Spikemoss patterns. Systematics and historical biogeography of Selaginellaceae

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1401 Spikemoss patterns Systematics and historical biogeography of Selaginellaceae STINA WESTSTRAND ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2016 ISSN 1651-6214 ISBN 978-91-554-9647-0 urn: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 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Professor Michael J. Donoghue (Department of Ecology and Evolutionary Biology, Yale University). Abstract Weststrand, S. 2016. Spikemoss patterns. Systematics and historical biogeography of Selaginellaceae. (Mosslummermönster. Systematik och historisk biogeografi hos Selaginellaceae). Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty 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. With an estimated age of some 350 million years, the family is historically important as one of the oldest known groups of vascular plants. Selaginellaceae is herbaceous with a worldwide distribution. However, the majority of the ca. 750 species in the single genus Selaginella are found 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 (plastid and single-copy nuclear) of one-third of the species richness in the group. Attention was paid to cover the previously undersampled taxonomic, morphological, and geographical diversity. Morphological features were studied and mapped onto the phylogeny. The results show an overall well-supported phylogeny and even more complex morphological patterns than previously reported. Despite this, many clades can be distinguished by unique suites of morphological features. With the phylogeny as a basis, together with the thorough morphological studies, a new subgeneric classification with seven subgenera, representing strongly supported monophyletic groups, is presented for Selaginella. By mainly using gross morphological features, easily studied by the naked eye or with a hand lens, the intention is that the classification should be useful to a broader audience. During the work with species determinations, it was revealed that the correct name for an endemic Madagascan Selaginella species is S. pectinata Spring, not S. polymorpha Badré as previously proposed. The robust phylogeny of Selaginellaceae allowed for a historical biogeographical analysis of the group. A time-calibrated phylogeny, together with extant species distribution data, formed the basis. The results show pre-pangean diversification patterns, Gondwanan vicariance, and more recent Cenozoic long-distance dispersals. The many inferred transoceanic dispersals during the last 50 million years are surprising considering Selaginella s large megaspores that are thought to have a negative effect on dispersal. In conclusion, this thesis presents a well-founded hypothesis of the evolutionary history of Selaginellaceae including its phylogeny, morphology, and historical biogeography. The thesis forms a firm basis for further studies on Selaginellaceae in particular, and gives us a better understanding 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 1651-6214 ISBN 978-91-554-9647-0 urn: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 heterosporous Selaginellaceae: A tale of pre-pangean diversifications, Gondwanan vicariance and Cenozoic long-distance dispersals. Manuscript. Paper III was reproduced with kind permission from the publisher. Note. To make clear that the nomenclatural novelties in Paper II are not validly published in this thesis, references to the place of publication of basionyms or replaced synonyms have been omitted.

Contents Preface... 9 1. Introduction... 11 1.1 Systematics... 11 1.2 Historical biogeography... 14 1.3 Selaginellaceae... 15 1.3.1 Morphology and reproduction... 18 1.3.2 Systematics... 20 1.3.3 Why study Selaginellaceae?... 21 2. Aims... 23 3. Materials and methods... 24 3.1 Studies of organisms... 24 3.2 Phylogenetic analyses... 26 3.3 Classification... 27 3.4 Historical biogeography... 27 4. Results and discussion... 29 4.1 Paper I and II: Phylogeny and subgeneric classification of Selaginella... 29 4.2 Paper III: Selaginella pectinata resurrected... 32 4.3 Paper IV: Historical biogeography of Selaginella... 32 5. Concluding remarks... 34 6. Svensk sammanfattning... 35 6.1 Jättekort... 35 6.2 Kort... 35 6.3 Lång... 36 6.3.1 Systematik... 36 6.3.2 Mosslumrar... 37 6.3.3 Avhandlingens fyra artiklar... 39 7. Acknowledgements... 42 8. References... 46

Abbreviations DNA ICN Ma nom. nov. PCR sp./spp. sp. nov. subg. deoxyribonucleic acid International Code of Nomenclature for algae, fungi, and plants million years ago nomen novum (new name) polymerase chain reaction species (singular/plural) species nova (new species) subgenus

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 biogeographical analyses. In this thesis, I present my work in four papers, all describing different aspects of the evolutionary patterns of spikemosses. 9

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 biogeography 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, especially not when some categories are viewed as worth more than others. However, 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 poisonous one. However, the notion of evolution, in contrast to the idea of the 11

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, especially 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 scientific 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 relationships. With a basis in the theoretical framework of phylogenetic systematics 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 phylogenies (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 systematics, and today molecular markers are the primary basis for phylogenetic analysis. Hypotheses about evolutionary relationships are continuously being evaluated 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 hypotheses 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 concerning 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 12

lycophytes vascular plants hornworts liverworts mosses spikemosses (Selaginellaceae) quillworts (Isoëtaceae) clubmosses (Lycopodiaceae) seed plants ferns Figure 1. Land plant phylogeny showing the major lineages of land plant groups: hornworts, liverworts, mosses, lycophytes, ferns, and seed plants.

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 evolutionary 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 evolutionary relationships, and the recognised units are monophyletic groups. However, how groups should be delimited is fairly arbitrary. Categories are human 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 depends 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 protect. 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 evolutionary 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 biogeography. Historical biogeography deals with larger time spans (often millions of 14

years) and evolutionary patterns on, at least, the level of species. Ecological biogeography deals with much shorter time spans and usually at the population 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 evolution 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 separate 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 longdistance 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 methodologies 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 hypothesis 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 lycopods) and not mosses even though the name erroneously implies so. Selaginellaceae is an herbaceous, worldwide family, but most of the ca. 750 species are found in the tropics and subtropics (Jermy, 1990). Some 10% of the 15

species are growing in temperate regions, but arctic and subarctic representatives are rare. One species, Selaginella selaginoides (L.) P.Beauv. ex Schrank & 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. selaginoides 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 example is the commercially sold S. lepidophylla (Hook. & Grev.) Spring (Fig. 2B). Selaginellaceae is one of three extant lineages of lycophytes, the other being Isoëtaceae Rchb. and Lycopodiaceae P.Beauv. ex Mirb. (Jermy and Øllgard, 1990; Fig. 1). Isoëtaceae, the quillworts, is the sister lineage to Selaginellaceae (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, Lycopodiaceae, 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, molecular 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 dating back to the Late Silurian some 420 Ma (Kenrick and Crane, 1997). Extinct 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 Selaginellaceae lineage is Selaginellites resimus N.P.Rowe, an isophyllous lycophyte 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). 16

& # ' $! % " Figure 2. A diverse collection of Selaginella species: (A) S. selaginoides (circumboreal), (B) S. lepidophylla (Southwest USA Mexico), (C) S. longipinna Warb. (Australia), (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). 17

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). However, these characters are definitely not the ones you may easily notice when out in the field. Here follows a description of the more apparent morphological features and their variability in the family. As mentioned above, all Selaginella species are herbaceous, but the general 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 common 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 monomorphic 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, respectively) 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 Hawaiian 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 strobili 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 anisophyllous strobili (bilateral strobili; Jermy, 1990). These can either be resupinate, 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 considerably in morphology, and several studies have shown a mosaic of morphological complexity (e.g., Minaki, 1984; Morbelli et al., 2001; Korall and Taylor, 2006; Zhou et al., 2015b). Another morphological feature traditionally 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 18

microsporangium with microspores microspore (n) sporophyll strobilus megasporangium with megaspores mature microgametophyte B A meiosis in sporangia sperms (n) megaspore (n) C rhizophore E mature megagametophyte protruding from within the megaspore wall D mature sporophyte (2n) young sporophyte attached to megaspore zygote (2n) archegonium with egg (n) water fertilisation Figure 3. Life cycle of Selaginellaceae: (A) mature sporophyte with anisophyllous shoots bearing vegetative leaves in four rows, rhizophores, and isophyllous, tetrastichous strobili, (B) strobilus showing mega- and microsporangia (where meiosis occurs), (C) maturing mega- and microspores, (D) fertilisation, and (E) young sporophyte. Inspiration taken from Evert and Eichhorn (2013).

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 vegetative 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 megagametophyte 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 selffertilisation in Selaginella (see one example in e.g., Valdespino, 1993)? How are Selaginella spores dispersed? Can a megaspore and a microspore be dispersed 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 Selaginella 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 classification 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 20

recent classifications, the one by Jermy (1986) has been most frequently referenced. All classifications listed above were based solely on morphological features, and not on phylogenetic analyses of Selaginella. The features most often used to distinguish groups in the classifications are: isophylly and anisophylly of both vegetative shoots and strobili, phyllotaxy, stelar arrangement, rhizophore position, habit, and spore ornamentation. The first classification presented in a phylogenetic framework, based on DNA sequence data, was the one proposed by Zhou and Zhang (2015). However, the first phylogenetic analysis of Selaginellaceae (based on DNA sequence data) was published already in 1999 by Korall and co-authors. Since then a few more studies have been conducted, extending both the taxon sampling and the molecular 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 problems consistently appearing in all previous studies on Selaginella phylogenetics are: finding clear morphological synapomorphies defining wellsupported 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 systematics is the morphological stasis seen in the genus. Selaginella is notorious for problems with species identification and unclear species delimitations. Previous 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 wellfounded 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 aspects to consider. We need to base our phylogenetic hypothesis on a repre- 21

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 lacking 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 Devonian. 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 interesting 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 assume would have a negative effect on dispersability. Compared to angiosperms, 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 understood, primarily, due to the lack of a phylogenetic framework on which we can base further studies. With a global, well-founded phylogenetic hypothesis, 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. 22

2. Aims The overall aim of this thesis is to get a better understanding of the systematics 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. Besides 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 studies 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 Selaginellaceae, with the large-scale phylogeny presented in Paper I forming the basis for the analyses. With their large megaspores and obligate outcrossing reproduction, Selaginella species have often been thought of as bad dispersers. We wanted to elucidate the historical biogeographical processes behind the worldwide distribution patterns seen in the family today. 23

3. Materials and methods Here follows a summary of the methodological choices in this thesis. Detailed descriptions of the materials and methods are presented in the respective 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 diversity 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 accessions from several species, to allow for an evaluation of within-species variation. DNA was extracted both from recently collected silica-dried tissue (e.g., from my field trips to Australia, Ecuador, and Scandinavia, Fig. 4), and herbarium 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. 24

Figure 4. Stina collecting S. selaginoides at the North Cape, Norway. Photo: J. Larsson. Selaginella is notorious for the difficulties associated with species determination, 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 regions 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 samples 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. pectinata Spring is the correct name for an endemic Madagascan species, and that the name S. polymorpha Badré from the late 1990s is superfluous and illegitimate (Paper III). For all accessions, morphological features were studied with the naked eye or under a stereo microscope. Gross morphological features used in previous 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, verified using literature data (for an account of references, see Paper I). In addition, information on megaspore ornamentation was taken from the literature (e.g., Minaki, 1984; Morbelli et al., 2001; Korall and Taylor, 2006). 25

3.2 Phylogenetic analyses My phylogenetic hypothesis is based on data from three different DNA regions: 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 multiple 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, subsequent 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 sequences were aligned, followed by single-region phylogenetic analyses in a Bayesian inference framework using MrBayes 3.2.4 (Ronquist and Huelsenbeck, 2003). The single-region phylogenies were then compared to elucidate possible well-supported conflicts between the data sets. No major conflicts 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 lineage to Selaginellaceae, was used as outgroup (Wikström and Kenrick, 1997; Korall et al., 1999). 26

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 deviate 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, despite 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 previously reported (e.g., Zhou et al., 2015a), and using morphology, we have problems to unequivocally assign species to the different subgenera proposed by Zhou and Zhang (2015). Further, the Zhou and Zhang classification is problematic in several additional aspects: (1) the use of the name Selaginella subg. Ericetorum which is incorrect given their circumscription, (2) the (unintentional) inclusion of a non-monophyletic group (Selaginella sect. Oligomacrosporangiatae), 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 historical 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 observed in the fossils were rigorously compared to the morphology seen 27

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 historical 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 speciation (J) (Matzke, 2014). The importance of including founder event speciation in historical biogeographical analyses have been thoroughly discussed by e.g., Matzke (2014). The resulting time-calibrated phylogeny with geographical range estimates 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 geological events such as the breakup of Pangea, subsequent breakups of Gondwana and Laurasia, as well as e.g., land bridge formations in the Northern hemisphere during the Cenozoic (following time spans given by, e.g., McLoughlin, 2001; Sanmartín et al., 2001; Rogers and Santosh, 2003; Sanmartín and Ronquist, 2004). 28

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 monophyletic 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 problematic 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 species, 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 phylogeny (see fig. 2 in Paper I). Our studies reveal even more complex morphological patterns than previously reported (Korall and Kenrick, 2002; Korall and Taylor, 2006; Zhou et al., 2015a), with most morphological characters showing reversals and/or parallelisms. 29

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 proposed correspond to monophyletic groups (Fig. 5), and despite the complex morphological patterns observed in the genus, each subgenus can be uniquely diagnosed by a suite of (mainly) gross morphological characters. The seven subgenera proposed in our classification are: Selaginella subg. Selaginella, 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, Lepidophyllae, Gymnogynum, Exaltatae, and Ericetorum), whereas clade B corresponds 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. 30

Isoëtes S. subg. Selaginella S. subg. Rupestrae S. subg. Lepidophyllae S. subg. Gymnogynum S. subg. Exaltatae S. subg. Ericetorum Rhizophoric clade S. subg. Stachygynandrum 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. 31