Reticulate Evolution in Diphasiastrum (Lycopodiaceae) - DocsLib

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Home , Diphasiastrum, Diphasiastrum alpinum, Diphasiastrum digitatum, Diphasiastrum sitchense, Diphasium, Lycopodiaceae

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In memory of Morten T.

Cover: Diphasiastrum alpinum from Northern Norway. Photo: B. Pettersen.

List of papers

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

I Aagaard, S.M.D., Vogel, J.C. & N. Wikström. Resolving maternal relationships in the clubmoss genus Diphasiastrum (Lycopodiaceae). In press. Taxon.

II Aagaard, S.M.D., Greilhuber, J., Zhang, X.-C. & N. Wikström. Oc- currence and evolutionary origins of polyploids in the club moss genus Diphasiastrum (Lycopodiaceae). In press. Molecular Phyloge- netics and Evolution.

III Aagaard, S.M.D., Greilhuber, J., Vogel, J.C. & N. Wikström. Re- ticulate phylogenetic patterns in diploid European Diphasiastrum (Lycopodiaceae). Manuscript

IV Aagaard, S.M.D., Gyllenstrand, N. & N. Wikström. Homoploid hybridization in Central European Diphasiastrum (Lycopodiaceae). Manuscript.

V Thulin, M., Aagaard, S.M.D., Wikström, N. & C. Jarvis. Revised lectotypification of Lycopodium complanatum L. (Lycopodiaceae). In press. Taxon.

Papers I, II and V are reproduced by kind permission of the publishers.

All papers included in the thesis are written by the first author, with additional comments and elaborations of technical procedures provided by the co-authors. The laboratory work and analyses were conducted by S. M. D. Aagaard with the exception of the Feulgen DNA image densitometry analyses in paper II and III, which were conducted by Professor Johann Greil- huber.

Contents

Introduction...... 9 Polyploidy ...... 11 Homoploid hybridization and speciation ...... 12 Aims ...... 15 Materials & Methods ...... 16 Taxa included in the phylogenetic analyses ...... 16 Investigated populations...... 17 Chloroplast sequence data (Paper I)...... 18 Chloroplast microsatellites (Paper I – IV)...... 18 Flow densitometry data (Paper II and Paper III)...... 18 Nuclear single and low copy regions (Paper II – Paper IV)...... 19 Patterns expected in polyploids (Paper II)...... 19 Patterns expected in homoploid hybrids (Paper III and Paper IV) ...... 19 Results & Discussion ...... 20 Intrageneric relationships and diversity (Paper I and Paper V)...... 20 Polyploidy in Diphasiastrum (Paper II) ...... 22 Homoploid hybridization in Diphasiastrum (Paper III and Paper IV).....24 Phylogenetic analyses...... 24 Admixture analyses ...... 26 Concluding remarks ...... 28 Svensk sammanfattning (Swedish summary) ...... 30 Acknowledgments...... 32 References...... 33

Introduction

“The taxonomist whose species concept is the result of experience princi- pally with plant taxa in which characters are more gross and differences between species are of a more qualitative nature is likely to be unimpressed by the subtle and quantitative differences he finds between L. complanatum and its relatives. He may feel that no matter how constant they are (and many of the individual characters do overlap), such differences hardly constitute sub- specific, let alone specific characters.” Joan H. Wilce (1961)

A hybrid is the product of a cross between two distinct species. Individuals of hybrid origin are assumed to be sterile, as the parental genomes in most cases are incompatible. But sometimes the merger of genomes between closely related species can give rise to a new distinct, evolutionary lineage. Species of hybrid origin are initially divided into two categories; those having the same number of chromosomes as their parent taxa, i.e. homoploid hybrids, and those established through chromosome doubling, i.e. allopolyploids (Mallet, 2007; Rieseberg and Willis, 2007). Both processes are now acknowledged to enable quick adaptation, and consequently ecological spe- cialisation (Lexer et al., 2003; Jiggins et al., 2008). Reticulate evolutionary events also constitute a major challenge in evolutionary as well as ecological studies of the club moss genus Diphasiastrum (Holub). The genus Lycopodium L. comprises about 40-50 species attributed to nine sections (Øllgaard, 1987). With 20-30 species Diphasiastrum, or Lyco- podium section Complanata Victorin, is by far the largest group. Most species in Diphasiastrum are widely distributed across the northern hemisphere (Wilce, 1965), but some are reported from tropical highlands with temperate climate. A number of endemics have also been described from islands in both the Atlantic and the Pacific Ocean (Wilce, 1961; Wilce, 1965). The most unique feature contributing to the species count in Diphasiastrum is that at least five diploid hybrid species are commonly recognized in the genus (Figure 1; Wilce, 1965; Holub, 1975; Stoor et al., 1996). Polyploids are less frequently reported, but at least two polyploid species have been considered in the literature (Wilce, 1961, Wilce, 1965). Clubmosses are characterized by having two independent generations; diploid sporophytes, producing wind born spores with a potentially unlim- ited dispersal range (Page, 2002), and haploid gametophytes. As the gametophytes are bisexual, three different breeding systems might be observed;

9 outbreeding, intergametophytic selfing or sib-mating, and intragametophytic selfing (Soltis and Soltis, 1988). Intragametophytic selfing, where two gam- etes from the same bisexual gametophyte fuse and produce a completely homozygous individual, could potentially aid the establishment and genetic ‘fixation’ of a new species of hybrid origin (Rieseberg and Willis, 2007). A number of factors make it problematic to investigate hybridization in Diphasiastrum. They have a simple morphology with few discrete morphological features that can be evaluated (Wilce, 1961; Wilce, 1965, Vogel and Rumsey 1999). Also, little is known about their reproductive biology. The mycorrhizal dependence of the gametophyte makes their spores difficult to germinate in controlled laboratory environments (Whittier, 1977), and cross- ing experiments are virtually impossible to accomplish. Counting chromosomes in Diphasiastrum is difficult, but in most cases a base chromosome number n=23-24 has been reported (Wagner, 1992). Even though diploid homoploid hybrid species are assumed to predominate in Diphasiastrum, two taxa, D. wightianum (Grev. et Hook.) and D. zanclophyllum (J. H. Wilce) Holub, have been recognized to be of polyploid hybrid origin (2n=4x=96). Diphasiastrum wightianum has been confirmed to be tetraploid by chromosome counts (Ninan, 1958). Diphasiastrum zanclophyllum has been considered to be a polyploid based on the observation that its spores are significantly larger than in known diploids (Wilce, 1961; Wilce 1965). Observing the morphological diversity among accessions identified as D. wightianum and D. zanclophyllum, Wilce (1965) discussed the possibility that the number of polyploids in Diphasiastrum has been underestimated. Nonetheless, reports on ploidal level of accessions identified to be putative homoploid hybrid species confirm chromosome numbers equal to that of the suggested parent taxa (Wagner, 1992).

10 Polyploidy Polyploidization is a relatively common mode of sympatric speciation among vascular plants, and is considered more frequent in homosporous ferns and lycophytes than other plant groups (Otto and Whitton, 2000). The occurrence of polyploids has been well accounted for in two other genera in Lycopodiaceae, Huperzia Bernh. and Lycopodiella Holub (Wagner et al., 1985), but reports from Diphasiastrum and Lycopodium sens. str. are less frequent (Wagner, 1992). Depending on the mode of origin and degree of divergence between the parents, polyploids are generally classified as either allopolyploids or autopolyploids. Autopolyploids are formed as a result of genome doubling within a single population or species, whereas allopolyploids are the result of interspecific hybridization and genome doubling (Thompson and Lumaret, 1992; Soltis and Soltis, 1993; Ramsey and Schemske, 1998; Soltis and Soltis, 1999; Ramsey and Schemske, 2002). An increasing number of studies conclude, however, that complex evolutionary processes affect the genome in polyploids more severely than previously believed (Soltis and Soltis, 1993; Doyle et al., 2003; Slotte et al. 2008). Thus, strict allopolyploids and autopolyploids should be considered as theoretical extremes in a continuum, and the observed evolutionary patterns might be more ambiguous (Ramsey and Schemske, 1998, 2002; Popp et al., 2005; Slotte et al., 2006; Soltis et al. 2007; Slotte et al., 2008). Diphasiastrum wightianum is, as mentioned, the only species in Diphasi- astrum previously confirmed by chromosome counts to be tetraploid (Ninan, 1958). Morphology indicates that one of the parents is round branched and isophyllous, i.e. D. sitchense (Rupr.) Holub or D. veitchii (Christ) Holub, whereas the other is more or less flat branched and heterophyllous (Wilce, 1965). Alternative parental combinations, based on morphology and biogeographical distribution, have been suggested to be D. veitchii and D. alpinum (L.) Holub, D. sitchense and D. multispicatum (J. H. Wilce) Holub or D. sitchense and an undescribed “Chinese species” (Wilce, 1965). An- other hypothesis is that D. wightianum is a polyploid D. sabinifolium (Willd.) Holub (cf. Wilce, 1965), a putative diploid hybrid species originat- ing from a cross between D. sitchense and D. tristachyum (Pursh) Holub. All suggested parental combinations are listed in Table 1. Diphasiastrum zanclophyllum is confined to Madagascar and South Af- rica. Speculations that this species should have a polyploid origin have been based on observations that the spores in this species are significantly larger than in known diploids (Wilce, 1961). Suggested parents are D. tristachyum and/or the North American species D. digitatum (A. Br.) Holub (Wilce, 1961; Wilce, 1965).

11 Table 1. Hypothesized origins of D. wightianum and D. zanclophyllum as discussed by Wilce (1965). The combination D. sitchense x D. tristachyum corresponds to the putative homoploid hybrid D. sabinifolium. D. zanclophyllum is less debated; consequently the two taxa Wilce (1965) mentions as putative parents are listed separately. Polyploid Diploid parent Diploid parent -round branched -flat branched -isophyllous -heterophyllous D. wightianum D. sitchense D. multispicatum D. sitchense ”Chinese plant” D. veitchii D. alpinum D. sitchense D. tristachyum

D. zanclophyllum ? D. digitatum ? D. tristachyum

Homoploid hybridization and speciation Considered somewhat more controversial, and with fewer documented ex- amples in the literature, homoploid hybridisation is now fully recognized as an important evolutionary mechanism (Rieseberg and Willis 2007, Mallet, 2007). The most studied complexes being the sunflowers, Helianthus L. (Rieseberg, 1997), and the butterfly genus Heliconius Kluk (Mavárez et al., 2006). In Helianthus, a rapid adaptation to an extreme environment has provided a selective advantage as well as functioned as effective isolation mechanisms, enabling the establishment of several hybrid taxa within this genus (Lexer et al. 2003). In Heliconius it seems that switches in morphological imprinting have had similar effects (Mavárez et al., 2006; Jiggins et al., 2008). Incidents of homoploid hybridization have been reported from several genera among homosporous plants (Haufler, 2008), whereas homoploid hybrid speciation has first and foremost been discussed with reference to the genus Diphasiastrum. Homoploid hybrid speciation is not easily detected (Rieseberg and Ell- strand, 1993; Rieseberg, 1997). Recombination randomly redistributes the parental genomes during meiosis (Linder and Rieseberg, 2004; Nakhleh et al., 2004; Hegarty and Hiscock, 2005), and the initially heterozygous pattern displayed by the homeologous chromosomes in the F1 offspring will be disrupted within one generation. Subsequent backcrosses with either parent species are also assumed to favour more parental like genotypes due to fit- ness advantages (Gow et al., 2006). Homoploid hybrid speciation is therefore dependent on the rapid formation of genomic barriers, such as severe chromosome rearrangements, to prevent backcrossing (Rieseberg, 1997; Buerkle et al., 2000; Lexer et al., 2003).

12 At least five species are considered to be of homoploid hybrid origin in Diphasiastrum (Figure 1; Wilce, 1965; Holub, 1975; Wagner and Beitel, 1993; Stoor et al., 1996). In Europe, Diphasiastrum issleri (Rouy) Holub is considered to be the result of the cross between D. alpinum and D. complanatum L., D. zeilleri (Rouy) Holub the result of the cross between D. complanatum and D. tristachyum and D. oellgaardii Stoor et al. the result of the cross between D. tristachyum and D. alpinum (Figure 1A). Note that Wilce (1965) refers to D. issleri from the U.S. as a cross between D. alpinum and D. tristachyum (Figure 1B), which is not coherent with the view presented in European literature (e.g. Stoor et al., 1996). Two additional homoploid hybrid taxa are considered from North America; D. habereri (House) Holub, recognized as the result of the cross between D. digitatum and D. tristachyum, and the aforementioned D. sabinifolium (D. sitchense x D. tristachyum; Figure 1B).

D. tristachyum D. tristachyum

D. zeilleri D. sabinifolium

D. zeilleri D. oellgaardii D. complanatumD. habereri D. issleri D. sitchense

D. complanatumD. issleri D. alpinum D. digitatum D. alpinum

A; Europe B; North America

Figure 1. Hypothesized reticulate evolution in A) European (Stoor et al., 1996) and B) North American (Wagner et Beitel, 1993) Diphasiastrum. Putative parent species are annotated with black diamonds, whereas putative hybrid species are annotated with white diamonds.

The existence of specimens with apparent intermediate morphology was acknowledged already in the early 20th century, which led several authors to discriminate putative hybrid entities as neohybrids or separate varieties of one of the suggested parent species (Issler, 1910; Victorin, 1925). Wilce (1965), in her revision of the group, discussed the occurrence of hybrids at great length. However, by this time, the idea that some of these hybrids represent independent evolutionary entities was firmly established, and she

13 clearly accepted the species status of the North American hybrid D. sabinifolium. The status of the other hybrid taxa she considered more uncertain, yet North American floras (Wagner and Beitel, 1993), European floras (Jermy, 1993; Frey et al., 2006) and regional assessments of endangered species (Holub and Procházka, 2000; Horn et al., 2001) still tend to treat them as species. Despite the increasing acceptance of species with homoploid hybrid origins in Diphasiastrum, there has been only one attempt to investigate this further using molecular data. Stoor et al. (1996) used allozyme data as evidence for the hybrid origin of D. oellgaardii. This work was, however, considered inconclusive by Vogel and Rumsey (1999). Their criticism was part- ly based on the limited utility of some allozyme loci with regards to taxon delimitation. Also, the interpretation by Stoor et al. (1996) of the obtained allozyme banding pattern as additive, i.e. ‘fixed heterozygosity’, left several alternative explanations unexplored. ‘Fixed heterozygosity’, where the homeologous chromosome pairs inherited from the different diploid parent species do not recombine after the gene duplication event, is as a rule displayed by allopolyploids (Linder and Rieseberg, 2004; Small et al., 2004). Thus the observed alleles represent different paralogs, developing and func- tioning independently of each other. If taxa displaying such patterns are confirmed by chromosome counts or flow cytometry to be polyploid, their evolutionary and taxonomic integrity is rarely questioned (Linder and Riese- berg, 2004). However, the few studies published report specimens identified as D. issleri and D. zeilleri to display the same chromosome number as their assumed parents, 2n=46, implying that they are putative homoploid hybrids and not polyploids (Wagner, 1992). Considering that the Central European hybrids are reported to be diploids, the observed pattern of ‘fixed heterozygosity’ could also imply that the specimens analyzed by Stoor et al. (1996) are F1 hybrids. F1 hybrids would display a congruent pattern of alleles inherited from both parent species in each analyzed region, as they have not yet undergone recombination during meiosis (Vogel and Rumsey, 1999). The observed morphological variation would then simply represent reoccurring neohybrids in a hybrid swarm (Ar- nold, 1997).

14 Aims In this thesis, the origins of polyploids and putative homoploid hybrid taxa recognized in Diphasiastrum are investigated. The study is divided into three main consecutive parts to elucidate the observed interspecific variation in the genus, with the following specific aims:

• To establish an intrageneric phylogeny of Diphasiastrum based on a combined dataset of non-recombinant, maternally inherited chloroplast loci. • To infer the ploidal levels and evolutionary origins of putative polyploids and homoploid hybrids using data obtained from Feulgen DNA image densitometric analyses and phylogenetic analyses of multiple low copy nuclear loci. • To investigate the genetic integrity between putative homoploid hybrids and their parents through admixture analyses on an expanded dataset.

15 Materials & Methods

Taxa included in the phylogenetic analyses Included in the phylogenetic analyses are accessions representing both wide- spread species and species with more narrow endemic distributions in Di- phasiastrum. All putative parent species in the hybrid complexes, i.e. D. alpinum, D. complanatum, D. digitatum, D. multispicatum, D. sitchense, D. veitchii and D. tristachyum, were represented in the analyses. Additional, non-hybrid taxa included were D. nikoense (Franch. et Savat.) Holub from Japan, D. thyoides (Willd.) from South America and the two island endemics D. madeirense (J. H. Wilce) Holub and D. henryanum (E. Brown) Holub. One accession from China not previously associated with any known taxa was also included in the nuclear analyses. Two accessions of African polyploid D. zanclophyllum and one accession identified as the Asian polyploid D. wightianum were included (Paper I, Paper II). Accessions identified as one of the five homoploid hybrids D. habereri, D. issleri, D. oellgaardii, D. sabinifolium and D. zeilleri, were included in the initial study (Paper I) in order to cover the morphological variation recognized in the genus. In the nuclear study (Paper II and Paper III) the number of putative homoploid hybrid accessions is reduced, com- prising only D. sabinifolium and accessions from putative hybrid populations in Central Europe.

16 Investigated populations Accessions included in the population study were collected from six localities where one or more of the putative hybrid species had been previously reported (Figure 2). Among them the type localities of D. issleri and D. oellgaardii, Le Tanet and Champ du Feu, both situated in the Vosges Moun- tains, France (Lawalrée, 1957; Stoor et al., 1996). Other localities visited were Zwieselberg (Horn and Bennert, 2002) in Austria and Sankt Andreas- berg (Stoor et al. 1996), Weiden and Bayerischer Wald (Gaggermeier, 1993) in Germany. Accessions from allopatric populations in Scandinavia and North America were used as references, representing unambiguously identified parent lineages from different geographical regions.

Figure 2. Map over Central Europe. Putative hybrid populations are marked with diamonds.

17 Chloroplast sequence data (Paper I) Plastid genomes in most plants are uniparentally inherited (Vogel and al., 1998; Birky, 2001), and are therefore not exposed to recombination. Identi- fying incongruence between a chloroplast DNA based dataset and a dataset based on nuclear markers, such as low copy genes or microsatellites, is currently one of the best ways to detect hybridization (Linder and Rieseberg, 2004). Initial knowledge regarding the intrageneric phylogeny, covering most of the observed diversity, is crucial when initiating a study involving a taxon where several incidents of reticulate evolution are recognized. In Paper I, five plastid regions were sequenced for further analyses; rps4 and rps4- trnS intergenic spacer (ca 850 base pairs), rbcL-atpB intergenic spacer (ca 700-950 bp), trnG intron (ca 800 bp) and trnG-trnR intergenic spacer (ca 1500 bp), rpl20-rps12 intergenic spacer (ca 3200 bp) and petG-petL intergenic spacer, petL and petL-psbE intergenic spacer (ca 1950 bp).

Chloroplast microsatellites (Paper I – IV) Two microsatellites were identified as potentially informative in the chloroplast regions rbcL-atpB intergenic spacer and trnG-trnR intergenic spacer. These microsatellites were used in a larger screening for additional diversity in the six putative hybrid populations from Central Europe. Microsatellite length and motifs were also used in order to infer the maternal lineage in putative polyploids and homoploid hybrids.

Flow densitometry data (Paper II and Paper III) Feulgen DNA image densitometry using the CIRES system (Kontron, Mu- nich; compare Vilhar et al., 2001) was applied in order to infer ploidal level on included accessions. Ordinary and more commonly used flow cytometry yielded unsatisfactory results with the herbarium and silica dried material available. The analysed material included a subset of the accessions collected from the Central European populations as well as single accessions of D. madeirense (J. H. Wilce) Holub, D. multispicatum, D. veitchii and the unclassified Asian taxon. All putative polyploid accessions in Paper II and the single accessions representing putative homoploid hybrids included in the phylogenetic analyses in Paper III were analysed.

18 Nuclear single and low copy regions (Paper II – Paper IV) Genus specific primers were developed for three single or low copy regions of the nuclear genome; RNA polymerase II (RPB2, ca 450 base pairs), LEAFY (ca 500 bp) and Lycopodium annotinum MADS-box gene 4 protein (LAMB4, ca 1400 bp). Phylogenetic analyses were performed separately on sequence data obtained from the different regions in order to infer reticulate evolutionary events among confirmed polyploid and diploid accessions. Furthermore, admixture analyses were conducted in the program NewHy- brids using sequence data obtained from an expanded dataset including putative homoploid hybrid populations.

Patterns expected in polyploids (Paper II) In a polyploid, sequences from two homoeologous loci for a given gene are expected to resolve with either the same or different parental lineages representing the diploid parent species. This depending on whether the polyploid is an autopolyploid or an allopolyploid. No recombination is expected between the homeologous chromosomes in an allopolyploid, which should display a strict disomic inheritance pattern. Hence, single copy nuclear loci in an allopolyploid should display haplotypes representing both parental orthologs, i.e. ‘fixed heterozygosity’.

Patterns expected in homoploid hybrids (Paper III and Paper IV) Sequence data from multiple independent single and low copy regions in combination with information from chloroplast markers has successfully been used to infer reticulate evolutionary events in diploids (e.g. Doyle et al., 2003; Kronforst et al., 2006; Mavárez et al., 2006). Establishing patterns of reticulate evolution in putative hybrid specimens is crucial before initiating larger, population based studies addressing the consequences of such evolutionary processes. However, the initially heterozygous pattern displayed by the homeologous chromosomes in a F1-offspring will be disrupted due to meiotic recombination within one generation. Hence admixture analyses of obtained sequence data were conducted in the program NewHybrids (Anderson and Thompson, 2002) on an expanded dataset from the six putative hybrid populations. Without requiring any prior information of the parent populations, or in this case parent taxa, NewHybrids estimates the posterior probability of each individual belonging to the genotype frequency classes ‘parent species 1’, ‘parent species 2’, ‘F1 hybrid’, ‘F2 hybrid’, ‘backcross with parent 1’, ‘backcross with parent 2’. These estimations are based on frequency differences between alleles not necessarily fixed for either parent.

19 Results & Discussion

Intrageneric relationships and diversity (Paper I and Paper V) Phylogenetic analyses based on a combined dataset of the five chloroplast markers, including eight outgroup taxa from other sections in Lycopodium (sensu Øllgaard 1987), confirm a monophyletic origin of Diphasiastrum (Paper I). It also supports an internal rooting point within Diphasiastrum, separating D. tristachyum and D. multispicatum from the remaining species included in the analyses. Despite low levels of sequence variability, the phylogenetic analyses confirmed the delimitation of at least eight lineages in Diphasiastrum; D. alpinum, D. complanatum, D. digitatum, D. madeirense, D. multispicatum, D. sitchense, D. tristachyum and D. veitchii (Figure 3). Inferred maternal lineages comprise all putative parental taxa combinations suggested in the Central European hybrid complexes, the North American hybrid complexes and for the two polyploid taxa. With one exception, all accessions identified as homoploid hybrids resolve with one of the putative parent taxa. The one exception, D. zeilleri (D. complanatum x D. tristachyum) from the U.S., resolves with D. digitatum. This accession might be misidentified and represent an unrecognized parental combination, or alternatively the origin of D. zeilleri in North America has been misinterpreted. The chloroplast microsatellite screening of allopatric and putative hybrid populations revealed that additional variation in the putative hybrid populations occurred in very low frequencies. Only one haplotype is shared between different localities, and they are best interpreted as representing local intraspecific variability at the level of single populations (Paper I). Hence no variability representing historical individuality beyond the putative parents could be documented. The application of scientific names is determined by types, usually a type specimen. Upon investigation the currently accepted type specimen of Lyco- podium complanatum (= D. complanatum), a name proposed by Linnaeus, turned out to represent L. tristachyum (= D. tristachyum). This means that L. complanatum is the correct name for L. tristachyum, whereas no name is available for L. complanatum. Having established L. tristachyum and L. complanatum as two distinct lineages (Paper I), a revised typification of L.

20 complanatum is proposed (Paper V), so that the traditional usage of this well-known name can be maintained.

D. multispicatum CN D. tristachyum Diphasiastrum zeilleri AT

Diphasiastrum tristachyum NO 1.00 100 Diphasiastrum tristachyum US group Diphasiastrum1 habereri US

Diphasiastrum madeirense PT D. veitchii Diphasiastrum wightianum MY 1.00 88 1.00 Diphasiastrum veitchii CN 91 group Diphasiastrum henryanum US D. digitatum 0.92 Diphasiastrum zeilleri US − Diphasiastrum zanclophyllum ZA 1.00 94 Diphasiastrum thyoides EC group

Diphasiastrum digitatum US

Diphasiastrum complanatum ssp. montellii RU D. complanatum 1.00 Diphasiastrum complanatum NO 89 1.00 1.00 Diphasiastrum issleri FR 75 98 0.99 Diphasiastrum complanatum DE

63 group Diphasiastrum complanatum US D. sitchense

0.98 Diphasiastrum sitchense US 61 0.99 Diphasiastrum sabinifolium US 60 Diphasiastrum nikoense JP group

0.96 D. alpinum − Diphasiastrum oellgaardii FR

1.00 Diphasiastrum complanatum x alpinum US

87 Diphasiastrum alpinum CA group

Diphasiastrum1 alpinum NO

substitutions per site 0.001

Figure 3. Diphasiastrum (Lycopodium Section Complanata). Bayesian majority rule consensus phylogram of Diphasiastrum only. Numbers above and below branches are Bayesian posterior probabilities and parsimony bootstrap frequencies, respectively. Groups supported by less than 50% in the parsimony bootstrap analyses are annotated “-“. With reference to putative parent species in the different hybrid complexes, six “species groups” are indicated to the right. Putative polyploid species D. wightianum and D. zanclophyllum are represented with one accession each. Three accessions identified as the three homoploid hybrid species reported from Central Europe, D. issleri, D. oellgaardii and D. zeilleri, are included. Three accessions identified as homoploid hybrid species reported from North America, D. habereri, D. sabinifolium and D. zeilleri, are also represented. Note that Wilce (1965) defines D. issleri from North America as the same cross as D. oellgaardii in Europe. We were not able to obtain material representing North American D. issleri, but another accession identified as a cross between D. alpinum and D. complanatum was included. Country of origin is denoted after each accession, abbreviations being as follows; Austria = AT, Canada = CA, China = CH, Ecuador = EC, France = FR, Germany = DE, Japan = JP, Malaysia = MY, Norway = NO, Portugal/Azores = PT, Russia = RU, South Africa = ZA, Sweden = SE.

21 Polyploidy in Diphasiastrum (Paper II) The two polyploid taxa recognized in Diphasiastrum have somewhat different geographical distributions and parental origins. Diphasiastrum zanclophyllum is the only taxon in Diphasiastrum documented from South Africa and Madagascar, which has made it difficult to elucidate its evolutionary origin. Based on morphological similarities, the North American taxon D. digitatum was suggested to be close to at least one of the original parents (Wilce, 1961; Wilce, 1965). This hypothesis is corroborated by chloroplast data (Figure 3; Paper I), and one paralog that displays corresponding relationships in the nuclear regions RPB2 and LEAFY (Figures 4 A and B). The relationships of the other paralog, observed in both regions, are less clear. The two paralogs obtained are resolved separately with good support and autopolyploidy is thus rejected as a possible explanation for the observed pattern. The included accession from Malaysia identified as D. wightianum pos- sesses paralogs that in all three nuclear regions are resolved with D. veitchii and D. multispicatum, respectively (Figures 4 A-C). The other polyploid accession from Asia, included in the analyses as Diphasiastrum sp., pos- sesses one paralog that resolves with D. veitchii (Figures 4 A and C). As in D. zanclophyllum, the relationship of the other paralog is less clear (Figures 4 A and B), but it seems to display close relationship with D. tristachyum. Despite this uncertainty, we firmly establish that the two Asian accessions have originated separately. However, none of the parental combinations corresponds to those previously discussed by Wilce (1965) (Table 1). All polyploids collected in Asia have until now been identified as D. wightianum. Our results support the observations of Wilce (1965) based on morphology, which suggest that the diversity in Diphasiastrum with respect to Eastern Asia is quite unexplored.

Figure 4. Bayesian majority rule consensus phylograms for the regions RPB2 (A), LEAFY (B) and LAMB4 (C). Numbers above branches are Bayesian posterior probabilities. When multiple gene copies are present, these are annotated with the letters A (1 and 2) or B after the taxon name. The letters A and B denote paralogs or alleles showing respectively congruent and incongruent relationships with maternally inherited markers obtained from the chloroplast (Figure 3; Paper I). Country of origin is denoted after each accession, abbreviations being as follows; Austria = AT, Canada = CA, China = CH, Ecuador = EC, France = FR, Germany = DE, Japan = JP, Ma- laysia = MY, Norway = NO, Portugal/Azores = PT, Russia = RU, South Africa = ZA, Sweden = SE , Vietnam = VN.

22 D. tristachyum FR 1.00 D. tristachyum A1 CA 0.50 D. tristachyum A2 CA FR 0.65 D. tristachyum D. alpinum A2 CA D. tristachyum US 1.00 D. alpinum A1 CA Diphasiastrum sp. B CN ← Paralog B 1.00 D. alpinum FR D. alpinum A1 FR D. alpinum NO D. alpinum A2 FR 1.00 0.97 1.00 D. zanclophyllum 1 B ZA ← Paralog B D. sabinifolium 1 A US ← Allele A D. zanclophyllum 2 B ZA ← Paralog B D. sabinifolium 14 A US ← Allele A D. alpinum SE D. sitchense 8102 A1 US 0.86 D. nikoense JP D. nikoense JP D. sitchense 1 US 1.00 D. sitchense 8102 A2 US 0.98 0.99 D.sitchense 1 US 0.55 D. sabinifolium 1 A US ← Allele A 0.73 D. digitatum B US ← Allele B D. alpinum SE 1.00 D. alpinum CA D sitchense 8102 US

D. alpinum NO 1.00 D. veitchii CN D. zanclophyllum 2 B ZN ← Paralog B D. wightianum A MY ← Paralog A D. veitchii CN D. zanclophyllum 2 A ZA ← Paralog A 1.00 Diphasiastrum sp. A CN ← Paralog A 0.97 D. digitatum A US ← Allele A 0.61 0.63 D. wightianum A MY ← Paralog A D. zanclophyllum 1 A ZA ← Paralog A

0.98 D. madeirense 5 PT D. sabinifolium 1 B US ← Allele B D. maderiense 4 PT D. madeirense 4 PT 0.55 0.97 0.95 0.99 D. zanclophyllum 2 A ZN ← Paralog A D. madeirense 3 PT D. thyoides EC D. madeirense 5 PT 0.97 D. sabinifolium 1 B US ← Allele B D. complanatum US 0.97 D. digitatum US 0.99 D. complanatum A2 NO D. sabinifolium 14 B US ← Allele B 0.61 D. complanatum A1 NO 0.96 D. complanatum US 0.50 D. complanatum DE D. complanatum DE 1.00 D. complanatum ssp. montellii RU D. complanatum NO 0.99 D. thyoides A2 EC D. complanatum ssp. montellii RU D. thyoides A1 EC D. multispicatum A1 VN D. wightianum B MY ← Paralog B D. multispicatum A2 VN D. multispicatum VN D. wightianum B MY ← Paralog B D. multispicatum CN D. multispicatum CN A 0.01 substitutions per site B 0.01 substitutions per site

D. wightianum B MY ← Paralog B

Diphasiastrum sp. B CN ← Paralog B 1.00

1.00 D. tristachyum US

Diphasiastrum sp. A CN ← Paralog A

1.00 1.00 D. wigthianum A MY ← Paralog A 1.00

D. veitchii CN

0.87 D. madeirense 4 PT

D. thyoides EC

0.97 D. complanatum NO

D. complanatum US

D. alpinum CA

1.00 D. alpinum NO

0.67 D. alpinum FR 0.66

D. alpinum SE

D. multispicatum A1 VN

D. multispicatum A2 VN C 0.01 substitutions per site

Figure 4.

23 Homoploid hybridization in Diphasiastrum (Paper III and Paper IV) Phylogenetic analyses Three species of homoploid hybrid origin are commonly recognized from Central Europe, and all three putative parental combinations were identified among the six hybrid populations investigated; Diphasiastrum alpinum x D. complanatum, D. alpinum x D. tristachyum and D. complanatum x D. tristachyum (Figure 1 and Figures 5 A-D; Wilce, 1965; Stoor et al., 1996). Altogether six accessions included in the phylogenetic analyses display a reticulate phylogenetic pattern in all three regions, sharing alleles with different isolated evolutionary lineages, i.e. taxa, in the investigated loci (Fig- ures 5 A-C; Paper III). The observed allele distribution is also congruent among all three investigated regions (Figures 5 A-C). Such a pattern of 'fixed heterozygosity' would, as mentioned, either imply that the analysed specimen is allopolyploid, or if the specimen is a diploid, a neohybrid (Arnold, 1997; Vogel and Rumsey, 1999). Feulgen DNA image densitometry data verified that accessions included from Central Europe displaying a reticulate phylogenetic pattern are diploid (Paper III). Polyploidy, as well as pheno- typic plasticity, are therefore excluded as possible explanations for the observed morphological and molecular variation in the Central European populations.

Figure 5. Bayesian majority rule consensus phylograms from the analyses of RPB2 (A) LEAFY (B) and LAMB4 (C). Numbers above branches are Bayesian posterior probabilities. Alleles from a heterozygous individual that resolve with different groups are annotated A (maternal) and B (paternal). Alleles from a heterozygous individual that resolve within the same group are annotated A1 and A2 (both maternal). The maternal pattern is inferred by comparing the present results with that obtained in Paper I for maternally inherited plastid markers. Putative hybrid accessions from Central European populations are referred to as LT1 (Le Tanet), R9 (Weiden), Z1 and Z6 (Zwieselberg), CdF1, CdF3and CdF9 (Champ du Feu). Coun- try of origin is denoted after each accession, abbreviations being as follows; Austria = AT, Canada = CA, China = CH, Ecuador = EC, France = FR, Germany = DE, Japan = JP, Norway = NO, Portugal/Azores = PT, Russia = RU, Sweden = SE , Vietnam = VN. In D, accessions from Central Europe included in this study found to display reticulate phylogenetic patterns are put next to the arrow pointing from the maternal lineage.

24 Z6 A AT ← Allele A CdF1 B FR ← Allele B D. veitchii CN 0.92 D. complanatum ssp. montellii RU Z1 B AT ← Allele B 0.63 CdF9 FR 0.94 D. digitatum US 0.98 D. thyoides EC D. tristachyum A2 CA D. tristachyum A1 CA 0.99 D. complanatum US 1.00 0.96 CdF3 FR 0.99 D. madeirense 4 PT D. madeirense 5 PT D. alpinum A1 CA ‘D. tristachyum’ B NO ← Allele B R9 A DE ← Allele A D. complanatum NO LT1 B FR ← Allele B

CdF1 A FR ← Allele A 0.61 D. alpinum A2 CA 1.00 1.00 LT1 A FR ← Allele A D. alpinum SE R9 B DE ← Allele B Z1 A AT ← Allele A Z6 B AT ← Allele B D. alpinum NO D. complanatum DE D. veitchii CN ‘D. tristachyum’ A NO ← Allele A 0.70 D. sitchense 8102 US D. tristachyum CA D. sitchense 1 US Z6 A AT ← Allele A 0.81 D. nikoense JP CdF1 B FR ← Allele B 1.00 D. madeirense 5 PT CDF9 FR 0.95 D. madeirense 4 PT Z1 B AT ← Allele B D. madeirense 3 PT D. alpinum SE LT 1 A FR ← Allele A D. alpinum CAN 0.67 D. thyoides A2 EC D. alpinum NO 1.00 D. thyoides A1 EC CdF3 A1 FR D. complanatum US 0.94 D. nikoense JP 0.72 D. complanatum ssp. montellii RU R9 A DE ← Allele A D. complanatum DE CdF3 A2 FR 0.99 R9 B DE ← Allele B LT1 B FR ← Allele B CdF1 A FR ← Allele A Z1 A AT ← Allele A 0.95 D. sitchense 8102 A2 US D. complanatum A1 NO

0.86 D. complanatum A2 NO 1.00 D. sitchense 1 US D sitchense 8102 A1 US Z6 B AT ← Allele B D. multispicatum A2 VN D. multispicatum VN D. multispicatum A1 VN D. multispicatum CN D. multispicatum CN 0.01 substitutions per site 0.01 substitutions per site A B CdF1 B FR ← Allele B

‘D. tristachyum’ A NO ← Allele A 1.00 Z6 A AT ← Allele A

D. tristachyum CA 0.98

Z1 B AT ← Allele B

1.00 D. veitchii CN

D. complanatum NO

LT1 A FR ← Allele A

D. alpinum SE

0.59 LT1 B FR ← Allele B

CdF3 FR 1.00

D. alpinum CAN D. tristachyum Z1 A AT ← Allele A

0.98 D. alpinum NO

R9 B DE ← Allele B Z6 0.98 ‘D. tristachyum’ B NO ← Allele B ‘D. tristachyum’ NO 1.00 D. zeilleri Z6 B AT ← Allele B D. oellgaardii

D. complanatum US CdF1 Z1 D. thyoides EC

D. madeirense 4 PT LT1 R9 D. multispicatum A1 VN D. complanatumD. issleri D. alpinum D. multispicatum A2 VN C 0.01 substitutions per site D Figure 5.

25 Admixture analyses In the NewHybrids analyses of the expanded dataset, the majority of the accessions included from both allopatric and hybrid populations were assigned to be pure bred D. alpinum, D. complanatum or D. tristachyum. In each analysis several accessions from allopatric populations also displayed an ambiguous assignment (Figures 6 A-C). Whether the occurrence of am- biguously assigned accessions is due to interspecific gene flow between the three species or retention of ancestral polymorphisms, we cannot answer on the basis of this dataset. However, as these accessions are collected from putative allopatric populations, this result is most likely due to lack of diagnostic loci (Anderson and Thompson 2002), odd recombination events in the region LAMB4, old introgression events or a biogeographical sampling ef- fect. In the “D. alpinum x D. complanatum” dataset (Figure 6A) ten accessions from the three putative hybrid populations included displayed posterior probabilities >0.95 of being a F1 hybrid. The hybrid taxon D. issleri has previously been reported from all three populations (Lawalrée, 1957; Bennert, 1999). However, maternally inherited plastid sequence and microsatellite data (Paper I) combined with biparentally inherited sequence data from nuclear single or low copy regions (Paper III, Paper IV) clearly shows that F1 hybrids from Weiden and Le Tanet represent different and reciprocal crosses. Haplotype variability, as documented in the present analyses, similarly indicates that inferred F1 hybrids from Sankt Andreas- berg may represent a third, separate hybridization event. A similar pattern implying reciprocal crosses was also detected in the D. complanatum x D. tristachyum dataset (Paper III), including three accessions from respectively Champ du Feu, Zwieselberg, and Southern Norway. These accessions were all assigned as first generation backcrosses with D. tristachyum in the NewHybrids analyses (Figure 6C).

Figure 6. Posterior probability assignments of (A) inferred D. alpinum, D. complanatum and putative first and second generation hybrids (B) inferred D. alpinum, D. tristachyum and putative first and second generation hybrids and (C) inferred D. complanatum and D. tristachyum and first and second generation hybrids. In (A) and (B), inferred F1 hybrids are denoted with an asterisk, whereas in (C) an asterisk denotes accessions displaying a combined posterior probability >0.80 belonging to either one of the hybrid genotype frequency classes. Arrows denote accessions displaying ambiguous assignments, and in (B) this includes accessions assigned to be F2 hybrids with a posterior probability >0.80.

26 ↓↓ ↓ ** * * *↓ ** ** * ↓ ↓ ↓

100%

90%

80%

70% Backcross with D. alp. 60% Backcross with D. co. F2 50% F1 40% D. alpinum D. complanatum 30%

20%

10%

0% 1 4 7 101316192225283134374043464952555861646770737679

1- 29 Allopatric D. alpinum 30 - Champ du Feu 31-45 Sankt Andreasberg 46-59 Weiden 60-68 Le Tanet A 69-81 Allopatric D. complanatum

↓ ↓↓↓ ↓ ↓ ↓ ↓ ↓ ↓↓ ↓ * ↓

100%

90%

80%

70% Backcross with D. alp 60% Backcross with D. trist. F2 50% F1 40% D. alpinum D. tristachyum 30%

20%

10%

0% 1 5 9 131721252933374145495357616569737781

1-29 Allopatric D. alpinum 30-57 Champ du Feu 58-64 Bayerischer Wald 65-77 Zwieselberg 78-81 Allopatric D. tristachyum B

↓ * * * * ↓ ↓ ↓ ↓ ↓↓

100%

90%

80%

70% Backcross with D. co. 60% Backcross with D. trist. F2 50% F1 40% D. complanatum D. tristachyum 30%

20%

10%

0% 1 3 5 7 9 11131517192123252729313335373941434547

1-17 Champ du Feu 18-22 Zwieselberg 23-30 Bayerischer Wald 31-43 Allopatric D. complanatum 44 - D. complanatum x D. tristachyum from Norway 45-48 Allopatric D. tristachyum C Figure 6.

27 Concluding remarks Simulation studies have shown that a minimum of 60 generations are needed in order for a diploid hybrid lineage to be effectively differentiated from its parent (Ungerer et al. 1998). Complete stabilisation is similarly shown to rarely be obtained for even hundreds of generations (Buerkle and Rieseberg, 2007). These models demonstrate that substantial isolation is required to establish an independent hybrid lineage. Gene flow from the parent taxa will disrupt the stabilisation process and consequently result in the formation of a hybrid zone (Buerkle et al., 2000; Buerkle and Rieseberg, 2001; Buerkle and Rieseberg, 2007). In this study, available evidence taken together indicates that the investigated populations represent reoccurring hybrid zones. In Pa- per I we identified at least two different maternal lineages associated with different parent taxa in five of the six hybrid populations investigated (Sankt Andreasberg, Zwieselberg, Bayerischer Wald, Champ du Feu and Weiden). This pattern indicates that each of these populations consists of several of the parent taxa growing in sympatry. Also, the inferred number of accessions assigned as one of the parent taxa, F1 hybrids, F2 hybrids and first generation backcrosses in the presented datasets suggest that we are indeed looking at neohybrids in a hybrid zone rather than hybrid taxa (Figures 6 A-C). Intragametophytic selfing could potentially aid the establishment and genetic ‘fixation’ of a new species of hybrid origin (Haufler, 2002). A hybrid could hypothetically produce viable spores, each germinating into a gametophyte which fertilizes itself, thereby becoming effectively isolated from its parent species. Being entirely homozygous, but displaying alleles diagnostic for the different taxa at different, unlinked loci, such individuals should be possible to identify. However, the observed intraspecific variation (Paper IV) strongly suggests that intragametophytic selfing is not a common phenome- non in Diphasiastrum, corroborating previous reports that intergametophytic mating predominates among homosporous lycopods (Soltis and Soltis, 1988). Presented results (Paper I, Paper III, and Paper IV) fail to verify the presence of evolutionary isolated lineages of hybrid origin. Hence, future studies addressing evolutionary as well as ecological questions in Diphasiastrum should not assume the presence of taxa of homoploid hybrid origin. Also, Central European conservation management plans should primarily consider the parent taxa, not the recognized hybrid entities. Further investigations of natural hybrids would nonetheless be valuable among long-lived and difficult to propagate organisms, such as Diphasias- trum, because of the availability of highly recombinant hybrid genotypes (Rieseberg et al., 2000). Also, hybrid speciation as the result of introgression of single genes controlling traits with adaptive potential may cause reproductive isolation and consequently speciation (Jiggins et al., 2008), and such a possibly are not accounted for in this study. Acknowledging the observed

28 number of observed reticulate events in Diphasiastrum, future studies should focus on the consequences of interspecific gene flow with respect to the integrity of the parent taxa.

29 Svensk sammanfattning (Swedish summary)

En hybrid uppstår när två olika arter korsar sig. Botanister har länge sett att växter hybridiserar. Evolutionen kan därmed liknas vid ett nätverk där arvsmassa utbyts mellan arter. Ibland leder hybridisering till att helt nya arter uppstår. De kombinerar alltså arvsmassan från två föräldraarter men skiljer sig från dem både utseendemässigt och ekologiskt. Lummerväxter hör till en grupp växter som dominerade växtligheten på jordklotet under karbontiden för lite mer än 300 miljoner år sedan. Men bland annat klimatförändringar var en av orsakerna till att de blev utkonkur- rerade av ormbunksgrupper och tidiga fröväxter. Av de överlevande grup- perna bland lummerväxterna finns en hög frekvens av artbildning genom hybridiseringar. Släktet Diphasiastrum är inte något undantag, och minst sju av släktets 25 arter antas ha hybridursprung. I artikel I använder jag information från DNA-sekvenser för att förstå släktskapet mellan arterna i Diphasiastrum som inte är hybrider. Det har tills nu varit okänt. Jag visar också att Diphasiastrum är en enhetlig grupp. De flesta arterna finns i Nordamerika, Europa och Asien. Några arter är unika för öar i Stilla havet och Atlanten. Det finns också enstaka arter i Sydafrika och Sydamerika. I Skandinavien är fjällummer, plattlummer och cy- presslummer de vanligaste arterna. De har en relativt stor utbredning och de antas (i olika kombinationer) vara föräldraarter till flera av de beskrivna hybriderna. Resultatet från Artikel I är viktigt eftersom hybriderna kombinerar anlag som är karakteristiska för de olika föräldraarterna. Genom vidare släktskapsanalyser kan jag identifiera vem som är förälder till de olika hybriderna, och i Artikel II, III och IV analyserar jag flera olika hybrider med olika typer av data. Det finns två typer av hybrider: de som har samma antal kromosomer som föräldraarterna och de som har dubbelt så många kromosomer som föräldra- arterna, dvs. polyploider. Jag har funnit exempel på båda typerna hos Dipha- siastrum. Hybrider som har samma antal kromosomer som föräldrarna är väl de mest kända, t ex mula och mulåsna. De är som regel sterila. Men under vissa betingelser kan ändå nya arter uppstå. När mycket avkomma pro- duceras kan en liten fraktion av den första generationen hybrider vara fertila. Om föräldraarterna är relativt genetisk lika och hybriden växer isolerad i en miljö där den inte konkurrerar med föräldraarterna har den en potentiell chans att etablera sig som en egen art. Mina resultat i Artikel III och IV bekräftar att det finns flera hybrider i Diphasiastrum med samma antal kromosomer som föräldraarterna. Jag har påvisat alla de tre möjliga kombinationerna av de tre skandinaviska arterna. Mina resultat indikerar också att dessa i huvudsak är första generations- hybrider och att det är osäkert om de påvisade hybriderna faktiskt repre- senterar egna arter. Alla individerna jag studerat finns i samma område som

30 en eller båda av föräldraarterna. Det är alltså sannolikt att de relativt ”nya”, första generationens, hybrider jag har identifierat har grundat ”populationer” – hybridzoner – och att dessa har uppstått flera gånger på olika ställen. Tätheten av liknande hybridzoner i Europa och Nordamerika har lett till att flera av hybriderna blivit beskrivna som egna arter. Ur ett evolutionärt per- spektiv är inte detta så intressant i sig. Men om en av hybriderna korsar sig tillbaka med en av sina föräldrar kan det leda till att gener förs mellan föräldraarterna. Jag har sett indikationer på detta i mina data, och konsek- venserna av den här processen är spännande. Om en art får tillgång till delar av en arvsmassa som redan fungerar bra för en annan art kan arten snabbare anpassa sig till nya betingelser än vid en process som beror på slumpmässiga mutationer. Slutprodukten kan också i det här fallet vara uppkomsten av en helt ny art. Polyploidi, kromosomfördubbling, är ett ganska vanligt fenomen ute i na- turen. Det finns också många polyploider bland kulturväxter, till exempel hos våra vanliga sädesslag, majs, ris, bomull, tobak, soja, bananer och orkidéer. Hybrider som har genomgått kromosomtalsfördubbling är ganska stabila och korsar sig sällan tillbaka med föräldraarterna eftersom skillnaden i kromosomantal effektivt förhindrar detta. Det är följaktligen lättare att tala om ”hybridarter” när det gäller polyploida hybrider. Två arter i Diphasiastrum, en från Afrika (D. zanclophyllum) och en från Asien (D. wightianum), har tills nu antagits ha dubbelt så många kromosomer som de andra arterna i släktet. I Artikel II använder jag information från genetiska markörer kombinerat med uppskattningar av deras kromosomantal för att bekräfta att de två arterna faktiskt är polyploider och för att identifiera deras ursprung. Den afrikanska polyploiden är produkten av en korsning mellan en art främst känd från Nordamerika och en hittills oidentifierad art. Från Asien blev två polyploider med olika ursprung upptäckta, och de är båda ett resultat av en korsning mellan andra arter som framförallt har sin utbredning i Asien. Hybrider antogs länge vara en evolutionär återvändsgränd. Om en individ producerar en steril avkomma slösar den energi på något som inte bidrar till artens fortlevnad. Skulle avkomman mot förmodan vara fertil ansågs det vara ett hot mot den biologiska mångfalden. Man antog att en sällsynt art kunde bli ”mer lik” en vanligare, närbesläktad art. Forskningsresultat som i allt större grad talar för att hybridisering är en effektiv anpassningsmekanism i en kontinuerligt föränderlig miljö sätter nu tidigare uppfattningar på prov.

31 Acknowledgments

I am first and foremost grateful to my supervisor Niklas Wikström – it has been inspiring and great fun working with you. Many thanks to Johannes Vogel for initiating the project, Martin Lascoux for enthusiastic input, Niclas Gyllenstrand for being an effective and patient motivator in the lab, Johann Greilhuber and Eva M. Temsch for contributing the flow densitometry data and Mats Thulin for continuous feedback and for instigating the last article. Alan Currier, Arthur Gilman, Bente Eriksen, Eli Fremstad, David Lor- ence, Richard Milne, Ingar Pareliussen, Bjørnar and Ingunn Pettersen, Bjørn Sivertsen, Torbjørn Stokke, Elisabeth Stur, Andreas Tribsch and Xian-Chun Zhang generously provided locality information and plant material. Anders Larsson kindly supplied the map shown in the thesis summary. Lots of thanks to Lars Nilsson and Nahid Heidari for help and support at the Systematic' lab, and to Kerstin Santesson, Susanne Gustafsson and eve- rybody else at the Evolutionary Functional Genomics' lab for answering my many questions. I am grateful to Katarina Andreasen, Catarina Ekenäs, Per Erixon, Petra Korall, Åsa Kruys and Magnus Popp for giving me quick and constructive feedback on my manuscripts. Gemma Atkinson, Sandra Baldauf, Bengt Ox- elman and Leif Tibell also contributed useful comments. Thanks to Jesper Kårehed for translating the Swedish summary from Norwegian. Additional thanks to Per for confirming that the office I have occupied the last few years actually is situated in another timezone... Thanks to all PhD students, master students, post docs, senior researchers and administrative staff I have encountered at the Department of Systematic Biology – and in the corridors here at EBC – it has been quite the experience. Special thanks to Anja Rautenberg and Tanja Slotte for an infinite number of discussions! Sigurd M. Såstad is posthumously acknowledged for convincing me that Uppsala was the place to go. Thanks to Ingeborg, Marte, Linda, Camilla, Mali, Sølvi and all other friends back home in Trondheim, without whom I could definitely not have done this. My extraordinary parents - cave et aude.

32 References

Anderson, E.C. & Thompson, E. A. 2002. A model-based method for identifying species hybrids using multilocus genetic data. Genetics 160: 1217-1229. Arnold, M.L. 1997. Natural hybridization and evolution. Oxford Univ. Press, New York. Bennert, W.B. 1999. Die seltenen und gefährdeten Farnpflanzen Deutschlands. Biologie, Verbreitung, Schutz. Landwirtschaftsvlg Münster-Hiltrup. Birky Jr., C.W. 2001. The inheritance of genes in mitochondria and chloroplasts: Laws, mechanisms and models. Annu. Rev. Genet. 35: 125-148. Buerkle, C.A., Morris, R.J., Asmussen, M.A. & Rieseberg, L.H. 2000. The likeli- hood of homoploid hybrid speciation. Heredity 84: 441-451. Buerkle, C.A. & Rieseberg, L.H. 2001. Low intraspecific variation for genome isolation between hybridizing sunflower species. Evolution 55: 684-691. Buerkle, C.A. & Rieseberg, L.H. 2007. The rate of genome stabilization in homoploid hybrid species. Evolution 62: 266-275. Doyle, J.J., Doyle, J.L., Rauscher, J.T. & Brown, A.H.D. 2003. Diploid and polyploid reticulate evolution throughout the history of the perennial soybeans (Gly- cine subgenus Glycine). New Phytol. 161: 121-132. Frey, W., Frahm, J.-P., Lobin, W. & Fischer, E. 2006. Liverworts, mosses and ferns of Europe. Harley Books, Colchester. Gaggermeier, H. 1993. Zur aktuellen Verbreitung der Flachbärlappe Diphasium alpinum (L.) Rothm. und Diphasium issleri (Rouy) Holub im Vorderen Bayerischen Wald. Der Bayerischer Wald 7: 7-11. Gow, J.L., Peichel, C.L. & Taylor, E.B. 2006. Contrasting hybridization rates between sympatric three-spined sticklebacks highlight the fragility and reproductive barriers between evolutionary young species. Mol. Ecol. 15: 739-752. Haufler, C. 2002. Ferns and other homosporous vascular plants have highly polyploid chromosome numbers, but they express traits following diploid models and, although capable of extreme inbreeding, are predominantly outcrossing. BioScience 52(12): 1081-1093. Haufler, C. 2008. Species and speciation. Pp. 303-324 in Ranker, T.A. & Haufler C.H. (eds.), Biology and evolution of fern and lycophytes. Cambridge University Press, Cambridge. Hegarty, M.J. & Hiscock, S.J. 2005. Hybrid speciation in plants: new insights from molecular studies. New Phytol. 165: 411-423. Holub, J. 1975. Diphasiastrum, a new genus in Lycopodiaceae. Preslia 47: 97-110. Holub, J. & Procházka, F. 2000. Red list of vascular plants of the Czech Republic. Preslia 72: 187-230. Horn, K., Strobel, C. & Bennert, H.W. 2001. Die Bestandssituation gefährdeter Farnpflanzen (Pteridophyta) in Bayern – ein erster Bericht über Planung und Durchfürung von Schutz – und Pflegemanahmen. Bay. L. f. U. 156: 139-174. Horn, K. & Bennert, H.W. 2002. Diphasiastrum oellgaardii Stoor & al. (Lycopodiaceae, Pteridophyta), eine neue Flachbärlapp-Art fur die Flora von Östterreich. Phyton 42: 123-148.

33 Issler, E. 1910. Über 3 in den Vogesen vorkommende Lycopodien-Formen aus der complanatum-Gruppe. Mitt. Philomat. Ges. Elsass-Lothr. 4: 438-442. Jermy, A.C. 1993. Diphasiastrum J. Holub. In: T.G. Tutin, N.A. Burges, A.O. Chater, J.R. Edmonson, V.H. Heywood, D. M. Moore, D.H. Valentine, S.M. Walters and D.A. Webbs, (eds.), Flora Europaea. Vol. 1. Cambridge. Jiggins, C.D., Salazar, C., Linares, M. & Mavárez, J. 2008. Hybrid trait speciation and Heliconius butterflies. Phil. Trans. R. Soc. B. 363: 3047-3054 Kronforst, M.R., Young, L.G., Blume, L.M. & Gilbert, L.E. 2006. Multilocus analyses of admixture and introgression among hybridizing Heliconius butterflies. Evolution 60(6): 1254-1268. Lawalrée, A. 1957. Un Lycopode ardennais méconnu, Lycopodium issleri. Bull. Soc. Roy. Bot. Belg. 90: 109-120. Linder, C.R. & Rieseberg, L.H. 2004. Reconstructing patterns of reticulate evolution in plants. Amer. J. Bot. 91: 1700-1708. Lexer, C., Lai, Z. & Rieseberg, L.H. 2003. Candidate gene polymorphisms associated with salt tolerance in wild sunflower hybrids: implications for the origin of Heli- anthus paradoxus, a diploid hybrid species. New Phytol. 131: 255-233. Mallet, J. 2007. Hybrid speciation. Nature 446: 279-283 Mavárez, J., Salazar, C.A., Bermingham, E., Salcedo, C. & Jiggins, C.D. 2006. Speciation by hybridization in Heliconius butterflies. Nature 441: 868-871. Nakhleh, L., Warnow, T. & Linder, C.R. 2004. Reconstructing reticulate evolution in species: theory and practice. Pp. 337-346 in: Proceedings of the Eighth An- nual International Conference on Research in Computational Molecular Biol- ogy. San Diego. Ninan, C.A. 1958. Studies on the cytology and phylogeny of the Pteridophytes II. Observations on the genus Lycopodium. Proc. Natl. Inst. Sci. India 24: 54-66. Øllgaard, B. 1987. A revised classification of the Lycopodiaceae s. lat. Opera Bot. 92: 153-178. Otto, S.P. & Whitton, J.W. 2000. Polyploid incidence and evolution. Annu. Rev. Genet. 34: 401-437. Page, C.N. 2002. Ecological strategies in fern evolution: a neopteridological over- view. Rev. Palae. Paly. 2428: 1-33. Popp, M., Erixon, P., Eggens, F. & Oxelman, B. 2005. Origin and evolution of a circumpolar polyploid species complex in Silene (Caryophyllaceae) inferred from low copy nuclear RNA polymerase introns, rDNA, and chloroplast DNA. Syst. Bot. 30(2): 302-313. Ramsey, J. & Schemske, D.W. 1998. Pathways, mechanisms and rates of polyploid formation in flowering plants. Annu. Rev. Ecol. Syst. 29: 467-501. Ramsey, J. & Schemske, D.W. 2002. Neopolyploidy in flowering plants. Annu. Rev. Ecol. Syst. 33: 589-639. Rieseberg, L.H. & Ellstrand, N.C. 1993. What can molecular and morphological markers tell us about plant hybridization? Crit. Rev. Plant. Sci. 12(3): 213-241. Rieseberg, L.H. 1997. Hybrid origins of plant species. Ann. Rev. Ecol. Syst. 28: 359-389. Rieseberg, L.H., Baird, S.J.E. & Gardner, K.A. 2000. Hybridization, introgression and linkage evolution. Pl. Mol. Biol. 42: 205-224. Rieseberg, L.H. & Willis, J.H. 2007. Plant speciation. Science 317: 910—914. Slotte, T., Ceplitis, A., Neuffer, B., Hurka, H. & Lascoux, M. 2006. Intrageneric phylogeny of Capsella (Brassicaceae) and the origin of tetraploid C. bursa- pastoris based on chloroplast and nuclear DNA sequences. Am. J. Bot. 93: 1714-1724.

34 Slotte, T., Huang, H., Lascoux, M. & Ceplitis, A. 2008. Polyploid speciation did not confer instant reproductive isolation in Capsella (Brassicaceae). Mol. Biol. Evol. 25(7): 1472-1481. Small, R.L., Cronn, R.C. & Wendel, J.F. 2004. Use of nuclear genes for phylogeny reconstruction in plants. Austr. Syst. Bot. 17: 145-170. Soltis, D.E. & Soltis, P.S. 1993. Molecular data and the dynamic nature of polyploidy. CRC Crit. Rev. Plant. Sci. 12 : 243-273. Soltis, D.E. & Soltis, P.S. 1999. Polyploidy: recurrent formation and genome evolution. Trends Ecol. Evol. 14(9): 348-352. Soltis, D.E. & Soltis, P.S., Schemske, D.W., Hancock, J.F., Thompson, J.N., Hus- band, B.C. & Judd, W.S. 2007. Autopolyploidy in angiosperms: have we grossly underestimated the number of species? Taxon 56: 13-30. Soltis, P.S. & Soltis, D.E. 1988. Estimated rates of intragametophytic selfing in Lycopods. Amer. J. Bot. 75: 248-256. Stoor, A.M., Boudrie, M., Jérôme, C., Horn, K. & Bennert, H.W. 1996. Diphasias- trum oellgaardii (Lycopodiaceae, Pteridophyta), a new lycopod species from Central Europe and France. Feddes Rep. 107: 149-157. Thompson, J.D. & Lumaret, R. 1992. The evolutionary dynamics of polyploid plants: origins, establishment and persistence. Trends Ecol. Evol. 7(9): 302-307. Ungerer, M.U., Baird, S.J.E., Pan, J. & Rieseberg, L.H. 1998. Rapid hybrid speciation in wild sunflowers. Proc. Natl. Acad. Sci. USA 95: 11757-11762. Victorin, F.M. 1925. Les Lycopodinées du Quebec et leurs formes mineures. Contrib. Lab. Bot. Univ. Montreal 3. Montreal. Vogel, J.C., Russel, S.J., Rumsey, F.J., Barrett, J.A. & Gibby, M. 1998. Evidence for maternal transmission of chloroplast DNA in the genus Asplenium (Aspleni- aceae, Pteridophyta) Bot. Acta 111: 247-249. Vogel, J.C. & Rumsey, F.J. 1999. On the status of Diphasiastrum oellgaardii (Ly- copodiaceae, Pteridophyta). Feddes Rep. 110: 439-445. Wagner, F.S. 1992. Cytological problems in Lycopodium sens. lat. Ann. Missouri Bot. Gard. 79: 718-729. Wagner, W.H., Wagner, F.S. & Beitel, J.M. 1985. Evidence for interspecific hybridization in pteridophytes with subterranean mycoparasitic gametophytes. Pp. 273-281 in A.F. Dyer & C.N. Page (eds.), Biology of pteridophytes. Proc. Roy. Soc. Edinburgh 86B. Wagner, W.H. & Beitel, J.M. 1993. Lycopodiaceae, vol. 2, Pteridophytes and Gym- nosperms. In: Flora of North America Editorial Committee (eds.), Flora of North America: North of Mexico. Oxford University Press, Oxford. Whittier, D.P. 1977. Gametophytes of Lycopodium obscurum as grown in axenic culture. Canad. J. Bot. 55: 563-567. Wilce, J.H. 1961. Lycopodium complanatum L. and four new allied species of the Old World. Nova Hedwigia. 3: 93-117. Wilce, J.H. 1965. Section Complanata of the genus Lycopodium. Beih. Nova Hed- wigia 19: 1-233.

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