ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2016

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1401

Spikemoss patterns

Systematics and historical biogeography ofSelaginellaceae

STINA WESTSTRAND

ISSN 1651-6214ISBN 978-91-554-9647-0urn:nbn:se:uu:diva-300734

Dissertation presented at Uppsala University to be publicly examined in Zootissalen,Evolutionsbiologiskt centrum, Villavägen 9, Uppsala, Friday, 30 September 2016 at 09:15 forthe degree of Doctor of Philosophy. The examination will be conducted in English. Facultyexaminer: Professor Michael J. Donoghue (Department of Ecology and Evolutionary Biology,Yale University).

AbstractWeststrand, S. 2016. Spikemoss patterns. Systematics and historical biogeographyof Selaginellaceae. (Mosslummermönster. Systematik och historisk biogeografi hosSelaginellaceae). Digital Comprehensive Summaries of Uppsala Dissertations from theFaculty of Science and Technology 1401. 50 pp. Uppsala: Acta Universitatis Upsaliensis.ISBN 978-91-554-9647-0.

Selaginellaceae, spikemosses, is a heterosporous plant family belonging to the lycophytes. Withan estimated age of some 350 million years, the family is historically important as one ofthe oldest known groups of vascular plants. Selaginellaceae is herbaceous with a worldwidedistribution. However, the majority of the ca. 750 species in the single genus Selaginella arefound in the tropics and subtropics.

This thesis aims at elucidating the systematics and historical biogeography of Selaginellaceae.The evolutionary relationships of the family were inferred from DNA sequence data (plastidand single-copy nuclear) of one-third of the species richness in the group. Attention was paidto cover the previously undersampled taxonomic, morphological, and geographical diversity.Morphological features were studied and mapped onto the phylogeny. The results showan overall well-supported phylogeny and even more complex morphological patterns thanpreviously reported. Despite this, many clades can be distinguished by unique suites ofmorphological features.

With the phylogeny as a basis, together with the thorough morphological studies, a newsubgeneric classification with seven subgenera, representing strongly supported monophyleticgroups, is presented for Selaginella. By mainly using gross morphological features, easilystudied by the naked eye or with a hand lens, the intention is that the classification should beuseful to a broader audience.

During the work with species determinations, it was revealed that the correct name for anendemic Madagascan Selaginella species is S. pectinata Spring, not S. polymorpha Badré aspreviously proposed.

The robust phylogeny of Selaginellaceae allowed for a historical biogeographical analysis ofthe group. A time-calibrated phylogeny, together with extant species distribution data, formedthe basis. The results show pre-Pangean diversification patterns, Gondwanan vicariance, andmore recent Cenozoic long-distance dispersals. The many inferred transoceanic dispersalsduring the last 50 million years are surprising considering Selaginella’s large megaspores thatare thought to have a negative effect on dispersal.

In conclusion, this thesis presents a well-founded hypothesis of the evolutionary history ofSelaginellaceae including its phylogeny, morphology, and historical biogeography. The thesisforms a firm basis for further studies on Selaginellaceae in particular, and gives us a betterunderstanding of early land plant evolution in general.

Keywords: classification, historical biogeography, lycophytes, nomenclature, phylogeny,Selaginella, Selaginellaceae, systematics

Stina Weststrand, Department of Organismal Biology, Systematic Biology, Norbyv. 18 D,Uppsala University, SE-75236 Uppsala, Sweden.

© Stina Weststrand 2016

ISSN 1651-6214ISBN 978-91-554-9647-0urn:nbn:se:uu:diva-300734 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-300734)

Till farmor Maj-Lis som visade mig naturen.

Cover: “Spikemoss pattern”, by S. Weststrand

List of papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Weststrand, S., Korall, P. Phylogeny of Selaginellaceae: There is value in morphology after all! Submitted to American Journal of Botany.

II Weststrand, S., Korall, P. A subgeneric classification of Selaginella (Selaginellaceae). Submitted to American Journal of Botany.

III Smith, A.R., Weststrand, S., Korall, P. 2016. Selaginella pectinata resurrected: The correct name for an unusual endemic spikemoss from Madagascar. American Fern Journal 106: 131–134.

IV Weststrand, S., Korall, P. Historical biogeography of the heterospor-ous Selaginellaceae: A tale of pre-Pangean diversifications, Gond-wanan vicariance and Cenozoic long-distance dispersals. Manu-script.

Paper III was reproduced with kind permission from the publisher.

Note. To make clear that the nomenclatural novelties in Paper II are not val-idly published in this thesis, references to the place of publication of basio-nyms or replaced synonyms have been omitted.

Contents

Preface ............................................................................................................. 9

1. Introduction .............................................................................................. 111.1 Systematics ....................................................................................... 111.2 Historical biogeography .................................................................... 141.3 Selaginellaceae .................................................................................. 15

1.3.1 Morphology and reproduction ................................................... 181.3.2 Systematics ................................................................................ 201.3.3 Why study Selaginellaceae? ...................................................... 21

2. Aims ......................................................................................................... 23

3. Materials and methods ............................................................................. 243.1 Studies of organisms ......................................................................... 243.2 Phylogenetic analyses ....................................................................... 263.3 Classification .................................................................................... 273.4 Historical biogeography .................................................................... 27

4. Results and discussion ............................................................................. 294.1 Paper I and II: Phylogeny and subgeneric classification of

Selaginella ........................................................................................ 294.2 Paper III: Selaginella pectinata resurrected ...................................... 324.3 Paper IV: Historical biogeography of Selaginella ............................ 32

5. Concluding remarks ................................................................................. 34

6. Svensk sammanfattning ........................................................................... 356.1 Jättekort ............................................................................................. 356.2 Kort ................................................................................................... 356.3 Lång .................................................................................................. 36

6.3.1 Systematik ................................................................................. 366.3.2 Mosslumrar ............................................................................... 376.3.3 Avhandlingens fyra artiklar ....................................................... 39

7. Acknowledgements .................................................................................. 42

8. References ................................................................................................ 46

Abbreviations

DNA deoxyribonucleic acid ICN International Code of Nomenclature for algae, fungi,

and plants Ma million years ago nom. nov. nomen novum (new name) PCR polymerase chain reaction sp./spp. species (singular/plural) sp. nov. species nova (new species) subg. subgenus

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Preface

During the last years I have been doing genealogy research. Not on my own family though, but rather on the plant family Selaginellaceae, spikemosses. I have been digging into the long history of these plants, a history starting some 350 million years ago in a world that looked very different to the world we see today. It has been challenging, but most of all fascinating to follow these plants from present to past, and back again. The work has involved several different activities, all grading into each other, within the broad field of plant systematics: field work, molecular lab work, morphological studies, phylogenetic analyses, taxonomy, nomenclature, and historical biogeograph-ical analyses. In this thesis, I present my work in four papers, all describing different aspects of the evolutionary patterns of spikemosses.

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1. Introduction

Nature can appear as an indistinguishable green mass, or it can be a place with well-known and named companions. Systematics provides us with the names, the structure, and the history of the organisms around us. For me, there is an intrinsic worth in knowing what I see when I go outside; it makes for richer and happier walks.

In this section I will give you an overview of what systematics is, how a phylogenetic study can serve as a backbone for studies on the historical bio-geography of an organism group, and what spikemosses, the plant group of this thesis, are.

1.1 Systematics People like categorising. This is of course not always a good thing, especial-ly not when some categories are viewed as worth more than others. Howev-er, when it comes to e.g., plants, there is a lot to gain from knowing and naming the diversity seen and from understanding the evolutionary history of different plant groups.

Systematics, or systematic biology, is a discipline in which we study the biological diversity on Earth and trace evolutionary relationships. There are several definitions of systematics, but I follow the broad one presented by Judd et al. (2016):

[T]he fundamental aim of systematics is to discover all the branches of the evolutionary tree of life, to document changes that have occurred during the evolution of these branches, and to the greatest extent possible, to describe all species—the tips of the branches. Systematics is therefore the study of the biological diversity that exists on Earth today and its evolutionary history. (Judd et al., 2016, p. 2)

Even though Linnaeus was one of those who put the field of systematics on the map in the mid-1700s, systematics did not start with him. People have in all times made use of naming, and communicating what they see in nature, not the least to be able to for instance distinguish an edible plant from a poi-sonous one. However, the notion of evolution, in contrast to the idea of the

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world’s species being created by God as they are today and never changing, was for long unheard of. Luckily, the pioneers in evolutionary biology, espe-cially Charles Darwin and Alfred Russel Wallace, changed this picture and in the mid-1800s the idea of the world’s biological diversity being shaped by millions of years of evolution became established. However, the modern “tree view” of evolution, where organisms are grouped together based on common ancestry in a tree-like manner, was not readily accepted by the sci-entific community. Instead, an incorrect “ladder thinking”, where some groups of organisms are seen as more advanced than others (humans usually at the top), was dominating. Sadly, this kind of progression metaphors can still be seen in some popularisations of evolution, e.g., the iconic picture where a human develops in stages from a chimpanzee, instead of depicting them as two extant species living side by side.

Today, systematists use analytical methods for testing evolutionary rela-tionships. With a basis in the theoretical framework of phylogenetic system-atics introduced by Hennig (1966), we base our phylogenetic hypotheses on homology, “the sharing of features due to common ancestry” (Baum and Smith, 2013, p. 446). From the phylogenetic trees, or more simply, the phy-logenies (Fig. 1), we identify monophyletic groups (an ancestor and all of its descendants) supported by synapomorphies (shared derived character states). A synapomorphy can for instance be an observed morphological feature (as the seed for seed plants) or a nucleotide in the DNA. During the last 30 years, DNA sequence data have become more and more important to sys-tematics, and today molecular markers are the primary basis for phylogenetic analysis.

Hypotheses about evolutionary relationships are continuously being eval-uated using new data and/or analytical methods, giving us better and better inferences about the evolutionary patterns observed. The phylogeny of the major land plant lineages, as illustrated in Fig. 1, is a good example. Land plant evolution has been studied for a long time and several conflicting hy-potheses have been presented. Today, there is a consensus considering the major groupings, we for example agree that spore dispersed vascular plants (ferns and lycophytes) do not form a monophyletic group, and that ferns instead is the sister lineage to seed plants (Kenrick and Crane, 1997; Pryer et al., 2001; Fig. 1). However, there are still contradicting hypotheses concern-ing e.g., the relationship between the different moss groups (hornworts, liverworts, and mosses; Qiu et al., 2006; Cox et al., 2014; Wickett et al., 2014). In the end, we can of course never claim to have the true story of the evolution of land plants; what we have are well-supported hypotheses.

The field of systematics can today be viewed as much broader than it has been traditionally conceptualised. Following Judd’s definition quoted above, systematics is the basis for understanding history, not “only” a descriptive science focusing on describing the biological diversity we see; systematics today is a field grounded in theories, a discipline using analytical methods to

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study the evolutionary history of the current and former biological diversity. This includes e.g., how organisms have evolved morphologically and the historical biogeographical patterns that can be observed. Nonetheless, the “traditional” work of systematics, such as collecting material in the field, comparative morphological studies, delimiting and describing species, and classification, is still important and the basis for answering larger evolution-ary questions. If we do not know the organisms we are working with, we cannot hope to correctly understand the evolutionary patterns observed.

But how should the biological diversity be classified? The basic scheme for classification is a hierarchy; a species belongs to a genus, which belongs to a family, which belongs to an order, etc. For plants, the International Code of Nomenclature for algae, fungi, and plants (ICN, McNeill et al., 2012) provides us with the nomenclatural rules, the rules for naming, but when it comes to delimiting and classifying taxa, it is up to the respective researcher. Today, the majority of classifications are based on hypotheses of evolution-ary relationships, and the recognised “units” are monophyletic groups. How-ever, how groups should be delimited is fairly arbitrary. Categories are hu-man constructs, there are no “real” plant families out there. What is out there are “just” different organisms with different evolutionary relationships, and in the end all life is related and has descended from a common ancestor. How the boundaries are drawn, and a classification presented, mainly de-pends on what is considered useful. A classification is a tool for structuring the biological diversity observed.

In conclusion, the research field of systematics is the discipline that gives us an understanding of the biological diversity around us and its evolutionary history. Thanks to systematics we collect more and more pieces of the giant puzzle that is the evolutionary history of all organisms (extant and extinct) on Earth. The work done by systematists is also essential for protecting the biodiversity that still exists; without named organisms and knowledge on e.g., distribution and abundance, we cannot know what is out there to pro-tect. Last but not least, systematics is a major cornerstone for the work done in many other disciplines, in particular in other fields of biology such as ecology and developmental biology, where placing a work in an evolution-ary context is the basis for a thorough understanding of biological systems.

1.2 Historical biogeography Biogeography is the field that studies the distribution patterns of organisms in time and space; a field that is particularly important for understanding the biodiversity on Earth and the threats it is facing. Traditionally, biogeography has been subdivided into historical biogeography and ecological biogeogra-phy. Historical biogeography deals with larger time spans (often millions of

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years) and evolutionary patterns on, at least, the level of species. Ecological biogeography deals with much shorter time spans and usually at the popula-tion level (Sanmartín, 2012). However, the two subfields are grading into each other today with e.g., studies defined as phylogeography (Avise et al., 1987). Nevertheless, when talking about historical biogeographical studies, as I do in this thesis, one still generally intend larger time spans and the evo-lution of groups of species or higher taxa, rather than populations. The field of historical biogeography is old, even predating the theories of evolution (Lomolino et al., 2004), and observed biogeographical patterns served as a basis for the evolutionary theories presented by Darwin and Wallace in the mid-1800s (Donoghue, 2014).

Historical biogeography fits within the broad definition of systematics used in this thesis (p. 11). However, since the two subjects, systematics and historical biogeography, traditionally (and commonly) are seen as two sepa-rate research fields, I have chosen to treat them separately here.

The current distribution observed for an extant group of organisms is shaped by a whole range of evolutionary, geological, and ecological factors and events. Depending on the age of the organism group studied, possible explanations of, for instance, disjunct distribution patterns, can be long-distance dispersal (e.g., transoceanic dispersal), migration over landmasses, or vicariance scenarios due to e.g., continental fragmentation (Sanmartín, 2012).

For a well-founded hypothesis of the historical biogeography of a group, space and time should be incorporated already in the analyses, as compared to a post-analysis discussion of patterns seen in a phylogeny. “Time” is most often incorporated by the use of fossil data as calibration points. Ideally, incorporating the fossilized organisms into the actual analyses would strengthen the hypotheses (Ronquist et al., 2012). “Space” is the distribution patterns observed for the extant taxa included in the study. The methods available for estimating divergence times and historical biogeographical patterns are developing fast, and during the last years many new methodolo-gies have been presented (e.g., Landis et al., 2013; Matzke, 2013, 2014; Heath et al., 2014). While the field of historical biogeography in the past was largely a narrative discipline, it is today a field based on analysis and hy-pothesis testing.

1.3 Selaginellaceae This thesis focusses on spikemosses, which is the common name for the plant family Selaginellaceae Willk.; they are lycophytes (also called lyco-pods) and not mosses even though the name erroneously implies so. Selagi-nellaceae is an herbaceous, worldwide family, but most of the ca. 750 spe-cies are found in the tropics and subtropics (Jermy, 1990). Some 10% of the

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species are growing in temperate regions, but arctic and subarctic representa-tives are rare. One species, Selaginella selaginoides (L.) P.Beauv. ex Sch-rank & Mart., can be found in Sweden (Fig. 2A). The family has been shown to be monophyletic (Wikström and Kenrick, 1997; Korall et al., 1999), and it is monotypic, including the single genus Selaginella P.Beauv., with S. sela-ginoides as type. The different Selaginella species can vary considerably in morphology, even though the majority are delicate and adapted to humid conditions (Jermy, 1990; Fig. 2). A few drought-tolerant species are very conspicuous since they, during drought, form tight balls that slowly re-open when moistened; they are often referred to as resurrection plants and an ex-ample is the commercially sold S. lepidophylla (Hook. & Grev.) Spring (Fig. 2B).

Selaginellaceae is one of three extant lineages of lycophytes, the other be-ing Isoëtaceae Rchb. and Lycopodiaceae P.Beauv. ex Mirb. (Jermy and Øll-gard, 1990; Fig. 1). Isoëtaceae, the quillworts, is the sister lineage to Selagi-nellaceae (Wikström and Kenrick, 1997; Korall et al., 1999), and the two families share a heterosporic condition, i.e., they produce two kinds of spores, mega- and microspores, in two separate sporangia. Among land plants, heterospory is not common outside the seed plant lineage; it is only seen in Selaginellaceae, Isoëtaceae, and water ferns (Marsileaceae and Salviniaceae, Smith et al., 2006). The third lycophyte family, Lycopodi-aceae, is homosporous. Since lycophytes, like ferns, disperse with haploid spores, and not diploid seeds as seed plants, they have for a long time been grouped together with ferns and referred to as “fern allies”. However, mo-lecular studies have shown that lycophytes is the sister group to both ferns and seed plants (Kenrick and Crane, 1997; Pryer et al., 2001; Fig. 1).

Lycophytes is a historically important plant group with a fossil record da-ting back to the Late Silurian some 420 Ma (Kenrick and Crane, 1997). Ex-tinct members of this land plant lineage dominated parts of the flora during the Late Carboniferous (ca. 300 Ma, Kenrick and Crane, 1997), and during that time, many lycophytes formed large trees, which today make up for a large part of our ceasing coal deposits. Hence the name Carboniferous. The lycophyte-dominated flora during the Carboniferous can be compared to the small portion of the extant flora that this plant lineage constitutes today, less than 1% of the vascular plant species (Smith et al., 2006). Among the three extant lycophyte families, Selaginellaceae with its ca. 750 species is the largest (Jermy, 1990).

The oldest known fossil that unequivocally can be assigned to the Selagi-nellaceae lineage is Selaginellites resimus N.P.Rowe, an isophyllous lyco-phyte from the Early Carboniferous, some 345 Ma (Rowe, 1988). However, there are isoëtalean fossils from the Late Devonian–Early Carboniferous, some 370 Ma, implying that the two sister lineages diverged already by that time (Bateman and DiMichele, 1994; Kenrick and Crane, 1997).

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Figure 2. A diverse collection of Selaginella species: (A) S. selaginoides (circumbo-real), (B) S. lepidophylla (Southwest USA–Mexico), (C) S. longipinna Warb. (Aus-tralia), (D) S. watsonii Underw. (USA), (E) S. longipinna habitat (Australia), (F) S. australiensis Baker (Australia), and (G) S. umbrosa Lem. ex Hieron. (Central and South America). Photos: A–C, E–F, S. Weststrand; D, G, Hieronymus and Sadebeck (1901).

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1.3.1 Morphology and reproduction There are two morphological synapomorphies known for Selaginellaceae: megasporangia that contain only four megaspores, and a stele that is found in an air-filled cavity and is connected to the surrounding tissue by special “threads” called trabeculae (Jermy, 1990; Kenrick and Crane, 1997). How-ever, these characters are definitely not the ones you may easily notice when out in the field. Here follows a description of the more apparent morphologi-cal features and their variability in the family.

As mentioned above, all Selaginella species are herbaceous, but the gen-eral appearance can vary considerably among species (and even within): creeping, mat-forming, rosette-forming, ascending, erect, and even long and scandent, are all habits seen in the family (Fig. 2). However, the most com-mon morphology, found mainly in humid tropical forests where species richness within the genus is the highest, is delicate plants with anisophyllous flattened shoots bearing four rows of dimorphic vegetative leaves; two rows of smaller leaves on the upper (dorsal) side of the shoot, and two rows of larger leaves on the lower (ventral) side (Jermy, 1990; Figs. 2C and 3A). Some 50 species, mostly found in temperate and dry areas, have monomor-phic vegetative leaves that are helically arranged (except for three species where the leaves are decussately arranged; Jermy, 1990; Fig. 2D).

The mega- and microsporangia (enclosing mega- and microspores, re-spectively) are each subtended by sporophylls and arranged in strobili at branch tips (Jermy, 1990; Fig. 3A–B). The strobili are tetrastichous in all Selaginella species but two, the circumboreal S. selaginoides and the Hawai-ian S. deflexa Brack., in which the sporophylls are helically arranged like the vegetative leaves (Jermy, 1986). Similar to the vegetative shoots, strobili in Selaginella are either isophyllous or anisophyllous, where isophyllous strobi-li are most common and found in all species with isophyllous vegetative shoots, as well as in most of the species with anisophyllous vegetative shoots (Jermy, 1990). Some 60 species with anisophyllous shoots also have aniso-phyllous strobili (bilateral strobili; Jermy, 1990). These can either be resupi-nate, with the smaller sporophylls in the same plane as the larger vegetative leaves, or less commonly, non-resupinate, with the smaller sporophylls in the same plane as the smaller, vegetative leaves (Quansah and Thomas, 1985).

Megasporangia usually contain four megaspores, whereas microsporangia enclose over a hundred microspores. Both mega- and microspores vary con-siderably in morphology, and several studies have shown a mosaic of mor-phological complexity (e.g., Minaki, 1984; Morbelli et al., 2001; Korall and Taylor, 2006; Zhou et al., 2015b). Another morphological feature traditional-ly studied in Selaginella is stelar arrangement. The stem stele is a protostele, commonly a simple, circular to elliptic monostele, but other arrangements also occur in the genus, with the most conspicuous being the lobed

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actino-plectostele of the Central and South American species S. exaltata (Kunze) Spring (Mickel and Hellwig, 1969).

Root-like structures called rhizophores are seen in all Selaginella species, except S. selaginoides and S. deflexa (Karrfalt, 1981; Schulz et al., 2010, and references therein). Rhizophores most commonly emerge from the ventral or dorsal sides in branch dichotomies of aerial shoots (Fig. 3A), but there are also species where they are confined to a creeping rhizome. Another vegeta-tive morphological feature found in a group of mainly Central and South American species, is articulations, swellings below branch dichotomies, that usually appear as dark constricted segments in dried specimens (Jermy, 1990).

Due to their heterosporous reproduction, Selaginella species are obligate gametophytic outcrossers; a megaspore develops into a megagametophyte, and a microspore into a microgametophyte (Judd et al., 2016; Fig. 3C). Both the mega- and microgametophytes develop within the spore wall; they are endosporic. During development, the megaspore wall ruptures and the mega-gametophyte with its archegonia (with egg cells) protrudes (Fig. 3D). At microgametophyte maturation, the microspore wall ruptures and sperms are released. Fertilisation in Selaginella is water-dependent, the sperms need water to swim to the eggs. The need for water is a major difference between reproduction in lycophytes (and also ferns) as compared to seed plants. After fertilisation, a new sporophyte starts to grow from the megagametophyte (Fig. 3E).

Today, very little is known about the reproduction and dispersal biology in Selaginella. Some of the less studied questions are: How common is self-fertilisation in Selaginella (see one example in e.g., Valdespino, 1993)? How are Selaginella spores dispersed? Can a megaspore and a microspore be dis-persed as a unit (synaptospory, see e.g., Filippini-De Giorgi et al., 1997)? Can an already fertilised megaspore be dispersed, or will it be too vulnerable to e.g., dehydration? Can Selaginella spores be dispersed by animals? By wind? As you can see from this long list of questions, further studies on Sel-aginella reproduction and dispersal biology are much needed.

1.3.2 Systematics Palisot de Beauvois (1804) was the first to describe the genus Selaginella, and during the 200 years that have passed since he coined the name, the clas-sification of Selaginellaceae has been debated. Some authors have proposed that a single genus Selaginella should be recognised (e.g., Spring, 1840, 1849; Braun, 1858; Baker, 1883; Hieronymus and Sadebeck, 1901; Walton and Alston, 1938; Tryon and Tryon, 1982; Jermy, 1986), others have divided the family into several genera (e.g., Palisot de Beauvois, 1804; Rothmaler, 1944; Soják, 1993; Tzvelev, 2004), and Reichenbach (1828) even wanted to include all species in different subgenera under Lycopodium L. Of the more

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recent classifications, the one by Jermy (1986) has been most frequently referenced.

All classifications listed above were based solely on morphological fea-tures, and not on phylogenetic analyses of Selaginella. The features most often used to distinguish groups in the classifications are: isophylly and an-isophylly of both vegetative shoots and strobili, phyllotaxy, stelar arrange-ment, rhizophore position, habit, and spore ornamentation. The first classifi-cation presented in a phylogenetic framework, based on DNA sequence data, was the one proposed by Zhou and Zhang (2015). However, the first phylo-genetic analysis of Selaginellaceae (based on DNA sequence data) was pub-lished already in 1999 by Korall and co-authors. Since then a few more stud-ies have been conducted, extending both the taxon sampling and the molecu-lar markers used (Korall and Kenrick, 2002, 2004; Arrigo et al., 2013; Zhou et al., 2015a). Overall, the different phylogenetic studies agree, showing a small clade of S. selaginoides and S. deflexa being the sister group to all other species, the so-called rhizophoric clade (sensu Korall et al., 1999), i.e., including the species having rhizophores. The rhizophoric clade is further subdivided into clades A and B (sensu Korall and Kenrick, 2002). Two prob-lems consistently appearing in all previous studies on Selaginella phyloge-netics are: finding clear morphological synapomorphies defining well-supported clades, and unequivocally resolving the phylogenetic positions of some enigmatic groups, e.g., S. sinensis (Desv.) Spring and close allies.

Something that has hampered our understanding of Selaginella systemat-ics is the morphological stasis seen in the genus. Selaginella is notorious for problems with species identification and unclear species delimitations. Pre-vious studies (and also this thesis, Paper I) show that there is an obvious problem with non-monophyletic species and probable species complexes. Thus, further studies on the alpha taxonomy of the group are needed (but see e.g., Valdespino, 1993, 2015; Gardner, 1997; Stefanović et al., 1997; Jermy and Holmes, 1998; Mickel et al., 2004; Zhang et al., 2013; Valdespino et al., 2015).

1.3.3 Why study Selaginellaceae? Despite the 200 years that have passed since Palisot de Beauvois recognised the genus Selaginella in 1804, there are still many questions waiting to be answered regarding the evolutionary history of this group of lycophytes.

First, predictions of the evolutionary history have to be based on well-founded hypotheses. Thanks to the phylogenetic studies of Selaginellaceae presented during the last 15 years (Korall et al., 1999; Korall and Kenrick, 2002, 2004; Arrigo et al., 2013; Zhou et al., 2015a), we have increased our knowledge of the evolutionary history of this group. However, to get better hypotheses about Selaginellaceae evolution, there are still many more as-pects to consider. We need to base our phylogenetic hypothesis on a repre-

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sentative global sample of the extant species richness. Previous studies have in particular left out much of the African and Pacific diversity in the family. Thorough morphological studies, in combination with a well-corroborated phylogenetic hypothesis, would also serve as a basis for a new, and much needed, classification of the group. In addition, a well-supported, global phylogeny would serve as a basis for alpha-taxonomical work, which is lack-ing for many regions of the world.

Second, Selaginellaceae is a historically important plant group. It is one of three extant lineages of lycophytes, a plant group dating back to the De-vonian. Selaginellaceae is estimated to be at least 350 Ma, an age predating the origin of angiosperms with some 200 million years (Judd et al., 2016). Thus, Selaginellaceae is representing one of the earliest groups of vascular plants, and can give us better insights into e.g., early land plant evolution and adaptations.

Third, the fact that Selaginellaceae is heterosporous makes it an interest-ing study system, where one of the diaspores, the megaspore, is large (0.2–1.2 mm in diameter, Korall and Taylor, 2006), something that we can as-sume would have a negative effect on dispersability. Compared to angio-sperms, we have little insight today about the historical biogeography of spore dispersed vascular plants, and especially about heterosporous spore dispersed plants such as Selaginellaceae. Despite a good and lengthy fossil record, the evolutionary history of Selaginellaceae has been poorly under-stood, primarily, due to the lack of a phylogenetic framework on which we can base further studies. With a global, well-founded phylogenetic hypothe-sis, we could ask questions on i.e., the historical biogeography of the group. Have the geographical patterns observed today been shaped by i.e., historical geological events like plate tectonic movements? Are Selaginella species “bad dispersers” as often believed?

Doing systematic studies (in a broad sense) on Selaginellaceae will give us a better understanding, not only of the evolutionary history of this plant group, but also on a broader scale, getting insights into the early evolution of land plants.

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2. Aims

The overall aim of this thesis is to get a better understanding of the systemat-ics and historical biogeography of the lycophyte family Selaginellaceae.

In Paper I we present a large-scale phylogeny of Selaginellaceae, covering the morphological, geographical, and taxonomic diversity in the family. Be-sides giving us new insights about the evolutionary history of this plant group, the resulting phylogeny should serve as a robust framework for the subsequent studies presented in this thesis, and as a backbone for future stud-ies on Selaginellaceae.

In Paper II we propose a revised subgeneric classification of Selaginella, based on the phylogenetic and morphological work presented in Paper I. In Paper I we show that the existing classifications of Selaginella poorly reflect the phylogenetic relationships in the genus, and/or recognise groups difficult to unequivocally identify based on morphology. The aim was to present a robust classification that can easily be used both by experts in the field and by a broader audience.

In Paper III we thoroughly review the nomenclature of the Madagascan species S. polymorpha Badré following the rules of the ICN. While working with Paper I, we realised that a nomenclatural act concerning the species was erroneously made in the late 1990s.

In Paper IV we investigate the historical biogeography of Selaginell-aceae, with the large-scale phylogeny presented in Paper I forming the basis for the analyses. With their large megaspores and obligate outcrossing re-production, Selaginella species have often been thought of as “bad dispers-ers”. We wanted to elucidate the historical biogeographical processes behind the worldwide distribution patterns seen in the family today.

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3. Materials and methods

Here follows a summary of the methodological choices in this thesis. De-tailed descriptions of the materials and methods are presented in the respec-tive papers, which will be referred to below.

3.1 Studies of organisms The backbone of my studies is a phylogenetic tree based on DNA sequence data from extant species of Selaginella (Paper I). There are ca. 750 species of Selaginella around the world, known to science (Jermy, 1990). I sampled approximately one third of those (340 accessions representing 223 species, see appendix 1 in Paper I). My aim was to collect as representative a sample as possible, covering the geographical, morphological, and taxonomic diver-sity in the genus. With previous studies on Selaginella phylogenetics as a starting point (e.g., Korall and Kenrick, 2002, 2004; Zhou et al., 2015a), I paid special attention to groups that had previously been undersampled, such as the many African and Pacific species, as well as to enigmatic groups with a previously unresolved phylogenetic position, as for instance, the Asian species S. sinensis and close allies. In addition, I sampled multiple acces-sions from several species, to allow for an evaluation of within-species var-iation.

DNA was extracted both from recently collected silica-dried tissue (e.g., from my field trips to Australia, Ecuador, and Scandinavia, Fig. 4), and her-barium material, the oldest collected in 1921. Apart from this, I made use of DNA sequence data obtained from previous studies on Selaginella, available in GenBank. However, this was done with caution since my preliminary analyses revealed potential problems with misidentifications, as shown by many non-monophyletic species.

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Figure 4. Stina collecting S. selaginoides at the North Cape, Norway. Photo: J. Lars-son.

Selaginella is notorious for the difficulties associated with species determi-nation, and a common sight when visiting the Selaginellaceae section in herbaria is a large pile of undetermined Selaginella spp. Furthermore, floras and taxonomic treatments are lacking, or in need of revision, for many re-gions of the world. Due to these problems, I was cautious when choosing accessions for my study. In addition to evaluating the species determinations myself, I have made sure to use herbarium specimens identified by experts in the field as far as possible, or, if that proved impossible, compared my sam-ples to specimens identified by experts. For species identification I used floras, species descriptions, Reed (1965–1966), and online checklists (e.g., Hassler and Schmitt, 2001).

During the work with species determinations, we concluded that S. pecti-nata Spring is the correct name for an endemic Madagascan species, and that the name S. polymorpha Badré from the late 1990s is superfluous and illegit-imate (Paper III).

For all accessions, morphological features were studied with the naked eye or under a stereo microscope. Gross morphological features used in pre-vious classifications (leaf heteromorphism, phyllotaxy, growth form, stelar arrangement, rhizophore position, and presence of articulations) were noted for all accessions at hand (see morphological characters mapped onto the phylogeny in fig. 2, Paper I). My observations were, as far as possible, veri-fied using literature data (for an account of references, see Paper I). In addi-tion, information on megaspore ornamentation was taken from the literature (e.g., Minaki, 1984; Morbelli et al., 2001; Korall and Taylor, 2006).

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3.2 Phylogenetic analyses My phylogenetic hypothesis is based on data from three different DNA re-gions: the chloroplast region rbcL, and the two single-copy nuclear regions pgiC and SQD1. Molecular markers used for phylogenetic work need to be orthologous (homologous gene sequences descended through speciation), and in addition, they need to have the “right” amount of variation for the questions asked. The plastid region rbcL has been used in plant systematics for a long time (see e.g., Hasebe et al., 1995 on ferns), and it is generally considered to evolve slowly, which has made it useful for phylogenetic work on e.g., the family level or above. However, previous studies on Selaginella have shown tremendously high substitution rates for rbcL (37% informative characters reported in Korall and Kenrick, 2004). Thus, for Selaginella, rbcL can be used for within-family phylogenetic analyses, and has formed the basis for all previous phylogenetic studies of the group (Korall et al., 1999; Korall and Kenrick, 2002, 2004; Arrigo et al., 2013; Zhou et al., 2015a).

With the aim to assemble a DNA data set covering both the plastid and nuclear genomes, I evaluated the two nuclear regions pgiC and SQD1, as possible molecular markers for phylogenetic analysis in Selaginella. Neither of the two regions have previously been used for Selaginella systematics, but both have been shown to be single-copy in ferns (Rothfels et al., 2013). A first estimation of the variability of the two regions was made based on mul-tiple sequence alignments of transcriptome data of eight Selaginella species obtained from the 1000 Plants Initiative (1KP, onekp.com). The multiple sequence alignments were subsequently used for finding conserved regions for primer design. Based on my analyses of the transcriptome data, subse-quent lab work, and phylogenetic analyses, I concluded that both genes (pgiC and SQD1) are single-copy in Selaginella. All the settings for the PCR amplification of the three different regions used for phylogenetic analyses can be found in Paper I.

For each of the three DNA regions analysed, the assembled DNA se-quences were aligned, followed by single-region phylogenetic analyses in a Bayesian inference framework using MrBayes 3.2.4 (Ronquist and Huel-senbeck, 2003). The single-region phylogenies were then compared to eluci-date possible well-supported conflicts between the data sets. No major con-flicts were observed, and the three single-region data sets were combined to a partitioned data set with each partition assigned its own model for DNA substitution. A phylogenetic analysis using MrBayes was then performed on the combined three-region data set. Samples from Isoëtaceae, the sister line-age to Selaginellaceae, was used as outgroup (Wikström and Kenrick, 1997; Korall et al., 1999).

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3.3 Classification The resulting phylogeny, and the integrated morphological studies (Paper I), formed the basis for proposing a new subgeneric classification of Selaginella (Paper II). For the classification, I recognised seven major, well-supported monophyletic groups, each uniquely defined by a suite of morphological characters. For the classification to be useful, both for the experts in the field, as well as for a broader audience, I find the following criteria to be of special importance:

(1) stability – the groups named should be monophyletic and well-supported by phylogenetic analyses, that is, not expected to change in the near future, (2) morphological identification – the subgenera should, as far as possible, be recognizable by (gross) morphological features that are easily studied, and (3) a conservative approach – unless the evidence is convincing, we prefer to de-viate as little as possible from Jermy’s (1986) well-established classification, and the historical recognition of morphologically well-founded groups (i.e., series Articulatae (Spring) Hieron. & Sadeb.). (Weststrand and Korall, Paper II, p. 4)

In Paper II, we propose a new subgeneric classification of Selaginella, de-spite the recently published classification by Zhou and Zhang (2015). The main reasons for this are that our new data (Paper I) show an even more complex picture of the morphological evolution in Selaginella than previous-ly reported (e.g., Zhou et al., 2015a), and using morphology, we have prob-lems to unequivocally assign species to the different subgenera proposed by Zhou and Zhang (2015). Further, the Zhou and Zhang classification is prob-lematic in several additional aspects: (1) the use of the name Selaginella subg. Ericetorum which is incorrect given their circumscription, (2) the (un-intentional) inclusion of a non-monophyletic group (Selaginella sect. Oli-gomacrosporangiatae), and (3) the exclusion of a whole clade (S. sinensis and close allies).

3.4 Historical biogeography In Paper IV, we use our phylogenetic tree from Paper I as a basis for a his-torical biogeographical analysis of Selaginella. In this study, I made use of the taxon-wise most complete data set assembled for Paper I, the chloroplast rbcL data set.

Phylogenetic relationships and lineage divergence times were estimated in a Bayesian inference framework using BEAST 2.4.0 (Bouckaert et al., 2014). Three nodes were time-calibrated based on fossil data. To be able to place the fossils as accurately as possible, the morphological features ob-served in the fossils were rigorously compared to the morphology seen

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among extant taxa. The calibration points were assigned a lognormal prior distribution following the discussions in e.g., Ho and Phillips (2009).

The historical biogeographical analysis was performed in a maximum likelihood framework using the R package BioGeoBEARS (Matzke, 2013). I defined nine larger geographical regions based on both information on his-torical geographical and continental scenarios, as well as on ecology of the extant species included in the data set. The distribution data for the extant species, together with the time-calibrated tree obtained from BEAST, formed the basis for the historical biogeographical analysis, in which ancestral range evolution was simulated under the DEC+J model, i.e., a dispersal-extinction cladogenesis (DEC) model (Ree and Smith, 2008) with founder-event speci-ation (J) (Matzke, 2014). The importance of including founder event specia-tion in historical biogeographical analyses have been thoroughly discussed by e.g., Matzke (2014).

The resulting time-calibrated phylogeny with geographical range esti-mates for nodes, was carefully evaluated. The timing of cladogenetic events (including the 95% highest posterior density intervals), in combination with ancestral range estimates for the nodes in question, were compared to geo-logical events such as the breakup of Pangea, subsequent breakups of Gondwana and Laurasia, as well as e.g., land bridge formations in the North-ern hemisphere during the Cenozoic (following time spans given by, e.g., McLoughlin, 2001; Sanmartín et al., 2001; Rogers and Santosh, 2003; San-martín and Ronquist, 2004).

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4. Results and discussion

In the following pages I give a summary of and discuss the results from the four studies included in this thesis.

4.1 Paper I and II: Phylogeny and subgeneric classification of Selaginella

The first two papers of this thesis (Paper I and II) were done in parallel and are tightly connected, therefore I present and discuss them together here. The first phylogenetic analyses of Selaginellaceae, some 15 years ago, gave us the first insights into the evolutionary history of this old plant group with some 750 extant species (Korall et al., 1999; Korall and Kenrick, 2002, 2004). In particular, they showed a group with a complex morphological evolution and a phylogeny, based on DNA sequence data, with monophylet-ic groups not corresponding to the morphology-based classifications at hand.

With the earlier studies of Selaginella phylogenetics as a starting point, we aimed at presenting a large-scale phylogenetic analysis of the group. The resulting phylogeny, based on a representative global taxon sample, shows a topology in line with previous findings (Korall et al., 1999; Korall and Kenrick, 2002, 2004; Arrigo et al., 2013; Zhou et al., 2015a). Overall, we present a well-supported phylogeny, where the positions of earlier problem-atic groups (i.e., the Asian S. sinensis and close allies) are placed with strong support. In addition, we address the position of another enigmatic group, the sanguinolenta group (S. sanguinolenta (L.) Spring and S. nummularifolia Ching). Our phylogeny reveals some problems with non-monophyletic spe-cies, however, these problems are less prominent than found in previous studies (Zhou et al., 2015a).

Morphological studies were done in parallel to the phylogenetic work based on DNA sequence data. We studied morphological features used in previous Selaginellaceae classifications, and mapped them onto the phyloge-ny (see fig. 2 in Paper I). Our studies reveal even more complex morpholog-ical patterns than previously reported (Korall and Kenrick, 2002; Korall and Taylor, 2006; Zhou et al., 2015a), with most morphological characters show-ing reversals and/or parallelisms.

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In the light of our new phylogenetic hypothesis of Selaginella, together with our thorough morphological studies of the group, we propose a new subgeneric classification of the genus (Paper II). The seven subgenera pro-posed correspond to monophyletic groups (Fig. 5), and despite the complex morphological patterns observed in the genus, each subgenus can be unique-ly diagnosed by a suite of (mainly) gross morphological characters. The sev-en subgenera proposed in our classification are: Selaginella subg. Selaginel-la, S. subg. Rupestrae Weststrand & Korall, S. subg. Lepidophyllae (Li Bing Zhang & X.M.Zhou) Weststrand & Korall, S. subg. Gymnogynum (P.Beauv.) Weststrand & Korall, S. subg. Exaltatae Weststrand & Korall, S. subg. Ericetorum Jermy, and S. subg. Stachygynandrum (P.Beauv. ex Mirb.) Baker. In addition to a formal taxonomical treatment, a key to the different subgenera is provided (see Paper II).

The clade corresponding to subg. Selaginella is sister to the rest of the Selaginella species, the rhizophoric clade, which share the synapomorphy of having rhizophores. The rhizophoric clade is further subdivided into clades A and B, where clade A includes five of the subgenera (Rupestrae, Lepi-dophyllae, Gymnogynum, Exaltatae, and Ericetorum), whereas clade B cor-responds to subg. Stachygynandrum, the largest subgenus including ca. 600 of the 750 species known in the family. Our intention is to provide a useful and robust classification that could be used by both experts and the broader public, and with features that mainly can be recognised with the naked eye, or with a stereo microscope or hand lens.

The phylogeny (Paper I) gives us new insights into the evolutionary history of Selaginellaceae, and together with the proposed subgeneric classification (Paper II), we provide a framework for further studies of the group.

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Figure 5. A schematic overview of the phylogenetic relationships of Selaginella depicting the seven subgenera. All nodes are supported by a Bayesian posterior probability (PP) of 1.0, except for clade B (PP 0.97). Clade size is based on number of accessions, scaled logarithmically.

Isoëtes

S. subg. Selaginella

S. subg. Rupestrae

S. subg. Lepidophyllae

S. subg. Gymnogynum

S. subg. Exaltatae

S. subg. Ericetorum

S. subg. Stachygynandrum

Rhizophoricclade

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4.2 Paper III: Selaginella pectinata resurrected While working on species determinations for Paper I, we came across an erroneous nomenclatural act concerning a Selaginella species endemic to Madagascar. In the account of Selaginellaceae for “Flore de Madagascar et des Comores” (Stefanović et al., 1997), it was noted that the combination S. pectinata (Willd.) Spring is illegitimate as it was based on Lycopodium pec-tinatum Willd. from 1810, which is an illegitimate later homonym of L. pec-tinatum Lam. from 1792. However, according to Article 58.1 of the ICN (McNeill et al., 2012), S. pectinata can be treated as a new name (nom. nov.) for the replaced illegitimate synonym L. pectinatum Willd. and is then to be cited as S. pectinata Spring dating from 1843. This provision in the ICN was overlooked by Stefanović et al. (1997) when proposing the illegitimate and superfluous S. polymorpha Badré. The conclusion made in Paper III is that the correct name for this Madagascan endemic is S. pectinata Spring.

4.3 Paper IV: Historical biogeography of Selaginella The robust and well-supported phylogeny presented in Paper I allows for further studies on the evolutionary history of Selaginellaceae. The historical biogeography of the family, has not previously been analysed, and due to its old age (at least 350 Ma) and heterosporous reproduction, Selaginellaceae gives us a unique opportunity to study distribution patterns shaped by mil-lions of years of evolution, different geological events, and ecological fac-tors. In addition, Selaginellaceae has a good fossil record, a prerequisite for getting good estimates on divergence times.

How can the worldwide distribution patterns seen in Selaginellaceae to-day be explained? Are Selaginella species, due to their large megaspores, “bad dispersers” as often suggested?

With the inclusive plastid rbcL data set (from Paper I) as a basis, a diver-gence time estimation analysis was performed. The resulting tree corre-sponds well to the phylogeny presented in Paper I and shows a Late Devoni-an (364 Ma) origin of the Selaginellaceae lineage, i.e., an age even predating the formation of the supercontinent Pangea (Fig. 6). Our subsequent histori-cal biogeographical analysis indicates that, despite the ambiguous ancestral range estimates for the early divergences in the phylogeny, most of these early cladogenetic events occurred on Pangea, and a few also predate Pan-gea. Later range expansions can most probably be correlated to both the breakup of Pangea, and the subsequent breakup of Gondwana, during the Mesozoic (Fig. 6). That is, vicariance may be responsible for the observed patterns. Later cladogenetic events are possible to explain by migration across landmasses, mainly from Southeast Asia into e.g., India and Eurasia. The most surprising result, however, are the many (at least fifteen) long-

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distance (transoceanic) dispersals seen in the family in more “recent” Ceno-zoic times, approximately the last 50 million years (Fig. 6). The majority of the long-distance dispersals are seen in the large clade corresponding to subg. Stachygynandrum, and primarily in the mainly Southeast Asian sub-clades. Transoceanic dispersal events are inferred to have occurred from Southeast Asia to especially Africa, but also to the Pacific and South Ameri-ca.

Our results on the historical biogeography of Selaginellaceae change our view of what plants can disperse over long distances, and indicate that fur-ther studies on the dispersal and reproduction biology of Selaginella are highly needed to increase our understanding of this old heterosporous plant group.

Figure 6. Time scale showing eras (lower row) and periods (upper row) from 350 million years ago (Ma) until today. P, Pangea; G, Gondwana. Dashed lines illustrate the start of the breakup of the supercontinents. The solid globe with a P is placed to illustrate when Pangea had its maximum. The time scale follows Walker et al. (2012).

350 200250300 050100150Mesozoic CenozoicPaleozoic

Carboniferous Permian Triassic Jurassic Cretaceous Paleogene Neo.

Ma

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5. Concluding remarks

In this thesis, I discuss the systematics and historical biogeography of the lycophyte family Selaginellaceae, a historically important plant group dating back some 350 Ma. In four papers, I, together with colleagues, have ex-plored various aspects of the evolutionary history of this heterosporous group, with a worldwide extant species distribution.

Based on DNA sequence data, we present a well-founded hypothesis of the evolutionary relationships of Selaginellaceae, covering one-third of the species richness in the family (Paper I). Despite the morphological com-plexity of the group, thorough morphological studies show that all the major clades in the phylogeny can be uniquely diagnosed by suites of morphologi-cal features. There is value in morphology after all!

We propose a new subgeneric classification of Selaginella, including sev-en subgenera, based on phylogenetic relationships (Paper II). The subgenera can be identified by gross morphological features, and our intention is that the classification should be useful to a broad audience.

Following the ICN, we resurrect the name S. pectinata Spring for the Madagascan Selaginella species that was erroneously renamed S. polymor-pha Badré in the late 1990s (Paper III).

A historical biogeographical analysis, based on a time-calibrated phylog-eny and extant species distribution data, indicates that Selaginellaceae shows pre-Pangean diversification patterns, Gondwanan vicariance, and Cenozoic long-distance dispersals (Paper IV). Since Selaginella species, due to their large megaspores, have been considered “bad dispersers”, the large number of long-distance dispersals reconstructed are surprising, and gives us new insights about the evolutionary history of spore dispersed plants.

The work presented in this thesis forms a firm basis for future studies on Selaginellaceae. Work needed involves rigorous organismal studies includ-ing alpha-taxonomical work with a phylogenetic approach, comparative morphological studies on e.g., spore and stelar morphology, and a better understanding of Selaginella reproduction and dispersal biology in general. Further, the large historical biogeographical patterns, spanning hundreds of millions of years, should be compared to studies on other scales in time and space, e.g., of Southeast Asia where the geological history involves exten-sive land mass changes, or at the phylogeographical level.

Overall, there is a lot to gain from studying the evolutionary patterns of spikemosses.

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6. Svensk sammanfattning

Efter att ha jobbat med en avhandling i flera år är det lätt att fastna i detaljer. För mig har det varit viktigt att hela tiden ta ett steg tillbaka och reflektera över vad jag egentligen håller på med. Ett utmanande sätt att göra det på har varit att i olika sammanhang försöka förklara min forskning på ett så kortfat-tat sätt som möjligt. I den här sammanfattningen kan du välja hur mycket just du vill läsa om mitt doktorandprojekt om mosslummermönster.

6.1 Jättekort Mosslumrar är en 350 miljoner år gammal grupp av lummerväxter som idag finns i hela världen. I mitt doktorandprojekt har jag släktforskat på de här växterna för att förstå hur deras släkthistoria ser ut. Jag har bland annat kommit fram till att de har fått sin nutida världsomspännande utbredning genom att både ”lifta” med kontinenter (när superkontinenterna Pangea och senare Gondwana bröts upp för flera hundra miljoner år sedan) och genom att under de senaste 50 miljoner åren sprida sig över världshaven.

6.2 Kort Mosslumrar, växtfamiljen Selaginellaceae, hör till lummerväxterna. Gruppen har en lång evolutionär historia (utvecklingshistoria) som går tillbaka så långt i tiden som 350 miljoner år, en tid då jorden var långt ifrån lik hur den ser ut idag. Trots att de heter moss-lumrar så är mosslumrarna inte särskilt nära släkt med mossor. Istället är de närmare släkt med både ormbunksväx-ter (t.ex. stensöta) och fröväxter (t.ex. tall och tulpan, Fig. 1, s. 13). Moss-lumrarna är örtartade i sitt växtsätt (aldrig buskar eller träd) och finns fram-förallt i fuktiga tropiska områden, men arten dvärglummer växer i Sverige (Fig. 2, s. 17).

I mitt doktorandprojekt har jag undersökt mosslumrarnas släkthistoria. Jag har varit intresserad av hur olika mosslummerarter är släkt med varan-dra, om olika grupper av mosslumrar går att känna igen med hjälp av morfo-logiska (utseendemässiga) karaktärer och vilka processer och händelser som ligger bakom gruppens idag världsomspännande utbredning. Projektet har gjorts inom forskningsfältet växtsystematik. Som systematiker är jag intres-

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serad av att kartlägga den biologiska mångfalden vi ser idag och dess evolut-ionära historia.

Jag har analyserat mosslumrarnas släktskap med hjälp av DNA-sekvens-data (delar av arvsmassan). Ju mer av DNA-sekvenserna man delar via en gemensam förfader, desto närmare släkt är man. Det resulterande släktskaps-trädet är en väl underbyggd hypotes över släktskapet mellan olika moss-lummerarter (Fig. 5, s. 31). I släktskapsträdet ser vi flera tydliga mosslum-mergrupper som var och en kan skiljas ut genom en unik kombination av morfologiska karaktärer. Med detta som grund presenterar vi en ny klassifi-kation av mosslumrarna – vi har satt namn på de mosslummergrupper som vi kan urskilja för att få bättre ordning på alla de 750 mosslummerarter som finns i världen. Med släktskapsträdet som bas har jag också undersökt hur mosslumrarna idag kan finnas i hela världen. Hur har de fått en sådan stor utbredning? Jag har bland annat kommit fram till att de har ”liftat” med kon-tinenter när superkontinenterna Pangea och senare Gondwana bröts upp, men att de också har spridit sig över världshaven på ”senare” tid (de senaste 50 miljoner åren).

6.3 Lång Den här avhandlingen handlar om en grupp lummerväxter som på svenska kallas mosslumrar, men vars vetenskapliga namn är Selaginellaceae. Det övergripande målet med avhandlingen har varit att reda ut mosslumrarnas släkthistoria, så man kan säga att jag har släktforskat på mosslumrar. Min forskning har gjorts inom forskningsfältet växtsystematik. Här nedanför berättar jag mer om vad systematik är och varför det är viktigt, hur du känner igen en mosslummer och lite mer specifikt om vad de fyra artiklarna som ingår i min avhandling handlar om.

6.3.1 Systematik Att kategorisera och systematisera det vi ser omkring oss har människan hållit på med i alla tider. Detta är såklart inte alltid positivt, särskilt inte om olika kategorier anses vara olika mycket värda. Men det finns också mycket bra att få ut av systematik, inte minst när det kommer till växter! Systemati-ker studerar den biologiska mångfalden som finns på jorden idag och följer dess evolutionära historia (utvecklingshistoria) tillbaka i tiden. Vi beskriver inte bara det vi ser utan analyserar också evolutionära mönster och undersö-ker processerna som har format dessa mönster.

Ett sätt att tänka på den biologiska mångfalden och dess evolutionära historia är som ett stort träd där lövverket representerar nutiden med de nu levande arterna och grenarna visar hur allt hänger ihop och är släkt med varandra. Om vi vill förstå den evolutionära historien behöver vi studera

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grenarna. Problemet är att det enda vi ser av trädet är löven, det som lever idag. Vi kan inte se den evolutionära historien. Systematiker undersöker både trädets lövverk och grenverk.

Om vi inte har namn på växterna i naturen blir allt som en stor grön massa. För mig är det ett värde i sig att veta vilka organismer jag stöter på under min skogspromenad. Det blir som att möta gamla bekanta. Systemati-ken ger oss dessutom historien om hur olika organismer har evolverat och hur de hänger ihop i ett ”livets släktskapsträd”. Ett tydligt exempel rör gif-tiga växter. Om vi vet att en växt är giftig är det mycket troligt att dess närmaste släktingar också är giftiga – det giftiga är en egenskap som de har ärvt tillsammans från en förfader. Att veta vilka organismer vi har omkring oss är dessutom viktigt ur ett bevarandeperspektiv. Om vi inte vet vilka arter som finns i naturen, hur ska vi då kunna ta väl genomtänkta beslut om att skydda dem? Systematik är också grunden för många andra forskningsområ-den där det är avgörande att placera arbetet i ett evolutionärt ramverk.

Som systematiker använder jag information om de organismer som lever idag för att göra släktskapsanalyser och ta fram väl underbyggda hypoteser om organismers evolutionära historia. Grunden för släktskapsanalyserna är idag nästan alltid DNA-sekvensdata (arvsmassa). När jag sedan utvärderar det resulterande släktskapsträdet (fylogenin) letar jag efter monofyletiska grupper, d.v.s. grupper där alla ingående organismer härstammar från en och samma förmoder. Systematiker vill bygga evolutionära hypoteser utifrån gemensamt ursprung.

Dagens levande arter delar gemensamma förfäder och i slutändan har allt liv ett gemensamt ursprung. Ett felaktigt tankesätt om evolution som alltför ofta figurerar är en linjär utvecklingshistoria som antyder att evolutionen har en riktning och ett mål. Det klassiska exemplet är bilden av en människa som, med några mellansteg, utvecklats från en schimpans. Men vi har inte utvecklats från schimpansen! Schimpans (och dvärgschimpans) och männi-ska har en gemensam förmoder som varken var schimpans eller människa.

6.3.2 Mosslumrar Mosslumrarna tillhör växtgruppen lummerväxter (lumrar) och är alltså inte särskilt nära släkt med mossor. Lumrarna delas in i tre växtfamiljer: moss-lumrar (Selaginellaceae), braxengräs (Isoëtaceae) och de vanliga lumrarna (Lycopodiaceae), alla med ett fåtal representanter i Sverige (Fig. 1, s. 13). Dvärglummer (Selaginella selaginoides) är den enda art av mosslummer som finns i Sverige. Den ser nästan ut som en liten mossa och växer fram-förallt i de norra delarna av landet (Fig. 2A, s. 17). Braxengräsen är moss-lumrarnas närmaste nu levande släktingar. De är vattenlevande och sitter som små rosetter med sylformade blad på botten av sjöar och vattendrag. Den vanliga lummern är kanske den som flest har ett förhållande till. Sedan gamla tider har det t.ex. varit vanligt att ha revor av matt- eller revlummer

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som prydnad till jul. Idag ska man däremot undvika att plocka lumrar då de växer långsamt och är hotade till följd av bl.a. skogsbruk.

Förutom dvärglummer som växer vilt i Sverige kan några av de andra mosslummerarterna ibland dyka upp i handeln. Den sydamerikanska Sela-ginella martensii sitter ibland i julgrupper och Selaginella lepidophylla är en rolig torktålig art från sydvästra USA och Mexiko som i torrt tillstånd är hoprullad till en boll, men som sakta ”slår ut” när den blir fuktig. Ibland kallas den för uppståndelseväxt eller Jerikoros (Fig. 2B, s. 17).

Eftersom lummerväxter sprider sig med sporer, och har kärlsträngar (led-ningsvävnad), trodde man länge att de var nära släkt med ormbunksväxter (t.ex. stensöta). Senare studier har däremot visat att ormbunksväxter och fröväxter (t.ex. tall och tulpan) är varandras närmaste släktingar och att lummerväxterna är lika nära släkt med ormbunksväxter som med fröväxter (Fig. 1, s. 13).

Idag finns det ca. 1300 arter av lummerväxter, 750 av dem är mosslumrar. Lummerväxterna utgör alltså bara en liten del av de ca. 300 000 levande landväxtarter som vi känner till idag, varav den största delen, ca. 250 000 arter, är blomväxter. För ungefär 300 miljoner år sedan, under karbon, var det däremot annorlunda. Då var lummerväxterna en dominerande landväxt-grupp och det fanns till och med lummerträd höga som dagens granar. Dessa förhistoriska lummerträd utgör en betydande del av vårt sinande kollager och det är därför karbontiden ibland kallas för kolets tidsålder. Det äldsta fossilet som har hittats av en mosslummer är ca. 345 miljoner gammalt. Det kanske mest fascinerande med detta fossil är att det utseendemässigt liknar dagens mosslumrar, trots att över 300 miljoner år av evolution har passerat.

Idag finns det bara små lummerväxter. Alla mosslumrar är örtartade (de är aldrig buskar eller träd) och det allra vanligaste är att de har ett krypande växtsätt (Fig. 2F, s. 17) eller en upprätt stam som förgrenar sig en bit ovanför marken (Fig. 2E, G, s. 17). De flesta är anpassade till ett varmt och fuktigt regnskogsklimat. Ett utseende som återfinns hos majoriteten av alla moss-lumrar är att ha blad som sitter i fyra rader: två rader med mindre blad på ovansidan av skottet och två rader med större blad på undersidan (Fig. 2C och 3A, s. 17 och 19).

Som tidigare nämnts sprider sig mosslumrar med sporer (se Fig. 3, s. 19, för en skiss över mosslumrarnas livscykel). Det här är en stor skillnad från fröväxter (bl.a. blomväxter) som sprider sig med frön. Ett frö innehåller ett litet växtembryo som växer upp till en ny planta. En spor däremot innehåller inget embryo. Istället utvecklas en spor till något som kallas för gametofyt. Mosslumrar har två sorters sporer som båda behövs för fungerande repro-duktion: ”stora” megasporer (0,2–1,2 mm i diameter) och små mikrosporer (ca. 0,03 mm). Inuti megasporen utvecklas en megagametofyt med speciella strukturer som innehåller ägg, och inuti mikrosporen utvecklas en mikroga-metofyt med spermier. När sporerna är ”mogna” öppnar de sig och om det finns vatten kan spermierna från den ena sporen simma till äggen i den andra

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sortens spor och befruktning kan ske. Vid lyckad befruktning utvecklas ett embryo som kan växa upp till en ny mosslummerplanta (Fig. 3, s. 19).

Mosslumrar är intressanta att studera ur ett evolutionärt perspektiv av flera olika anledningar. Gruppens höga ålder (minst 350 miljoner år) skapar unika möjligheter att skaffa djupare förståelse för evolutionen hos de tidig-aste kärlväxterna. Att mosslumrarna dessutom behöver två olika sorters spo-rer för fungerande reproduktion är ytterligare en intressant aspekt som gör att de skiljer sig från de flesta andra landväxtgrupper.

6.3.3 Avhandlingens fyra artiklar Mitt avhandlingsarbete är uppdelat i fyra olika delarbeten/artiklar. Här be-skriver jag innehållet i var och en av dem och hur de hänger ihop med varandra.

Grunden för mitt arbete är ett släktskapsträd över mosslumrarna baserat på DNA-sekvensdata. För att kunna ta fram en väl underbyggd hypotes över mosslumrarnas släktskap behövde jag DNA från så många olika mosslum-merarter som möjligt. Förutom att åka runt i världen för att själv samla in växtmaterial (Fig. 4, s. 25) har jag haft enorm nytta av herbariematerial som en lång rad forskare före mig har samlat. Ett herbarium är som ett växtmu-seum/arkiv med torkade pressade växter, alltifrån flera hundra år gamla till mer nutida. När jag samlade mosslumrar till mitt projekt var jag noga med att få ett så representativt urval av arter som möjligt. Jag lade särskilt stor vikt vid att ta med arter från Afrika och Stillahavsöar, områden som har sak-nats i tidigare mosslummerstudier. Jag extraherade fram DNA ur de olika mosslumrarna och ”klippte ut” samma tre DNA-bitar (genetiska markörer) från alla inkluderade arter. DNA-sekvenserna jämfördes sedan med varandra och genom analyser med avancerade datoralgoritmer, som använder kunskap om hur DNA-sekvenser evolverar, fick jag fram en hypotes för ett släkt-skapsträd.

I artikel I presenterar vi ett stort släktskapsträd över mosslumrarna baserat på DNA-sekvensdata. Studien täcker in ca. en tredjedel av alla kända moss-lummerarter. Vi studerade också morfologin hos alla inkluderade arter. Föru-tom att ha tagit fram en väl underbyggd hypotes över mosslumrarnas evolut-ionära släktskap, visar vi att morfologin i gruppen är mer komplex än vad man tidigare har trott. Trots detta ser vi att de större grupperna i släktskaps-trädet går att skilja från varandra med hjälp av unika kombinationer av mor-fologiska karaktärer.

Systematiker klassificerar naturen enligt ett hierarkiskt system där en art ingår i ett släkte, som ingår i en familj, som ingår i en ordning, osv. Det är viktigt att komma ihåg att all kategorisering av naturen är ett mänskligt på-fund. Det finns inga ”riktiga” växtfamiljer eller växtsläkten ute i skogen, det

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som finns är en massa organismer som är mer eller mindre nära släkt med varandra. Hur gränserna ska dras, och grupperingar göras, är godtyckligt och beror framförallt på vad forskare tycker är lämpligt och användbart. Vad som alla däremot mer eller mindre är överens om är att alla indelningar som görs bör spegla evolutionära släktskap, d.v.s. motsvara grupper med ett ge-mensamt ursprung (monofyletiska grupper).

I mosslummerfamiljen Selaginellaceae ingår bara ett släkte: Selaginella. Vanligtvis ingår flera släkten i en familj, men i fallet med Selaginellaceae anser vi forskare som jobbar med mosslumrar att det är bäst att ha ett släkte. Däremot är det opraktiskt att inte ha någon ytterligare indelning av gruppen eftersom så många som 750 arter ingår i släktet Selaginella. Framförallt blir det svårt att referera till olika artgrupper om man inte har ytterligare katego-rier. I artikel II utgår vi därför från släktskapsträdet och de morfologiska studierna som vi presenterar i artikel I och föreslår en ny undersläktesklassi-fikation av släktet Selaginella. Vår klassifikation omfattar sju undersläkten och alla går att känna igen med en unik kombination av morfologiska karak-tärer, varav de flesta går att se med blotta ögat eller med en handlupp.

När vi arbetade med artikel I upptäckte vi att en av de mosslummerarter som finns på Madagaskar hade ett felaktigt vetenskapligt namn. I artikel III går vi igenom nomenklaturen (namnreglerna) för den här arten och konstaterar att det korrekta namnet är Selaginella pectinata Spring, där Spring står för den franska botanisten Antoine Frédéric Spring som gav arten namnet redan år 1843.

Ett sätt att använda “systematisk kunskap” är att ställa vidare frågor om den evolutionära historien hos en organismgrupp. I artikel IV använder vi släkt-skapsträdet över mosslumrarna som presenteras i artikel I och undersöker den historiska biogeografin för gruppen. Historisk biogeografi handlar om att i ett historiskt perspektiv studera relationer mellan olika arter och deras geografiska utbredning. Varför finns olika mosslummerarter där de finns idag? Hur har de hamnat där? Idag finns det mosslumrar i hela världen och genom att lägga till både tid och rum till släktskapsträdet så kan vi ta fram hypoteser om hur mosslumrarna har spridits. Tidsaspekten får vi genom att tidskalibrera vårt släktskapsträd med hjälp av fossil av känd ålder. Rums-aspekten fås genom att ta hänsyn till den geografiska utbredningen hos da-gens mosslummerarter. Genom att göra en historisk biogeografianalys kan vi sedan uppskatta vilken geografisk utbredning förfäder till dagens mosslum-merarter förmodligen hade. Tolkningar av våra analyser visar att mosslum-rarna har fått sin nutida världsvida utbredning genom att både ”lifta” med kontinenter (när superkontinenterna Pangea och senare Gondwana bröts upp) och genom flera åtskilda långdistansspridningar, d.v.s. att de har spridits över långa avstånd (framförallt över världshaven), under de senaste 50 mil-joner åren. Att mosslumrarna har spridits ett flertal gånger (minst femton!)

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över världshaven under senare tid är häpnadsväckande med tanke på deras stora megasporer som tros ha en negativ effekt på spridningsförmågan.

Sammanfattningsvis presenterar jag i den här avhandlingen en väl under-byggd hypotes för mosslumrarnas evolutionära historia, en historia som inte bara lär oss mer om mosslumrar, utan som också ger oss en bättre förståelse för hur de tidigaste landväxterna kan ha utvecklats och spridits.

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7. Acknowledgements

Från djupet av mitt hjärta: den största kramen och det största tacket till Petra, världens bästa handledare! Varje gång jag ser dig blir jag glad och efter att ha pratat med dig känns allting alltid bättre. Tack för att du är en sådan klok och fin människa, för att jag har fått vara din doktorand, för att du alltid har haft tid för mig, för att du har fått mig att växa, för alla diskussioner vi har haft om forskning, undervisning och livet, för ditt engagemang i alla mina tredje uppgiften-projekt och för alla roliga resor vi har gjort tillsammans. Jag ser fram emot många fortsatta gemensamma projekt!

Thanks to Sandie for all your support and for always believing in me. You really got me hooked on systematics with an inspiring Master’s project!

Tack Agneta för all värme, för att jag alltid kan gå till dig om jag behöver en kram, för allt kreativt och för allt du har hjälpt mig med på biblioteket. Tack Afsaneh för all din omtanke och för allt du har lärt mig om labbarbete. Det blev så tomt när du slutade på avdelningen! Tack Nahid för alla kloka och stöttande ord, för allt du har lärt mig på labbet och för alla goda maträt-ter du har tipsat mig om.

Thanks to all senior researchers that have helped and inspired me. Tack Mikael för allt ditt stöd, för alla goda råd och för att du i alla lägen har varit ett sådant bra bollplank. Tack Mats för ditt otroliga engagemang i mitt klass-ifikationsarbete, för allt du har lärt mig om nomenklatur och växter och för att du sprider ett tryggt lugn omkring dig. Tack Magnus för din omtanke, ditt engagemang i allt jag gör och all hjälp med manuskriptläsning. Du inspire-rar! Tack Martin för goda råd, kommentarer på avhandlingen och för alla fikastunder. Tack Åsa för att du aldrig har sagt nej till en kopp te och för den fina fruktskålen vi hade tillsammans. Tack Inga för ditt stöd och för trevliga samtal. Tack Bertil för en bra kurs i Ecuador. Tack Leif för allt du lärde mig om svampar och lavar under VEM-kursen. Tack Sanja för dina uppmunt-rande ord. Thanks to David for your support. Tack Hanna för att du sprider en sådan bra stämning omkring dig. Thanks to Baset for all the past and coming badminton games. Thanks to Paco for good collaboration with the RAD project. Tack Catarina för att du lånade material åt mig på herbariet i Bryssel. Tack Mariette för din uppmuntran kring mina tredje uppgiften-projekt och för alla bra böcker jag har fått av dig. Tack Katarina för att du alltid intresserar dig för vad jag gör. Tack Kristiina för alla trevliga lunch-samtal. Thanks to Jenni for your endless support! Thanks to Maria R for all your help during my Master’s project. Tack Alex A för att du inspirerar och

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för allt du har lärt mig om att samla växter. Tack Annika, Elisabeth och Per för all rolig floristikundervisning vi har gjort tillsammans. Thanks also to Thomas, Stefan, Anushree, Chengjie, Fabien, Sara, Mohammad, Eric, Aaron, Cécile, Ruxandra, Diem, Petr, and Omar for contributing to an inspi-ring working environment.

Thanks to Alan Smith for the good collaboration on the Selaginella pecti-nata project and for fruitful discussions on Selaginella in general.

Thanks to all my fellow PhD students during the years! Tack Anders L för allt du har lärt mig, för dina peppande ord, för alla bra program jag har fått från dig och för all rolig undervisning vi har gjort tillsammans. Tack John för att du alltid finns där för att svara på mina frågor, för dina klack-ande skor i korridoren och för att du alltid tittar in på mitt kontor för att se hur jag har det. Thanks to Allison for being such a great inspiration and for always supporting me. Tack Anneleen för all undervisningsinspiration. Also a big collective thanks to Allison and Anneleen for introducing me to to-matoes and efficiency excursions. I have grown a tomato farm by now and the efficiency excursions really have saved me during the last years of my PhD. Tack Sarina för allt ditt stöd, alla fikastunder och alla roliga badmin-tonmatcher. Thanks to Sanea for always cheering me up, for all the chats, and for having a GIF for all moments. Tack Karin S för din omtänksamhet, för all labbhjälp med dvärglummerprojektet och för allt trevligt umgänge. Thanks to Astrid for being such a great office mate; it got so empty when you left. Thanks to Raquel for good company, cookies, and for bringing good coffee to the lunch room. Tack Julia för att du är en så bra rumskompis som pratar om annat än bara jobb. Tack Henrik för din hjälpsamhet och för att du beställer så många tokiga grejer till vårt rum. Tack Hugo för allt du har lärt mig om att söka stipendier. Thanks to Ding for your enthusiasm and your support. Tack Maria L för din omtanke när jag var ny på avdelningen. Tack Anders R för ett superbt samarbete under vår premiär som floristiklä-rare. Thanks to Ping for your support. Thanks to Brendan, Jesper, Ioana, Lore, Veera, Roel, Stella, Juma, Vichith, and Chanda. It has been great to be a PhD student together with you!

Tack Anneli S för att du alltid sticker in huvudet hos mig och engagerar dig i mina projekt. Det är jätteroligt att du också ska jobba med Selaginella nu. Thanks also to Isabel for your support.

Tack till alla studenter som jag har haft under åren. Ni har varit en stor in-spirationskälla.

Tack Irene för ditt stöd och för allt ditt arbete som prefekt. Tack Karin L, Annette, Iva, Tove, Emma och Signe för all hjälp under åren och för alla trevliga luncher. Tack alla på intendenturen för ert arbete. Tack alla på IBG för ett bra samarbete med undervisningen. Tack alla herbarier som har lånat ut material till mig: A, BR, CNS, DUKE, GH, L, MEL, NSW, P, S, U och UPS. Ett särskilt tack till Mats, Monika och alla andra på herbariet i Uppsala

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som har hjälpt mig så bra. Thanks also to all of you that have sent me Selaginella specimens from all around the world.

Tack till mina fantastiska fältassistenter som har åkt med mig hundratals mil för att samla dvärglummer i myggrika trakter: Anders J, Johanna, Maja och Anders R. Jag älskar ju att samla och jag hoppas att ni också tyckte det var roligt. Thanks to Emma and Nathalie for all the help with the field work in Queensland. I suppose we will never forget that eye-loving leech.

Tack Charlotte för att du coachade mig så bra inför Forskar Grand Prix och för alla roliga projekt vi har gjort efter det. Tack Teknat Samverkan för alla tredje uppgiften-projekt som ni har involverat mig i under åren. Tack Anders och Fabian för ett roligt boksamarbete. Jag ser fram emot slutspur-ten.

Jag är mycket tacksam för allt finansiellt stöd som jag har fått genom åren: Helge Ax:son Johnsons stiftelse, Stiftelsen Lars Hiertas minne, Stiftel-sen Extensus, Linnéska stipendiestiftelsen, Liljewalchs stiftelse, Knigges stiftelse, Sernanders stiftelse, Anna Maria Lundins stipendiefond, K.V. Os-sian Dahlgrens botaniska stipendiefond, Stiftelsen Bergmangårdarna på Fårö, Anders Karitz stiftelse och Lennanders stiftelse.

Tack Tore för att du sa till mig på gymnasiet att du trodde att jag skulle tycka om att forska, jag hade kanske inte kommit på det själv.

Tack till alla mina fina vänner utanför jobbet i Uppsala. Tack Johanna, Henning och Ture för all er värme och omtanke och för att det är så kul att ha projekt ihop med er. Tack Maja E för att det alltid känns så bra att vara med dig och för att vi alltid har något härligt TV-program att titta på. Tack Fredrika och Emil för allt mys och för att ni alltid har en ledig gästsäng och en tandborste till mig trots att vi bor tre kilometer från varandra. Tack Hjal-mar för din omtanke, mysiga naturutflykter och alla goda mustiga grytor. Tack Fia, Aaron och Idunn för att ni är sådana bra grannar och för all god mat och gott kaffe. Tack Johan S för glada tillrop. Tack Lovisa W för trev-liga mat- och bakkvällar och för dina peppande ord. Tack Klara och Joel för skönt filmhäng. Tack Marianne, Maja B, Maja E, Johanna och Anders för att ni gör vår bokcirkel så trevlig.

Tack till alla mina fina vänner utanför Uppsala. Tack Karin M för att du alltid finns nära mig trots att du har varit långt borta, för att du alltid känner på dig när jag behöver en sms-kram och för att du och jag har delat världens bästa cyberkontor. Tack Axel för att du håller tipsen ständigt flödande i chat-ten. Tack Angelica för att det är så lätt att hänga med dig. Tack Anneli J för världens bästa arbetsvecka på Österlen och för allt annat mysigt och roligt. Tack Elisabet och Johan för allt trevligt häng och för att ni stöttade mig så bra under de sista stressiga veckorna med avhandlingen. Tack Lovisa G för ditt stöd. Thanks to Emma for all your energy and support. Tack Lena för all värme du utstrålar. Tack Anders R och Martin för alla roliga växtutflykter i

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när och fjärran. Tack familjen Jakobsson för att ni alltid finns där och bryr er om mig.

En stor kram till alla mina roliga fina lekkamrater som har hjälpt mig att koppla av under avhandlingsarbetet och tagit med mig till fantasins värld där jag trivs så bra: Björn, Maj, Pelle, Lucas, Nils, Ture, Julius, Mira och Idunn. Nu ska vi leka ännu mer!

Till sist en stor stor kram och puss till hela min familj som jag älskar så mycket! Tack för allt allt allt! En extra stor puss till Anders som alltid finns där med sina kloka tankar, lösningar och lugnande ord. Utan dig hade jag inte varit där jag är idag.

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8. References

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