling is autopolyploidy, distinguished from allopolyploidy in that the duplication of chromosomes occurs within a species (hybridization is not involved). Early estimates of polyploid speciation among angiosperms ranged from 30% (Stebbins, 1950) to 52% (Grant, 1981). Considerably higher estimates have been presented more recently. A study based on guard cell sizes estimate that at least 70% of extant angiosperms are the result of polyploid speciation (Masterson, 1994). Several taxa in Potentilla and Fragariinae have members that are polyploid (Goldblatt and Johnson, 2010) and both clades have been proposed to have an evolution shaped by hybridization and polyploidy (Schulze-Menz, 1964).

One way to screen a larger clade (e.g. Fragariinae and Potentilla) for potential allopolyploid speciation is to contrast phylogenies based on easily amplified DNA sequence regions from the plastid and the nucleus (e.g. Smedmark and Eriksson, 2002). Instances (nodes) in the separate phylogenies that display well supported incongruent patterns are potentially

12 reflecting allopolyploid speciation because plastid DNA is maternally inherited, hence reflecting maternal phylogenetic relationships while nuclear DNA is biparentally inherited potentially reflecting the paternal phylogeny. Based on primary results from the screening, further studies would focus on the most promising incongruences and by cloning and sequencing nuclear single/low copy DNA sequence regions it may be possible to trace the parental species of the allopolyploids by obtaining all potential copies of polyploid species. This method has been shown to be succesful in several studies (e.g. Sang, 1999; Smedmark, 2003; Mason-Gamer, 2001; Popp et al., 2005). A phylogeny based on all copies will depict a tree with allopolyploid species having different copies positioned in different places in the tree, as sisters to their respective parental species. When combining the multiple copies from a single taxon into a reticulate tree, hypotheses about allopolyploid speciation can be drawn. This method will also distinguish allopolyploid speciation (where DNA copies from a taxon are distantly related) from autopolyploidy (where DNA copies are sisters). With this method ancient allopolyploid speciation may also be detected. The results of studying nuclear single/low phylogenies will hopefully give better knowledge and understanding of the role allopolyploid speciation have had in larger clades such as Fragariinae and Potentilla. This will also give an indication of the potential impact allopolyploid speciation may have played in the evolution of other angiosperm groups.

The three Fragariinae genera, Alchemilla, Sibbaldia and Fragaria are of special interest to us because, (1) Alchemilla and Sibbaldia have been suspected to be polyphyletic due to complicated morphology, (2) intriguing geographical distributions and (3) all three clades have taxa with established polyploidy. Therefore we have performed more detailed investigations of these three groups/genera.

The first group (“genus”) in Fragariinae we have focused on is Alchemilla in the wide sense (i.e. Alchemilla, Lachemilla and Aphanes). Alchemilla is the largest genus in Fragariinae and is characterized by its small apetalous inconspicuous flowers and a basal chromosome number of x = 8. Alchemilla in strict sense is mainly distributed in the northern hemisphere and East Africa. While Lachemilla is restricted to montane South America and Aphanes is scattered in different regions of the world but primarily found in the Mediterranean area to central Europe, the west coast of South and North America and southern parts of Australia. Alchemilla in the wide sense has by earlier classifications been placed either in Sanguisorbeae ([Poterieae]

13 Hutchinson, 1964; Focke, 1894) or by on its own in the subtribe Alchemillinae, belonging in Potentilleae (Schulze-Menz, 1964; Soják, 2008).

The second genus we have focused on is Sibbaldia. Sibbaldia has consistently been placed in Rosoideae and often considered to be a separate genus because of a small number of stamens 5 (-10). Other morphological characters such as, trifoliate or pinnate leaves and relatively few carpels are common in Sibbaldia. Otherwise, large variation regarding e.g. leaf form and division, stamen placement, anther form, presence or absence of floral disc and flower colour are observed in the genus. Sibbaldia has mainly a northern temperate and alpine distribution and the centre of diversity is in the Himalayas. More than 40 species names of Sibbaldia have been described, but recent treatments lists 10-15 species (Dixit and Panigrahi 1981, Rajput et al. 1997, Dikshit and Panigrahi 1998, Li et al. 2003) or as few as two (Soják, 2008).

The third genus we have focused on is Fragaria. Based on the results from Lundberg et al. (2009; Paper I, in this thesis), Fragaria was considered one of the most promising groups for further investigation of allopolyploidy. Fragaria was suspected to have an evolution shaped by allopolyploidy due to incongruent phylogenies and established polyploid species. About 24 Fragaria species are described (Staudt, 2008) and mainly distributed in the northern hemisphere. Fragaria is distinguished from closely related genera by having white flowers and a swollen receptacle (i.e. the 'strawberry') and the ploidy level ranges from diploid (2x=2n=14) to decaploid (2x=10n=70). Different genomic composition models involving allopolyploidy have been proposed for the octoploid Fragaria (Fedorova, 1946; Senanayake and Bringhurst, 1967; Bringhurst, 1990) and molecular studies trying to elucidate relationships (Potter et al., 2000) and polyploidy (Rousseau-Gueutin et al., 2009) have been published but never with any strong support for the evolution of the polyploid Fragaria.

Aims

The overall aim for this thesis was to elucidate the systematics and evolution of the clade Potentilleae, with focus on allopolyploid speciation. Earlier studies have shown that polyploidy is widespread and recent molecular studies have indicated that both inter- and intrageneric relationships among Fragariinae genera/taxa and the sister clade Potentilla are intriguing and less than well understood. In this doctorate thesis, five different studies are

14 presented, together giving new insights towards understanding speciation, polyploidy and evolution in Potentilleae.

In paper I, the main aim was to identify potential allopolyploid speciation events in the clade Fragariinae and also discuss taxonomic implications of included genera/taxa.

The purpose of paper II was to identify potential allopolyploid speciation events in the clade Potentillineae. Identify major clades in Potentilla and evaluate the monophyly including phylogenetic position of segregated genera Argentina as well as Ivesia, Horkelia and associated genera.

In paper III we tested the relationships of segregated genera in the clade Alchemilla in the wide sense and presented a comprehensive phylogeny based on molecular sequence data.

Paper IV focused on testing the monophyly of genus Sibbaldia and the relationships of Sibbaldia species in the clade Potentilleae.

Paper V, the main purpose was to elucidate polyploid speciation in Fragaria. Differentiate between auto- and allopolyploids, and if relevant, erect a well supported and detailed hypothesis of the reticulate evolution in Fragaria.

Material and Methods

In Paper I, the sampling was based on the study of Eriksson et al. (2003) where Fragariinae was established to be monophyletic. Additional sampling intended to generally increase the number of taxa from each genus in Fragariinae, and taxa with established polyploidy were particularly sampled. In addition, Potentilla miyabei and P. cuneata, traditionally classified in Potentilla in strict sense were also sampled. These two species have been classified close to Potentilla tridentata, currently Sibbaldiopsis tridentata, a genus in Fragariinae (Eriksson et al., 2003). In total, 40 taxa were sampled of which 34 were part of the ingroup. Two nuclear ribosomal (ITS, ETS) and two plastid regions (trnL/F, trnS/G) were amplified and sequenced. Analyses using Bayesian inference (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003) were conducted. Topologies were compared in order to detect incongruences, and three criteria (see paper I for details) had to be fulfilled in order for the incongruences to be considered as potential allopolyploid speciation events.

15 In paper II, we extended the Potentilla data sets used in the studies of Eriksson et al. (1998 and 2003) with emphasis on sampling the major groups proposed by Wolf (1908). In addition, taxa proposed to be close relatives to Horkelia and Ivesia were sampled. Five representatives of the clade Fragariinae and two taxa outside Potentilleae served as outgroup. In total were 64 ingroup taxa and seven outgroup taxa sequenced for the two nuclear ribosomal regions, ETS and ITS, as well as the two plastid regions, trnL/F and trnS/G. Phylogenetic analyses were performed using MrBayes and topologies were compared by eye to detect incongruencies.

In paper III, four criteria were applied for sampling (see Paper III for details) reaching a total of 100 taxa. The ingroup consisted of 89 taxa: 71 Alchemilla, 6 Aphanes and 12 Lachemilla. The outgroup consisted of 11 taxa, sampled to cover a large part of the Fragariinae clade, with representatives from Potentilla in the strict sense and Rosa majalis. Amplification and sequencing were performed of the plastid trnL/F region and nuclear ribosomal ITS region. Parsimony and non-parametric bootstrap analyses to obtain support values, as well as Bayesian inference analyses were performed.

In paper IV, the intention was to sample as many taxa traditionally described in Sibbaldia as possible. In addition, at least two species from each genus of Potentilleae along with three outgroup taxa that represented the two clades most close to Potentilleae (Eriksson et al., 2003) were sampled. Diploids were chosen before polyploids and taxa shown to be resolved in early splits of each “genus” sampled were favoured whenever possible. Sibbaldia trullifolia and S. tenuis were not sampled because of no available material for DNA extraction. In total 43 ingroup and 3 outgroup taxa were sampled. Amplification and sequencing of nuclear ribosomal DNA and plastid trnL/F DNA regions were performed. DNA sequence data were analyzed by Bayesian inference and evaluated by convergence diagnostics.

The aim in paper V was to sample all 24 Fragaria species (Staudt, 2008) described and several closely related taxa in the clade Fragariinae (Lundberg et al., 2009) as outgroups. In addition were several subspecies sampled for F. vesca, F. virginiana and F. chiloensis (Staudt, 1999). For the first time was the nuclear low copy intergenic region between the two genes, Resistance Gene Analogue 1 (RGA1) and Subtilase (Subt) used to conduct phylogenetic analyses. Diploid species were directly sequenced while polyploid species were first cloned and subsequently sequenced in a sufficient number to obtain a 95% confidence to acquire all potential copies of each ploidy level.

16 Bayesian inference was used to obtain the complete phylogeny and the reticulate tree was obtained by hand.

Results and Discussion

Paper I: Phylogenies based on two regions of nuclear and two regions of plastid DNA sequence data, respectively, were compared and revealed five instances of conflict. In order to classify detected incongruences as potential allopolyploid speciation events, three criteria were defined and that had to be fulfilled: incongruences had to be (1) supported by a clade credibility value above 0.95, (2) strength of conflict should be at least 0.90 (see Paper I for explanation) and (3) convergence diagnostics were assessed to rule out the possibility that the incongruences were the result of inadequate Bayesian inference analysis. The five incongruences that fulfilled the defined criteria involved Aphanes arvensis, Potentilla cuneata, Potentilla miyabei, Fragaria vesca/moschata and the Drymocallis clade. Incongruences were evaluated and allopolyploid speciation appear to be a probable hypothesis for all conflicts except the last, involving the Drymocallis clade (see discussion Paper I). Several genera in Fragariinae were found to be monophyletic, Alchemilla in the wide sense, Sibbaldianthe, Chamaerhodos, Drymocallis and Fragaria. The two orphan Potentilla species, P. miyabei and P. cuneata were placed in a clade comprising Sibbaldia and close relatives, and both were involved in incongruences between topologies. The two monotypic genera Sibbaldiopsis and Potaninia were nested within Sibbaldia and Dasiphora respectively. The incongruences detected in Sibbaldia and Fragaria were considered to be of main interest and Fragaria were further studied using single/low copy sequence region (see Paper V).

Paper II: Six major clades, the Anserina-, Alba-, Fragarioides-, Reptans-, ivesioid Potentilleae and the Argentea clade were found in both phylogenies based on nuclear or plastid data. In addition were three supported backbone clades identified. When nuclear and plastid phylogenies were compared, five strongly supported incongruencies were found in Potentilla. The incongruencies were: 1) within the Anserina clade, P. peduncularis and P. microphylla are changing places. 2) Within the Alba clade, P. articulata is changing position. 3) The Fragarioides and Reptans clades are switching positions. 4) P. dickinsii is part of the Fragarioides clade in the nuclear phylogeny while positioned as sister to P. fragarioides/stolonifera, ivesioid Potentilleae and Argentea clades in the chloroplast phylogeny. 5) P. norvegica is part of the Argentea clade in the chloroplast phylogeny but sister to the ivesioid Potentilleae in the nuclear phylogeny. Four of the 17 incongruences are considered to be caused by potential allopolyploid evolution. The ivesioid Potentilleae clade was nested within Potentilla and should therefore be merged with Potentilla in order for Potentilla to stay monophyletic. Potentilla lignosa that have earlier been placed elsewhere (Wolf, 1908; Botschantzev, 1952; Soják 2008), were shown to be part of the Anserina clade according to both nuclear and chloroplast data. Two morphological characters were tested for systematic information but only style morphology was considered valuable.

Paper III: Analyses of plastid and nuclear phylogenies were largely congruent and four distinct clades were resolved. These four clades were African Alchemilla, Eurasian Alchemilla, South American Lachemilla and annual Aphanes, where each of the clades corresponds to either distinct geographical areas or life forms. Within the clade of African Alchemilla moderate support for some patterns of mainly geographical distribution could be found (in at least the combined; plastid + nuclear DNA sequence data) analysis: (1) Southern African taxa form a clade, (2) taxa from Madagascar group together and (3) A. kiwunensis and A. volkensii are sister taxa, otherwise the clade was poorly resolved. The Eurasian Alchemilla clade comprise two major clades that correspond to the morphology of leaves, dissected (or nearly so) and lobed (at least not entirely dissected), with a few taxa as exceptions. The Lachemilla clade displayed incongruent pattern between plastid and nuclear topology, but not well supported. Lachemilla was sister to the African Alchemilla clade in the plastid phylogeny while sister to a clade including both Aphanes and Eurasian Alchemilla in the nuclear phylogeny. The topology of the nuclear analysis was also reflected the combined phylogeny. The resolution within the Lachemilla clade was better than the other major clades. The Aphanes clade is strongly supported and sister to the Eurasian Alchemilla clade in all analyses. All analyses show that Alchemilla in the wide sense is monophyletic but in the more restricted sense (excluding Aphanes and Lachemilla), Alchemilla is paraphyletic. Based on three factors involving priority of monophyly, morphological synapomorphies and nomenclatural stability, it was suggested that the name Alchemilla should be used in the wide sense, i.e. including both the genera Aphanes and Lachemilla.

Paper IV: Based on molecular nuclear and plastid DNA data, Sibbaldia was shown to be polyphyletic. A conglomerate of taxa primarily based on a reduced number of stamens 5 (-10) and often with trifoliate leaves end up scattered in five separate clades of Potentilleae. Three clades ended up in Fragariinae, (1) Sibbaldia in the strict sense comprising four taxa including

18 the type species Sibbaldia procumbens, (2) S. adpressa is placed in Sibbaldianthe and (3) S. perpusilloides is considered to represent a new undescribed genus which is nested in a clade with the genera Chamaerhodos, Drymocallis, Dasiphora and Potaninia. The two remaining Sibbaldia clades are placed within Potentilla (Potentillinae sensu Soják [2008]), (4) a clade comprising four Himalayan species end up in a basal position and (5) Sibbaldia micropetala is nested within the Potentilla anserina clade. The monospecific genus Sibbaldiopsis is close to or congeneric with Sibbaldia (see discussion in paper IV) but the species should be named Sibbaldiopsis retusa. Our results are mostly congruent with the findings of Soják (2008) which separate Fragariinae from Potentilla (Potentillinae) based on anther structure.

Paper V: The low copy nuclear intergenic region between the two genes, Resistance Gene Analogue 1 (RGA1) and Subtilase (Subt) was used for the first time for phylogenetic studies. The octoploid Fragaria virginiana and chiloensis had copies positioned in three defined main clades. One of the main clades also consisted of the diploid species F. vesca and mandshurica, the tetraploid F. orientalis and hexaploid F. moschata along with the octoploids. The second main clade constituted the octoploids, decaploid F. iturupensis and copies of the hexaploid F. moschata while the last main clade only had gene copies of the octoploids and decaploids. In addition, a clade with diploid F. viridis and gene copies of F. moschata and a clade consisting of the Asian diploids F. pentaphylla and nipponica and tetraploids F. gracilis, tibetica and corymbosa were also found. When gene copies of the polyploid species were found in more than one clade, the process of allopolyploidization was considered likely to have occurred and by combining the copies found in multiple clades from a single taxon we obtained a reticulate tree. Our favoured scenario to explain the reticulate evolution of genus Fragaria was obtained by inferring six events of allopolyploidy: 1) between the diploids F. vesca and viridis, resulting in an undescribed tetraploid. 2) Between this undescribed tetraploid and a diploid of the F. iinumae lineage gave rise to the hexaploid F. moschata. 3) Between the hexaploid F. moschata and a diploid of the F. iinumae lineage again, creating the origin of the octoploid lineage. 4) Between the octoploid lineage and an unknown diploid close to the base of the Fragaria phylogeny (but close to F. daltoniana) resulting in the potentially new decaploid species earlier known as F. virginiana ssp. platypetala. 5) Between the octoploid lineage and a diploid of the F. iinumae lineage that gave rise to the decaploid F. iturupensis and 6) in the Asian Fragaria clade, between a diploid of the F. nipponica lineage and a unknown diploid sister to the F. nipponica lineage

19 resulting in the tetraploid F. corymbosa. The other tetraploid Fragaria sampled were considered to be of autopolyploid origin because all gene copies of each tetraploid formed a separate clade in the phylogeny. The diploid progenitor of autotetraploid F. orientalis was F. mandshurica, the diploid F. nipponica was progenitor of autotetraploid F. tibetica and the tetraploid F. gracilis was the result of autopolyploidization of a diploid of the F. nipponica lineage. The octoploid Fragaria species have one origin and the speciation event resulting in F. virginiana and chiloensis took place after the octoploid lineage was formed. It should be noted that the Fragaria root is differently placed in our study compared with the study of Rousseau-Gueutin et al. (2009).

Conclusions

New knowledge of the evolution and relationships of taxa in Potentilleae have emerged based on the results of the five studies presented in this thesis and will be a good framework for further studies. Our results reinforces the split in Potentilleae, into the clades Fragariinae and Potentilla (=Potentillineae). The only morphological character known to coincide with this split is the anthers with confluent thecae at apex in Fragariinae while Potentilla species have anthers with two distinct thecae.

Several genera in Fragariinae received further support of being monophyletic while others were shown to be polyphyletic. Two “orphan” Potentilla species, P. miyabei and P. cuneata was placed in Fragariinae and five events of potential allopolyploid speciation were detected.

Six major clades were found within Potentilla. The ivesioid Potentilleae were shown to be an ingroup in Potentilla. Four instances of potential allopolyploid speciation were found in Potentilla. Style morphology was the single most important morphological character in Potentilla.

Alchemilla in the wide sense is monophyletic but in the more restricted sense (excluding Aphanes and Lachemilla), Alchemilla is paraphyletic. Four distinct clades were resolved, African Alchemilla, Eurasian Alchemilla, South American Lachemilla and annual Aphanes, where each of the clades corresponds to either distinct geographical areas or life forms.

Sibbaldia was shown to be polyphyletic and scattered in five different clades in Potentilleae and S. perpusilloides was considered to represent a new undescribed genus. 20 The evolution of Fragaria was shaped by polyploidization. The hexaploid F. moschata is the result of two allopolyploidy events, first involving F. vesca and F. viridis, secondly by a diploid of the F. iinumae lineage. The octoploid lineage emerged after an allopolyploidization event between F. moschata and a diploid of the F. iinumae lineage. The decaploid F. iturupensis was the result of allopolyploidization between the octoploid lineage and a diploid of the F. iinumae lineage. The potentially new decaploid species known as F. virginiana ssp. platypetala originated after an allopolyploidization event between the octoploid lineage and an unknown diploid close to the base of the Fragaria phylogeny. The tetraploid Fragaria species mainly originated from autopolyploidization.

Svensk sammanfattning

I den här avhandlingen behandlas gruppen (kladen) Potentilleae som är en del av rosväxtfamiljen (Rosaceae) och gruppen består av ett tusental arter. De är vanligast förekommande på norra halvklotet i tempererade, alpina eller arktiska områden men återfinns också på södra halvklotet. Under de senaste hundra åren har det föreslagits ett antal olika hypoteser om vilka arter som ingår i Potentilleae och hur dessa arters släktskapsrelationer (systematik) ser ut. Traditionellt har utseendekaraktärer (morfologi) använts som grund för hur växterna är besläktade med varandra men eftersom det har visat sig att flertalet av de morfologiska karaktärer som använts (t. ex. blomfärg, antalet ståndare, behåring på frukterna, bladutseende etc.) har uppkommit eller förlorats vid flera tillfällen under evolutionen i Potentilleae har det varit svårt att tolka systematiken enbart baserat på morfologiska karaktärer. Det har resulterat i att inget konsensus nåtts.

När molekylära metoder utvecklades och framförallt när ”Polymerase Chain Reaction-tekniken” (PCR) slog igenom på 1980-talet, blev det möjligt att ta fram nya och stora mängder data i form av DNA-sekvenser. DNA- sekvenserna som används vid systematisk forskning kommer ursprungligen från växtens cellkärna (kärn-DNA) eller från plastiden (samlingsnamn för de strukturer i varje cell vars primära uppgift är att fotosyntetisera; kloroplasten är en plastid; plastid-DNA). Parallellt med de nya molekylära metoderna utvecklades också nya analysmetoder. Oftast presenteras resultatet från analyserna i form av ett släktträd (fylogeni) som består av grenar och noder (de punkter där grenar möts i trädet). Fylogenin är en hypotes om hur växterna är besläktade.

21 Baserat på DNA-sekvenser från plastiden och cellkärnan har tidigare studier funnit att Potentilleae består av två evolutionära grenar (klader), nämligen Fragariinae respektive Potentilla (=Potentillineae). Den här avhandlingen behandlar främst Fragariinae (artikel I) och delar av denna klad (artikel III, IV och V) men även Potentilla (artikel II).

Förutom släktskapsrelationerna i Potentilleae har den artbildningsprocess som har att göra med kromosomtalsfördubblingar studerats. Arter som är kromosomtalsfördubblade kallas gemensamt för polyploider och de ssa delas in i två huvudkategorier, allopolyploider och autopolyploider. Allopolyploida arter bildas genom att två olika arter hybridiserar (reproduktion mellan individer av olika arter) och därefter kromosomtalsfördubblas, med an autopolyploider kromosomtalsfördubblas inom arten (involverar inte hybridisering). Man kan spåra föräldraarterna vid allopolyploidisering eller föräldraarten vid autopolyploidisering genom att ta fram sekvensen från en kärn-DNA region som endast finns i en eller ett fåtal kopior och undersöka en fylogeni baserad på denna sekvens. Eftersom en autopolyploid art endast har en förälder kommer samtliga genkopior vara placerade tillsammans på samma gren som föräldraarten (närmast besläktad), medan en allopolyploid art med två olika föräldrar kommer ha genkopior placerade på två olika grenar i fylogenin, tillsammans med respektive föräldraart.

Den första studien (artikel I) i avhandlingen gav ytterligare stöd för delningen av Potentilleae i de två kladerna Fragariinae och Potentilla. Fragariinae är monofyletisk (ett gemensamt evolutionärt ursprung) och består av tio släkten, Alchemilla (t.ex. daggkåpa) i vid bemärkelse, Comarum (t.ex. kråkklöver), Sibbaldia (t.ex. dvärgfingerört), Sibbaldianthe (spetsfingerört), Sibbaldiopsis (tretandsfingerört), Drymocallis (t.ex. trollsmultron), Chamaerhodos, Dasiphora (t.ex. tok), Potaninia och Fragaria (t.ex. smultron). Dessutom visas att två arter som för tillfället är klassificerade i Potentilla, P. cuneata och P. miyabei tillhör Fragariinae och är nära besläktade med Sibbaldia. Genom att jämföra och identifiera olikheter mellan den fylogeni som var baserad på plastidsekvenser med den fylogeni som var baserad på kärn-sekvenser, togs hypoteser om möjliga allopolyploidihändelser fram. Resultaten från denna studie låg till grund för övriga studier i denna avhandling. Fem välstödda olikheter (inkongruenser) identifierades och fyra av dem betraktades som troliga allopolyploidiseringar. De två mest intressanta inkongruenserna fanns i Fragaria (grunden för artikel V) och Sibbaldia (pågående projekt, men inte inkluderat i denna avhandling).

22 I artikel II användes samma tillvägagångssätt som i den första studien, där fylogenier baserade på plastid-DNA respektive kärn-DNA jämfördes och inkongruenser identifierades. I detta fall var det Potentilla-kladen (fingerörter m.fl.) som studerades mer i detalj och fem välstödda inkongruenser identifierades. Några släkten som tidigare brutits ut ur Potentilla, Horkelia, Ivesia, Horkeliella och Duchesnea visade sig vara delar av Potentilla-kladen. Detta resultat gav ökat stöd till tidigare, mer översiktliga studier. Det utbrutna släktet Argentina (P. anserina, gåsört; inkl. nära besläktade arter) var väl stött som syster till övriga arter av Potentilla enligt plastidfylogenin men svagt stött som syster till Fragariinae enligt kärn- fylogenin. Sex välstödda klader identifierades: Anserina-, Alba-, Fragarioides-, Reptans-, ivesioid Potentilleae och Argentea kladen. Utseendet på blommans stift var den enda morfologiska karaktär som kunde identifieras som innehållande relevant systematisk information inom Potentilla.

Gruppen/släktet Alchemilla (daggkåpor) i vid bemärkelse vilket också inkluderar Aphanes (jungfrukammar) och Lachemilla studerades i artikel III. Fylogenier baserade på sekvenser från plastid- och kärn-DNA var i stort sett kongruenta och fyra distinkta klader identifierades, afrikanska Alchemilla, eurasiska Alchemilla, sydamerikanska Lachemilla och Aphanes. Analyserna visar att släktet Alchemilla är monofyletiskt om det klassificeras i vid bemärkelse inklusive alla fyra kladerna, men om det delas upp tre separata släkten Alchemilla, Lachemilla och Aphanes– som ofta har skett – är släktet parafyletiskt (En parafyletisk grupp är inte en hel gren på ett fylogenetiskt träd utan utesluter delar av grenen.). Vi föreslog att namnet Alchemilla bör användas i vid bemärkelse, och inkludera släktena Lachemilla och Aphanes.

Ett antal arter blivit förda till Sibbaldia huvudsakligen baserat på de morfologiska karaktärerna, färre ståndare än Potentilla (5 eller 10 i stället för 20) och oftast förekomst av trefingrade blad. Baserat på en kombination av sekvenser från plastid-DNA och kärn-DNA visades att Sibbaldia är ett polyfyletiskt (består av olika grenar i en fylogeni) släkte som hör hemma på fem olika ställen inom Potentilleae (artikel IV). Tre klader placerar sig i Fragariinae; (1) Sibbaldia i strikt bemärkelse, som består av fyra arter (bland annat dvärgfingerört), (2) Sibbaldia adpressa placerar sig i Sibbaldianthe och (3) Sibbaldia perpusilloides som anses representera ett nytt obeskrivet släkte, placerade sig i en klad med släktena Chamaerhodos, Drymocallis, Dasiphora och Potaninia. De två återstående Sibbaldia-kladerna återfanns i Potentilla varav en klad (4) som bestod av fyra arter från Himalaya och (5) Sibbaldia micropetala som är en del av ”Potentilla anserina-kladen”. En

23 morfologisk karaktär skiljer Fragariinae från Potentilla: ståndarknapparnas två pollensäckar flyter samman i toppen hos Fragariinae, medan hos Potentilla är pollensäckarna skilda från varandra även i toppen.

En ingående studie av släktet Fragaria (smultron) utfördes för att försöka spåra de polyploida arternas ursprung samt att försöka identifiera föräldrarterna (artikel V). De polyploida arterna klonades och kolonier sekvenserades i ett representativt antal för att med 95% säkerhet få med alla potentiella genkopior. Resultaten visade att tre tetraploida (dubbel kromosomuppsättning) Fragaria-arter var autotetraploider och deras potentiella föräldraarter identifierades. En tetraploid art ansågs vara allopolyploid och föräldraarterna föreslogs. Den hexaploida (trippel kromosomuppsättning) F. moschata (parksmultron) föreslogs vara fullt allopolyploid, med ursprung från F. vesca (smultron), F. viridis (backsmultron) och F. iinumae. De två oktoploida (fyra kromosom- uppsättningar) arterna F. virginiana och F. chiloensis visade sig ha ett gemensamt ursprung och allopolyploida med föräldraarterna F. vesca, F. viridis samt två separata allopolyploidiseringar med F. iinumae-linjen. Den dekaploida (fem kromosomuppsättningar) F. iturupensis var allopolyploid och arten bildades efter en hybridisering mellan den oktoploida linjen och F. iinumae linjen, följt av polyploidisering.

Tack/Thanks

I am grateful to my supervisor Torsten Eriksson for good discussions, support and feedback. I acknowledge my supervisor Jürg Schönenberger. I would also like to thank all colleagues at the Department of Botany for help and support. I acknowledge financial support from the Department of Botany, SU (salary), the Swedish Research Council (VR, to T. Eriksson), and FORMAS (to T. Eriksson) for lab work.

Thank you Tom Davis and all the people I met at UNH.

Mamma, Pappa, Syster, Ulla och Marielle.

Sist men inte minst, stort tack alla vänner för att jag har just er!

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