Cytogenetics and Spore Morphology of Struthiopteris Spicant Var. Fallax

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Cytogenetics and spore morphology of Struthiopteris spicant var. fallax

Jóhannes Bjarki Urbancic Tómasson

Líf- og umhverfisvísindadeild Háskóli Íslands 2017

Cytogenetics and spore morphology of Struthiopteris spicant var. fallax

Jóhannes Bjarki Urbancic Tómasson

12 eininga ritgerð sem er hluti af Baccalaureus Scientiarum gráðu í líffræði

Leiðbeinandi Kesara Margrét Anamthawat-Jónsson

Líf- og umhverfisvísindadeild Verkfræði- og náttúruvísindasvið Háskóli Íslands Reykjavík, maí 2017

Cytogenetics and spore morphology of Struthiopteris spicant var. fallax 12 eininga ritgerð sem er hluti af Baccalaureus Scientiarum gráðu í líffræði

Líf- og umhverfisvísindadeild Verkfræði- og náttúruvísindasvið Háskóli Íslands Sturlugötu 7 101 Reykjavík

Sími: 525 4000

Skráningarupplýsingar: Jóhannes Bjarki Urbancic Tómasson, 2016, Cytogenetics and spore morphology of Struthiopteris spicant var. fallax., BS-ritgerð, Líf- og umhverfisvísindadeild, Háskóli Íslands, 24 bls.

Prentun: Svansprent Reykjavík, maí 2017 Útdráttur

Ofan við Deildartunguhver vex tunguskollakambur (Struthiopteris spicant var. fallax), afbrigði burkna sem hvergi er að finna annars staðar í heiminum. Þrátt fyrir sérstöðu tunguskollakambs hefur hann lítið verið rannsakaður, sérstaklega eftir að kjarngerð hans var birt árið 1968. Á síðustu árum hefur komið í ljós að gera þarf nýjar rannsóknir á tunguskollakambinum og er þessi ritgerð fyrsta birtingin í því verkefni. Sýnum fyrir litningagreiningu og rannsóknir á gróum var safnað af tunguskollakambi, spicant- og homophyllum-afbrigðum skollakambs (Struthiopteris spicant). Niðurstöður úr kjarngerðarrannsókn sýna að tunguskollakambur er tvílitna en ekki fjórlitna eins og áður var talið. Önnur afbrigði skollakambs eru fjórlitna og önnur sýni í rannsókninni en þau af tunguskollakambi höfðu fjórlitna erfðamengi. Gró tunguskollakambs báru einstök mynstur á grókápunni sem kom á óvart þar sem hingað til hefur verið talið að gró skollakambs séu áreiðanleg flokkunareinkenni. Niðurstöðurnar úr báðum hlutum rannsóknarinnar gefa til kynna að tunguskollakambur gæti flokkunarfræðilegrar sérstöðu. Þær opna dyr fyrir rannsóknum á sviði stofnvistfræði og þróun tunguskollakambs og varpa nýju ljósi á tegundamyndun.

Abstract

On top of the most powerful hotspring in Europe grows a variety of fern that is local to Iceland. The sporadic studies of this variety, S. spicant var. fallax, came to an end in 1968 after its karyotype was published. For various reasons the need to reinvestigate S. spicant var. fallax has recently become evident. Samples for karyotyping and spore morphology were collected and examined for S. spicant var. fallax, var. spicant and var. homophyllum. Results from the karyotype analysis show that S. spicant var. fallax is diploid rather than tetraploid, which is the prevalent ploidy level in S. spicant and which was observed in S. spicant var. spicant samples. Under a scanning electron microscope, the spores of S. spicant var. fallax showed unique features in ornamentation when compared to spores of other S. spicant varieties. The karyotype results and the unique spore morphology of S. spicant var. fallax challenge the current taxonomy of S. spicant var. fallax. This opens up numerous questions regarding the population ecology and evolution of S. spicant var. fallax and the the process of speciation.

Table of contents

Útdráttur ...... iii

Abstract ...... iii

Table of contents ...... iv

List of figures ...... v

List of tables ...... vi

Abbreviations ...... vii

Þakkir ...... ix

1 Introduction ...... 1

2 Material and methods ...... 4 2.1 Karyotype analysis ...... 4 2.2 Spore morphology ...... 4

3 Results ...... 7 3.1 Karyotype analysis ...... 7 3.2 Spore morphology ...... 9

4 Discussion ...... 12 4.1 Karyotype analysis ...... 12 4.2 Spore size and ornamentation ...... 13 4.3 Spore abortion ...... 14 4.4 Considerations about S. Spicant var. fallax population dynamics ...... 16 4.5 Models of differentiation between S. spicant var. fallax and S. spicant var. spicant ...... 17

References ...... 19

Supplement ...... 22

iv List of figures

Figure 1: A composite image of some representative cellular elements of various S. spicant samples observed with a fluorescent microscope and DAPI...... 9

Figure 2: SEM images of S. spicant spores from different S. spicant varieties bear different ornamental patterns...... 11

Figure 3: A SEM image of spores of S. spicant var. spicant in the open sporangium...... 14

v List of tables

Table 1: Samples used for spore morphology imaging...... 5

Table 2: Microscopic evidence for various ploidy levels of S. spicant chromosome samples assorted by their origin. 7

Table 3: Results of an ANOVA of equitorial spore length in relation to sample groups...... 9

Table 4: Results of a Tukey‘s honest significance test for equitorial spore length in relation to the sample groups...... 10

Table 5: Summarization of spore discriptions from each sample...... 10

vi Abbreviations

°C Degrees celcius

°N Degrees north

°W Degrees west

μm Microlitre

B. Blechnum cm Centimetre ml Millilitre

Inc. sed. Incertae sedis

P:E ratio Polar-Equitorial raito

S. Struthiopteris

SEM Scanning electron microscope var. variety

vii

viii Þakkir

Ég vil þakka Kesöru fyrir þolinmæðina og öll þau tækifæri sem hún hefur boðið mér. Ég vil einnig þakka samnemendum mínum í líffræðinni fyrir frábærar stundir í BS-náminu. Engu síður vil ég þakka öllum þeim sem hafa þurft að hlusta á burknatalið í mér síðastliðið ár.

ix 1 Introduction

On top of the most powerful hotspring in Europe grows a little known variety of an otherwise common fern, Struthiopteris spicant (L.) F.W.Weiss., or deer fern. This hotspring is Deildartunguhver and the plant variety is S. spicant var. fallax, which taxonomical status has, perhaps erroneously, remained mostly uncontested since 1968.

Struthiopteris spicant belongs to the Blechnaceae family of leptosporangiate ferns. The classification of S. spicant is not well established and has remained especially disarranged after the sequencing of cpDNA of some Blechnaceae species. Traditionally S. spicant belonged to the genus Blechnum (L.), as Blechnum spicant (L.) Sm., which was divided into 8, 9 or 12 subgenera by taxanomists based on spores, frond morphology, rhizomes, dimorphism and habit (Passarelli et al., 2010; Gabriel y Galán et al., 2013). According to this classification, B. spicant was placed in the penna-marina group of Blechnaceae ferns. Sequencing of the cpDNA of some American ferns by Gabriel y Galán et al. (2013) revealed a position for B. spicant at the base of the tree, while the penna-marina group was highly supported and nested deep within the tree. It was therefore suggested that B. spicant were placed in a separate clade, apart from other Blechnum species (Gabriel y Galán et al. 2013). Subsequently, a similar study of ferns of Australasia by Perrie et al. (2014) found a similar position for B. spicant on the evolutionary tree. The paper mentioned the possibility of placing B. spicant in its former clade Struthiopteris Scop. but opted for a more modest solution of segregating Brainea J.Sm. and Sadleria Kaulf. from Blechnum and left the task of classifying B. spicant further to later taxonomists (Pierre et al. 2014). A recent paper has reinstated the issue. The paper by de Gasper et al. (2016) aimed to clarify and expand the analyses of Pierre et al. (2014) and Gabriel y Galán et al. (2013). De Gasper et al. (2016) concluded their paper by suggesting that B. spicant be placed in Struthiopteris as S. spicant (de Gasper et al., 2016). In this paper I will refer to this species as Struthiopteris spicant in accordance with its most recent classification.

Struthiopteris spicant is distributed throughout northern temperate and arctic regions (Cousens, 1981; Soltis & Soltis, 1988; Gabriel y Galán et al., 2013) being native to Pacific North America, Europe to the Caucasus, North Africa (Gabriel y Galán et al. 2013) and Iran (Mazooji & Salimpour 2014). Struthiopteris spicant has a compact erect stem with frond rosettes growing from the apex, creeping rhizomes and dimorphic fronds (de Gasper et al. 2016). The sterile fronds are pinnate, lanceolate in the proportions 1:5 and form a rosette. The fertile fronds are longer than the sterile fronds, erect with narrower and more dispersed pinnae (hence its Icelandic name ‘devil’s comb’) which underside bears sporangia in continuous bands. Typically, plants range from 8-70 cm tall, with slight variation in allometry depending on location. Some varieties of S. spicant are classified, most notably S. spicant var. spicant, the common deer fern, S. spicant var. homophyllum, a homophyllous variety found on the Iberian Peninsula and S. spicant var. fallax, considered to be a geothermal variety of S. spicant but it is little known and is commonly ignored in academic press (Hallgrímsson, 1968; Löve & Löve, 1968; Flora of Iceland, 2016; Icelandic Institute of Natural History, 2017). Struthiopteris spicant var. fallax is only found in Iceland (Wasowicz et al., 2017a).

Struthiopteris spicant is rather uncommon in Iceland with around a hundred sites of reported findings (Flora of Iceland, 2016). There are two varieties of S. spicant in Iceland, S. spicant var.

1 spicant, by far the dominant variety, and S. spicant var. fallax, found at only one location, Deildartunguhver (Wasowicz et al., 2017b). In Iceland S. spicant var. spicant generally grows to 10-50 cm (Löve & Löve 1968; Flora of Iceland, 2016), but most commonly it ranges between 25-40 cm (Löve & Löve 1968). It grows in lowland areas, usually below 200 m, often in basins or crevices frequented by heavy snows (Flora of Iceland, 2016). The S. spicant var. fallax grows on a slope above the hotspring Deildartunguhver – a completely different habitat. The ground there is hot, devoid of snows and soil is nearly absent. The two varieties differ in more than habitat alone. Struthiopteris spicant var. fallax is minuscule compared to the common variety. Measuring between 2-8 cm, it usually grows to mere 2-5 cm. The fronds of S. spicant var. fallax are prostrate but S. spicant var. spicant has raised and slightly draping leaves. Probably the most important characteristic of S. spicant var. fallax is its monomorphism. Instead of having architecturally different fertile and sterile leaves, S. spicant var. fallax has only one type of leaves which bears spores (Löve & Löve, 1968; Flora of Iceland, 2016).

The fallax variety of S. spicant was first noted by a Danish botanist, Christian Grönlund, in 1876 (Hallgrímsson, 1968; Löve & Löve, 1968). Grönlund collected the variety and grew it in his home country. He discovered that the variety held its characteristics despite the different climate and was therefore not an ecomorph of S. spicant (Hallgrímsson, 1968). This was repeated by later botanists and the plant was even grown in Akureyri Botanical Garden for some time (Hallgrímsson, 1968; Löve & Löve 1968). Consequently, some botanists claimed that the fallax varitety should be classified as a distinct subspecies or species due to its unique features and phenotypic consistency (Hallgrímsson, 1968). However, the classification of S. spicant var. fallax became less certain after the discovery of a second homophyllous variety of S. spicant on the Iberian Peninsula, S. spicant var. homophyllum. The idea that S. spicant var. homophyllum and S. spicant var. fallax were the same variety was immediately rejected due to obvious morphological differences between the two varieties (Hallgrímsson, 1968). Struthiopteris spicant var. homophyllum is 8-20 cm in size, its leaves are hanging, lanceolate in the proportions 1:10 and slightly curved while those of var. fallax are prostrate, lanceolate in the proportions 1:5 and straight (Löve & Löve, 1968). Despite botanists agreeing on the different taxonomical status of S. spicant var. fallax and S. spicant var. homophyllum, no explanation for the homophylly of these two varieties has been presented to date.

The chromosome number of S. spicant from various regions has repeatedly been reported as 2n = 68 for the sporophyte of S. spicant var. spicant (Manton, 1950 p. 122; Löve & Löve, 1961; Löve & Löve, 1968; Tryon & Tryon 1982, p. 678) and as n = 34 for the gametophyte of S. spicant var. homophyllum (Horjales et al., 1990). One report exists regarding the basic chromosome number of the genus Struthiopteris, which is inexplicitly reported there as x = 31 or 34 without further notes (de Gasper et al., 2016).

Only a single report of the karyotype of S. spicant var. fallax exists, from Löve & Löve (1968) in which they simply mention that the karyotype of S. spicant is known to be 2n = 68 and that they can confirm the same chromosome number for S. spicant var. fallax (Löve & Löve, 1968). No information about the sample origin, experimental methods nor further descriptions of their results were presented. For this reason I find it important to reinvestigate the chromosome number of S. spicant var. fallax using plant material collected in the present study.

Löve & Löve‘s karyotype report (1968) seems to have quelled the idea that S. spicant var. fallax be classified as a separate subspecies or species (Hallgrímsson, 1968), perhaps for ambiguous reasons. Löve & Löve (1968) point out that hybrids between the Iberian S. spicant var. homophyllum and the common deer fern, S. spicant var. spicant, had been observed and that

2 the reason why the two varieties could hybridize was because of their identical chromosome number. Consequently they argue that the same must apply to S. spicant var. fallax and S. spicant var. spicant, because the two varieties have the same chromosome number (Löve & Löve, 1968), but which hybrids have never been observed. They declared the explanation for the absence of S. spicant var. fallax x S. spicant var. spicant hybrids to be geographical barriers (Löve & Löve, 1968). Although this explanation appears to have been immediately accepted by botanists (Hallgrímsson, 1968), its validity was questionable at the time and in retrospect it is most definitely incorrect. Firstly, there could be reproductive barriers between S. spicant var. fallax and S. spicant var. spicant other than ploidy and geography, such as hybrid inviability, sterility, hybrid breakdown, other gametic incompatibilities, temporal isolation in spore production or various forms of habitat isolation (Riesberg & Willis, 2007). This criticism is especially important since S. spicant var. fallax and S. spicant var. homophyllum probably evolved independently, something that Löve & Löve themselves acknowledge (Löve & Löve, 1968), and therefore it is not obvious that the same reproductive barrier would apply to both varieties when crossed with S. spicant var. spicant. Secondly, the distribution of S. spicant in Iceland was probably either much less extensive or less known when Löve & Löve put forward their hypothesis (Friðriksson, 1932; Guðmundsson, 1940; Löve, 1948; Hallgrímsson, 1968; Löve & Löve, 1968; Flora of Iceland, 2016; Icelandic Institute of Natural History, 2017). The current distribution of S. spicant in Iceland does not indicate that geography played a role as a barrier to hybridization as S. spicant var. spicant grows in at least 2-3 other locations in Borgarfjörður, the fjord where Deildartunguhver is situated (Flora of Iceland, 2016; Icelandic Institute of Natural History, 2017). Struthiopteris spicant in North America has been shown to lack population genetic structure, likely due to its ability to disperse spores effectively and due to its outcrossing mating system. Despite some founder effect observed in small, isolated populations of S. spicant composed of 100-200 plants (Soltis & Soltis, 1988), the level of intragametophytic selfing does only reach 40.3% in the most extreme case (Soltis & Soltis, 1987, 1988). Additionally, recent research from de Gasper et al. (2016) has found closely related species of Blechnum at opposite sides of the Atlantic Ocean. The current theory is that these related species have arisen due to frequent wind dispersal of spores across the Atlantic (de Gasper et al., 2016). In light of this, geographical barriers should not be a significant obstacle to dispersal of spores and varietal interbreeding, as Löve & Löve (1968) indicate in their writings.

Previous research on the geothermal variety S. spicant var. fallax is scarce and fundamental questions about the nature of this variety remain unexplored. No author has yet hypothesized about the origins and evolution of this variety, let alone provided data on the subject. Neither have the spores of S. spicant var. fallax been described, but spores are an important diagnostic character of ferns (Brown, 1960; Wagner, 1974; Tryon & Tryon, 1982). This paper is part of a research project which I was lucky enough to be part of. The objective of this project is to answer the abovementioned elementary questions regarding the origin and taxonomy of S. spicant var. fallax. In this paper I will compare some characteristics of spores from S. spicant var. fallax with other S. spicant varieties and populations and attempt to confirm the results from Löve & Löve‘s karyotyping of S. spicant var. fallax made in 1968. Hopefully, I will be able to present novel ideas about the evolution and nature of S. spicant var. fallax.

3 2 Material and methods 2.1 Karyotype analysis

Methods for karyotype analysis were based on an article by Anamthawat-Jónsson (2003) with minor deviations as the method was developed for birch (Betula pubescens).

Samples for karyotype analysis were collected from five locations in Iceland between 5th and 9th of july 2016. These locations were under Mount Trölladyngja in Reykjanes Peninsula, Brennisteinsalda in Landmannalaugar, Deildartunguhver, Svanshóll in Bjarnarfjörður and in Héðinsfjörður (see supplement). Between two and four fiddleheads, depending og their size, were collected from individual plants and placed in 15 ml plastic tubes containing 10 ml of frozen tap water and 2-3 ml of liquid tap water in order to keep the sampled tissue alive and to prevent frost damage to the samples. Where fiddleheads were not available, young leaf tips were collected. The samples were placed in a styrofoam container filled with ice for 24-27 hours in order to arrest mitosis in metaphase before fixation.

The fixative was prepared from one part glacial acetic acid and three parts 96% ethanol. The ingredients were cooled down before fixative preparation. Fresh fixative was made daily and kept cool before use. After 24-27 hours samples were ready for fixation. Samples were transferred from the ice water, quickly dried on tissue paper and placed in a new 5 ml tube filled with fresh fixative, maintaining single plant collection. After transfer the fixed samples were kept at room temperature for two hours during which time the fixative was replaced once with a disposable pipette. After two hours at room temperature the samples were stored at -20°C until use. 2.2 Spore morphology

Spore samples were prepared from fertile leaves that had been dried in paper envelopes. One of the plants that were used for karyotype analysis of S. spicant var. fallax, DE_03 from Deildartunguhver in Iceland was also used for analysis of spore morphology. Another fertile leaf was collected from a plant in Bjarnarfjörður but that individual plant did not donate material for karyotype analysis. Three samples of S. spicant var. spicant were sent from the Azores and other 11 samples were sent from Spain, including spores from S. spicant var. spicant, S. spicant var. homophyllum and what is believed to be a new morphotype of S. spicant. Some plants from which spore samples were collected fit only partly with known variants of S. spicant. These samples are denoted by incertae sedis (table 1).

4 Table 1: Samples used for spore morphology imaging. Respective varieties, sampling locations and GPS- coordinates are presented as they were given by the sampling collectors. The abbreviation Inc. sed. stands for Incertae sedis.

Sample ID Variety Location GPS-Coordinates

Pawel 425 Spicant Terceira, Azores 38.750127°N 27.213083°W

Pawel 427 Spicant Terceira, Azores 38.750127°N 27.21308°W Pawel 430 Spicant Terceira, Azores 38.08940°N 27.182597°W Deild 02 Fallax Deildartunguhver, Borgarfjörður 64.66348°N 21.41075°W Bjarn 02 Spicant Svanshóll, Bjarnarfjörður 65.78971°N 21.55871°W

AS JND09 Spicant Valdés, Paladeperre 43.783331°N 6.56667°W

AS JND10 Inc. sed. Valdés, Paladeperre 43.783331°N 6.56667°W AS JND11 Inc. sed. Valdés, Paladeperre 43.783331°N 6.56667°W AS JND12 Inc. sed. Valdés, Paladeperre 43.783331°N 6.56667°W AS JND21 Inc. sed. Valdés, Paladeperre 43.783331°N 6.56667°W

AS JND22 Spicant Valdés, Paladeperre 43.783331°N 6.56667°W

GA JND04 Homophyllum Tabagón/Tomiño, Pontevedra 41.93333°N 8.78333°W SA JND02 Inc. sed.. San Miguel de Valera, Salamanca 40.55000°N 5.91667°W SA JND03 Spicant San Miguel de Valera, Salamanca 40.55000°N 5.91667°W

SA JND04 Inc. sed. San Miguel de Valera, Salamanca 40.55000°N 5.91667°W

SA JND11 Inc. sed. Las Betuecas, Salamanca 40.45000°N 6.13333°W

Spore morphology was observed with a scanning electron microscope (SEM), model JEOL JSM 6610LA. Preparation of SEM specimens was done by carefully scraping spores from the underside of dried leaves onto a sticky carbon dot that was later attached to a specimen stub. The samples were viewed without any coating at low vacuum (20Pa) and high voltage (10 kV), and an arbitrary site from each specimen was photographed at 50x magnification. Individual spores were observed and photographed at 500x and 1000x magnification. Some samples were photographed at 900x magnification. From each sample some individual spores were selected as representative spores for the whole sample.

The measurement of spores was made in a computer by using pixle coordinates and Pythagoras‘ theorem to calculate the length of the spores from SEM images magnified 500, 900 or 1000 times. In all samples the perispore (the outermost layer of the spore) was measured. The equitorial axis was measured for all spores as in Gómez-Noguez et al. (2016). The equitorial axis for irregularly shaped spores was defined as the length of the spore parallel to the laesura. Polar axis length was measured where visible from the images so that the polar axis were perpendicular to the equitorial axis. The P:E ratio (Polar-Equitorial ratio) was calculated from the measurements as applicable. Simple calculations were done in Microsoft Excel 365 Pro+

5 and statistics were done in R Studio version 0.99.491. Measurements for spore polar axis were not applied as it was rarely visible on SEM photographs from a proper angle.

Description of spore morphology was done from SEM images and the terminology from Passarelli et al. (2010) was used to describe ornamental patterns.

6 3 Results 3.1 Karyotype analysis

Results from karyotyping were quite unclear as few cells from the samples taken were actively dividing and samples with visible chromosomes usually had a large and tight metaphase. Precise chromosome number could therefore not be counted but rough estimates were possible in some cases. The number of nucleoli observed in interphase was recorded as an indicator of ploidy. The results for all sample origins was that the plants were tetraploid or likely tetraploid as they regularly demonstrated more than two nucleoli or they had a chromosome count above 50. This does not apply to S. spicant var. fallax from Deildartunguhver, which is diploid as it has roughly 30 chromosomes and one or two nucleoli (table 2; figure 1).

Table 2: Microscopic evidence for various ploidy levels of S. spicant chromosome samples assorted by their origin. Abbreviations are N/A=Not applicable, f.=figure. Notes on this table: Samples RE_02, BJ_02 and BJ_05 were prepared from fiddleheads but cellulase digestion was too long and chromosome preparation failed. Samples RE_03 and DE_03 were prepared from leaf buds that were too mature. No mitosis was visible but spores and differentiated cells were visible. Samples LA_01, DE_02 and DE_04 were also prepared from leaf buds that were too mature but no spores were visible. Samples JM_04 and JM_06 were prepared from leaf pieces that contained no mitosis and too few cells.

Sample Origin Ploidy Microscopic evidence RE_01 Reykjanes Probably tetraploid Chromosomes similar to other samples. Nucleoli 2-3 (f. 1a). RE_02 Reykjanes N/A N/A RE_03 Reykjanes N/A N/A (f. 1b) LA_01 Landmannalaugar Probably tetraploid Large, tight metaphase (f. 1c, 1d). Nucleoli 2-4 (f. 1e). DE_01 Deildartunguhver Not tetraploid Small cells. Nucleoli 1-2 (f. 1f). DE_02 Deildartunguhver Diploid Metaphase with ~30 chromosomes (f. 1g). Different chromosome morphology. DE_03 Deildartunguhver N/A N/A DE_04 Deildartunguhver N/A N/A BJ_01 Bjarnarfjörður Probably tetraploid Large, tight metaphase (f. 1h). Numerous nucleoli (f. 1i). BJ_02 Bjarnarfjörður N/A Tight metaphase. BJ_05 Bjarnarfjörður Possibly tetraploid Compact metaphase. BJ_06 Bjarnarfjörður Tetraploid Chromosome number >50 (f. 1j, 1k). At least 3 nucleoli (f. 1l). HE_01 Héðinsfjörður Probably tetraploid Large, tight metaphase. Nucleoli 2-4 (f. 1m, 1n). HE_02 Héðinsfjörður Tetraploid Chromosome number >50 (f. 1o). Nucleoli 2-4 (f. 1p). JM_04 Spain, Tabagón Probably tetraploid At least 3 nucleoli (f. 1q). JM_06 Spain, Mondariz Possibly diploid Nucleoli 1-2 (f. 1r).

8 Figure 1 (on the previous page): A composite image of some representative cellular elements of various S. spicant samples observed with a fluorescent microscope and DAPI. (a) Struthiopteris spicant var. spicant from Reykjanes, sample RE_01, showing three nucleoli, indicating polyploidy; (b) A spore from sample RE_03 has the typical bean shape of S. spicant. The perispore and the exospore are outlined; (c, d) Metaphase chromosomes of Landmannalaugar sample LA_01 showing a high number of chromosomes, indicating polyploidy; (e) An interphase cell in sample LA_01 reveals numerous nucleoli, indicating polyploidy; (f) An interphase cell from Deildartunguhver, sample DE_01, showing two nucleoli, indicating possible diploidy; (g) A metaphase cell from sample DE_01 has a somatic chromosome count of around 30 chromosomes, indicating a lower ploidy level from previously reported samples; (h) Metaphase chromosomes in Bjarnarfjörður sample, BJ_01, showing many tight chromosomes; (i) An interphase cell from BJ_01 revealing multiple nucleoli, indicating polyploidy; (j, k) Metaphase cells from sample BJ_06 show well over 50 chromosomes, indicating polyploidy; (l) An interphase cell from sample BJ_06 showing at least three nucleoli, indicating polyploidy; (m, n) Interphase cells of Héðinsfjörður sample HE_01 show three and four nucleoli, respectively, indicating tetraploidy; (o) Metaphase chromosomes in sample HE_02 are well over 50, indicating polyploidy; (p) An interphase cell in sample HE_02 shows three nucleoli, indicating polyploidy; (q) An interphase cell from S. spicant var. homophyllum sample JM_04 shows three nucleoli, indicating polyploidy; (r) Interphase cells from sample JM_06 revealing one or two nucleoli, indicating possible diploidy.

3.2 Spore morphology

A Shapiro-Wilks test on the equitorial length of the measured spores showed that the hypothesis that the samples come from a population that is normally distriputed can not be rejected (W = 0.98747; p = 0.1004). The spore sample sizes will therefore be regarded as normally distributed.

The average equitorial length of spores in this study was 44.94 μm. From a one way analysis of variance (Anova) of equitorial spore length and five ‚sampling origins‘ (Deildartunguhver, Bjarnarfjörður, var. homophyllum, Pawel, var. spicant other, Incertae sedis) there was difference between the residuals of means of these five groups (F = 10.92; Pr(>F) = 3.5e-9). Significant differences in size were found between the groups Pawel-Bjarnarfjörður (P = 0.0018), Incertae sedis-Deildartunguhver (p = 0.0028), Pawel-Deildartunguhver (p = 1.4e-07), var. spicant-incertae sedis (p = 0.0040) and var. spicant-Pawel (p = 3.5e-07; table 3). The results of the ANOVA are confirmed by a Tukey‘s honest significance test where an additional statistically significant difference was found between var. spicant and var. homophyllum (p = 0.0496; table 4).

Table 3: Results of an ANOVA of equitorial spore length in relation to sample groups. Bold letters indicate statistical significance (p < 0,05). Abbreviations are Bjarnarfj. = Bjarnarfjörður, Deildart. = Deildartunguhver, var. hom = var. homophyllum, Inc. sed. = Incertae sedis.

Bjarnarfj. Deildart. Var. hom. Inc. sed. Pawel Deildartunguhver 1.0000 - - - - Var. Homophyllum 0.8956 0.0691 - - - Incertae sedis 0.6029 0.0028 1.0000 - - Pawel 0.0018 1.4e-07 1.0000 0.1706 - Var. Spicant 1.0000 1.0000 0.0662 0.0040 3.5e-07

9 Table 4: Results of a Tukey‘s honest significance test for equitorial spore length in relation to the sample groups. Bold letters indicate statistical significance (p < 0,05). Abbreviations are Bjarnarfj. = Bjarnarfjörður, Deildart. = Deildartunguhver, var. hom = var. homophyllum, Inc. sed. = Incertae sedis.

Bjarnarfj. Deildart. Var. hom. Inc. sed. Pawel Deildartunguhver 0.9721 - - - - Var. Homophyllum 0.4086 0.0515 - - - Incertae sedis 0.3095 0.0025 0.9988 - - Pawel 0.0016 1e-07 0.7422 0.1134 - Var. Spicant 0.9582 1.0000 0.0496 0.0036 3e-07

The appearance of spores varied slightly with most spores having either smooth or irregular outlines and rugulate patterns. Notable differences were found in the ornamentation of the spores from S. spicant var. fallax and sample AS_JND22 (figure 2, table 5).

Table 5: Summarization of spore discriptions from each sample. n stands for the number of spores examined. Spore ornamentational patterns were based on descriptions by Passarelli et al. (2010). Abbreviations are Deildart. = Deildartunguhver, Bjarnarfj. = Bjarnarfjörður, Inc. sed. = Incertae sedis, Homoph. = Homophyllum.

Sample n Variety Size (μm) Outline Pattern Pawel 425 7 Spicant 50.53731 ± 2.91610 Smooth Rugulate, undulate Pawel 427 13 Spicant 47.04701 ± 1.30875 Smooth Rugulate Pawel 430 12 Spicant 50.12229 ± 2.70310 Smooth Rugulate SA JND03 13 Spicant 43.70086 ± 2.73006 Irregular Rugulate AS JND09 9 Spicant 40.53951 ± 2.15832 Irregular Rugulate AS JND22 13 Spicant 42.55843 ± 1.48256 Smooth Filamentous, undulate AS JND12 8 Inc. sed. 46.44236 ± 4.59356 Irregular Rugulate SA JND11 8 Inc. sed. 48.71234 ± 2.92892 Irregular Rugulate AS JND10 10 Inc. sed. 44.61252 ± 1.31120 Irregular Rugulate SA JND02 5 Inc. sed. 47.09203 ± 1.09131 Irregular Rugulate SA JND04 3 Inc. sed. 44.11608 ± 3.93933 Irregular Rugulate AS JND11 6 Inc. sed. 47.05793 ± 3.85375 Irregular Rugulate GA JND04 12 Homoph. 46.84872 ± 1.33769 Irregular Rugulate AS JND21 5 Inc. sed. 44.97616 ± 3.52157 Irregular Rugulate Deildart. 44 Fallax 42.60072 ± 1.33769 Smooth, Filamentous abortive? Bjarnarfj. 17 Spicant 43.60092 ± 0.00951 Irregular, Rugulate hexagonal

10 A B

C D

E F

Figure 2: SEM images of S. spicant spores from different S. spicant varieties bear different ornamental patterns. (A) Struthiopteris spicant var. spicant from the Azores with rugulate ornamentation, (B) Struthiopteris spicant var. spicant from Iceland with rugulate ornamentation, (C) Struthiopteris spicant of an uncertain variety (incertae sedis) from Spain has rugulate ornamentation resembling the typical S. spicant ornamentation, (D) Struthiopteris spicant var. fallax from Deildartunguhver in Iceland has unorthodox filamentous ornamentation and concave appearance, (E) Struthiopteris spicant var. homophyllum from Spain is the other classified homophyllous variety aside from S. spicant var. fallax and bears rugulate ornamentation, (F) The untypical onramentation of the sample AS_JND22 is of S. spicant var. spicant from Spain and has slightly smoother ornamentation, although not as smooth as S. spicant var. fallax. Note different size bars in (B) and (D).

11 4 Discussion 4.1 Karyotype analysis

In order to observe the chromosomes of S. spicant, fiddlehead and leaf bud samples were collected around Iceland. All samples were taken from S. spicant var. spicant, except for the samples taken at Deildartunguhver, which were collected from S. spicant var. fallax. Furthermore, two samples of S. spicant var. homophyllum were sent from Spain but those samples gave unclear results and will not be discussed further here.

Some general problems were encountered when preparing the samples. In order to count the chromosomes of a cell, the cell must be actively dividing, preferably in metaphase. Most of the samples prepared from leaf buds were mature and the cells were already differentiated when collected so few metaphase cells were encountered. The samples prepared from fiddleheads were quite demanding in respect to how long the cell wall of the samples should be digested so some fiddlehead samples did not provide useful protoplasts for chromosome analysis. Due to this, chromosome counting could not be performed, except for a few cases where rough estimates could be presented.

Nucleoli were used as an indicator of the ploidy level of the sampled plants. Nucleoli form around specific chromosomal loci, termed nucleolus organizing regions (NOR), the production site of ribosomes. The NORs contain clusters of 45S rRNA genes which are actively transcribed by polymerase I (Tucker et al., 2010). Analysis of the active NORs in prairie cordgrass (Spartina pectinata Link) of various ploidy levels has revealed a reliable correlation between interphase nucleoli count and ploidy (Kim et al., 2015). In this study nucleoli were counted by using DAPI to stain DNA in chromosomes. Since the nucleolus is largely composed of RNA and proteins, it does not stain with DAPI but leaves a blank in the stain of interphase cells (Hernandez-Verdun, 2005). These blanks were counted as nucleoli.

The karyotype of S. spicant var. spicant has been repeatedly reported as 2n = 68 (Manton, 1950 p. 122; Löve & Löve, 1961; Löve & Löve, 1968; Tryon & Tryon, 1982, p. 678). The gametophytic chromosome number of S. spicant var. homophyllum has similarly been reported as n = 34 (Horjales et al., 1990). Only a single karyotype report exists for S. spicant var. fallax in which the sporophytic chromosome number is reported as 2n = 68 (Löve & Löve, 1968) in accordance with reports of the S. spicant var. spicant and S. spicant var. homophyllum karyotypes.

The basic chromosome number for Struthiopteris has been reported once as x =31 or 34 (de Gasper et al., 2016) and a single hypothesis for Blechnum, then including Blechnum spicant, was presented as x = 33 (Walker, 1973). In light of the observations made above, these basic chromosome numbers can be manifestly rejected for Struthiopteris. Since the reported chromosome number for S. spicant as 2n = 68 is in accordance with the chromosomes observed above, except for the those of S. spicant var. fallax, and as the number of nucleoli observed in S. spicant var. spicant usually ranged between two and four, it can be concluded that S. spicant var. spicant is tetraploid with 68 chromosomes and thus 2n = 4x = 68 revealing a basic number of x = 17 for S. spicant. The basic number x = 17 has been forecasted by Löve & Löve (1968) for the genus Blechnum when reporting their confirmation of the karyotype 2n = 68 for S. spicant, probably based on the sheer number of chromosomes observed (Löve & Löve, 1968).

12 Although this basic number was reported for Blechnum, it fits well with the observations made here on Struthiopteris spicant.

Interestingly, S. spicant var. fallax did not show high numbers of nucleoli, with intermittently one or two nucleoli, as opposed to the S. spicant var. spicant samples which show between two and four nuceloli per interphase cell. The chromosome count for sporophytic metaphase chromosomes was roughly 30 chromosomes (figure 1f). On account of the low number of nucleoli observed and small number of metaphase chromosomes it is evident that S. spicant var. fallax is diploid, but not tetraploid as previously reported for S. spicant var. fallax (Löve & Löve, 1968). Struthiopteris spicant var. fallax therefore shows unique ploidy among S. spicant varieties.

Based on the above observations of basic chromosome number, it is predictable that the karyotype of S. spicant var. fallax is 2n = 2x = 34, but this is merely speculation. 4.2 Spore size and ornamentation

Results from the measurement of spore equitorial length does not indicate that there is a considerable difference between the spores of S. spicant var. fallax, labeled as Deildartunguhver, and the other varieties (tables 2 and 3). The groups Pawel and Incertae sedis show significantly larger spores than plants from Deildartunguhver, and S. spicant var. spicant and the Pawel group also shows significantly larger spores than plants from Bjarnarfjörður. As the measurements in this study were performed with the perispore intact, 2,0-4,0 μm should be retracted from the values given here to obtain the exospore size which is reported as 35-50 μm for S. Spicant (Passarelli et al., 2010). The samples observed here fit the normal spore size reported by Passarelli et al. for S. spicant (2010). A characteristic rule of thumb exists for the relationship between spore size and ploidy in the old Blechnum genus since diploid species generally have an exospore length of 30-40 μm and tetraploid species have measures between 40-60 μm (Passarelli et al., 2010). Althought this rule of thumb does not have much scientific value as too little is known about ploidy in Blechnum, this rule seems to be applicable for most or all of the samples in this study after the thickness of the perispore has been corrected for, given that S. spicant var. fallax is diploid and the other varieties are tetraploid.

Interestingly, the spores of S. spicant var. fallax exhibit some difference in ornamental patterns from the spores of other groups (table 4). Ornamentation on the perispore is an important diagnostic tool in fern taxonomy (Passarelli et al., 2010). Struthiopteris spicant has rugulate spores (Mazooji & Salimpour, 2014; Zenkteler, 2012) and so do all samples in this study with two exceptions. The spores of S. spicant var. fallax are obviously differently ornamented bearing filamentous and smooth patterns. The spores of sample AS_JND22, sent from the Iberian peninsula, also shows interesting undulating or slightly filamentous patterns (figure 3).

Figure 3: A SEM image of spores of S. spicant var. spicant in the open sporangium. To the left are spores of the sample AS_JND09 which show typical ornamentation for S. spicant var. spicant. To the right are spores of AS_JND22 which differ somewhat from the rugulate ornamental pattern typical of S. spicant spores, but bear slightly smoother and more filamentous patterns.

The diagnostic value of fern spores has been underappreciated in the classification of species from the old Blechnum genus, including Struthiopteris spicant (Tryon & Tryon, 1982). Spore ornamentation has distinct diagnostic value even at specific level for Blechnum species (Passarelli et al., 2010) and novel Blechnum species, such as B. areolatum and B. longipilosum, have been assorted into groups based on perispore ornamentation and their spore size. Their spore ornamentation was also used to support their distinction as separate species (Dittrich et al., 2012). Although Struthiopteris has recently been segregated from Blechnum, there are indications that spore ornamentation could also be a useful complementary tool in Struthiopteris taxonomy since S. spicant appears to have unique spores when size and ornamentation are considered together (Passarelli, 2007). The above results of S. spicant var. fallax spore ornamentation are therefore interesting as they indicate that the spore ornamentation of S. spicant var. fallax is not the same as for other S. spicant varieties. This challenges the validity of the usage of spore ornamentation for species distinction in Struthiopteris or forces the reconsideration of S. spicant var. fallax classification. 4.3 Spore abortion

The appearance of spores of S. spicant var. fallax was not only ornamentally different but differed from all other samples in shape. They had the typical smooth pea shape of S. spicant spores (Mazooji & Salimpour 2014) but were obviously concave as if lacking or having a reduced protoplast and appear to be abortive. While 10-20% of fern spores are abortive for unknown reasons (Wagner & Wagner, 1986), a much higher number of abortive-looking spores was observed in SEM-images for S. spicant var. fallax. According to Wagner & Wagner (1986) abortive spores are characterized by exaggerated variability in spore size, spore collapse due to turgor loss which may be related to a lack of protoplast, irregular development of the perispore in massive perispore genuses such as the old Blechnum genus (which then included Struthiopteris as well), variability in size or shape of the laesura due to hybridity, twisting of the exospore and some sporangium characteristics which were not examined here (Wagner & Wagner, 1986). Some of these symptoms describe the spores of S. spicant var. fallax rather accurately. Almost all of the spores were concave, appearing to lack or suffering a reduction in

14 protoplast size. Many had twisted spores, although not only the exospore was twisted but the perispore as well. The ornamentation of the spores from S. spicant var. fallax was also different from other S. spicant samples.

However, the abortive appearance of S. spicant var. fallax spores does not reveal much about the nature of the spores and neither does it confirm that the spores of S. spicant var. fallax are abortive nor that they are unable to germinate. To begin with, even fern hybrids considered to be sterile are believed to be able to form at least limited populations (Wagner & Wagner, 1986). Out of 11 hybrids of Dryopteris species, spores of only one hybrid showed a germination rate og 0.0%, while four had a 0.5% germination rate, and others had a germination rate of 1.4%, 1.5%, 3.9%, 8.3%, 34.3% and 36.0%. While fern spores have vast capacity for long distance dispersal, most of the spores spread only a few meters from the parental plant (Peck et al., 1990; Penrod & McCormick, 1996; Chung & Chung, 2013). In that case, a spore germination rate of only a portion of a percent could be sufficient for a population to establish. This could be even more applicable to S. spicant var. fallax since it does not appear to compete with many other species for habitat. In order to resolve the viability of S. spicant var. fallax spores a germination test is undoubtedly necessary.

Although some of the abovementioned symptoms of spore abortion from Wagner & Wagner (1986) refer to the spores of S. spicant var. fallax, others are either incompatible or contrary to the samples. To start with, no change in shape of laesura was observed and although laesura length variability could not be measured, no obvious extreme cases were observed. This indicates that S. spicant var. fallax is not a hybrid of S. spicant, a monolete fern, and a trilete fern species. Another symptom not observed is exaggerated variability in spore size, in fact the variability in spore size, represented by the variance of equitorial spore length, was even lower in spores from S. spicant var. fallax (var = 20.496) than in S. spicant var. homophyllum (var = 22.825) and S. spicant var. spicant samples from Bjarnarfjörður (var = 24.538). The irregular development of the perispore may not even fit the description as the spores of S. spicant var. fallax were quite consistent in their phenotype as almost all of the spores were bean-shaped, concave with the same ornamental pattern. As mentioned above there are also environmental sources of abortion, notably cold and drought. In the case of S. Spicant var. fallax it is difficult to comprehend whether spore abortion could be triggered by environmental factors. Further research would be needed to clarify this.

Even though the spores of S. spicant var. fallax would fit all the descriptions of abortive spores, explaining why they were abortive could be troublesome. While abortive spores of this type are most common in pteridophyte fern hybrids, genetic or environmental factors may influence spore abortion (Wagner & Wagner, 1986). If the source of spore abortion in S. spicant var. fallax were due to hybridity, then according to the phenomenon of intermediacy, its phenotype should range somewhere between the parental species (Wagner & Wagner, 1986). As S. spicant var. fallax resembles only S. spicant in its morphology and is unique in its ecology, finding the parental species could become a complex task. Few studies have been done on hybrid spore morphologies but spores may also submit to the phenomenon of intermediacy. This was the case of the species Polystichum Xbicknelli, a hybrid species between P. setiferum and P. aculeatum. The spores of this species show an intermediary phenotype in ornamentation between the parental species and many of the spores are ill-formed and irregular in size and shape (Cubas & Pardo, 1992). Possibly this idea could be used to narrow the search for possible parental species of S. spicant var. fallax in the unlikely case it turns out to be a hybrid. The karyotype reported here does not indicate that S. spicant var. fallax is a hybrid between S. spicant and another fern species.

15 Occasional triploidy can be a source of abortion but this was not observed in provisional karyotyping. The karyotyping results above indicate that this is not a cause of spore abortion in S. spicant var. fallax. Somatic changes in ploidy were not investigated. Environmental factors should be investigated as they could play a role in S. spicant var. fallax spore abortion since it grows in rather hostile conditions where the ground is hot with high concentrations of geothermal chemical compounds and the air is cold and steamy.

There is therefore a possibility that the spores of S. spicant var. fallax were not abortive at all, except perhaps an occasional abortive spore in the baseline 10-20%. The explanations for some of the morphological features could be that the spores were not mature when collected. Immature spores are quite easily confused with abortive spores (Wagner & Wagner, 1986) and this would explain the lack of irregularity in development and size and the consistency in shape. The spores of S. spicant var. fallax also seemed to fragment less readily which could be a sign of abortion since it is believed that abortive spores have altered amounts of oil produced by the protoplast (Wagner & Wagner, 1986). In many samples small framgents of the perispore were distributed throughout the sample but this does not apply to S. spicant var. fallax samples. This would also explain why spores with some similarities in morphology were observed in the sample AS_JND22. However, claiming that the spores of S. spicant var. fallax were immature when collected is premature since the spores did neither accurately fit the description for abortive spores presented in Wagner & Wagner (1986), nor has the morphology of mature S. spicant var. fallax ever been reported for comparison. Furthermore, the spore samples were collected from well developed sporangia from brown sori, an indicator of spore maturity. Further sampling during the summer months should shed light on the immature and mature morphology of S. spicant var. fallax spores. 4.4 Considerations about S. spicant var. fallax reproductive ecology

If S. spicant spores were immature in S. spicant var. fallax but mature in S. spicant var. spicant a question inevitably arises: Do the two varieties not produce spores at the same time, and if not, does that bias the reproductive possibilities of the two varieties so that they are more likely to mate with their own variety and less so with others? Such a situation is theoretically possible and has been observed at least twice in angiosperms.

Antonovics (2006) observed that the grass species Anthoxanthum odoratum growing on an old zinc mine had an earlier flowering time than the same species growing in a pasture a few meters away. This difference in flowering had also been observed in a previous measurement in 1979 but no measurements had taken place between 1979 and 2005. The most plausible explanation for this is that the population growing on the mine tailings is tolerant to high concentrations of zinc and lead in the soil but the population growing on the pasture is not. This trait has led to reproductive hindrance between the population and in 2005 the isolation index was measured 0.43, meaning that even if the populations were intermixed, they were still 43% less likely to interbreed than to breed within themselves (Antonovics, 2006). McNeilly and Antonovics (1968) had made similar observations in 1968 with A. odoratum from another zinc mine and Agrostis tenuis from a copper mine. The mine populations of both species flowered about a week earlier than the pasture populations and they maintained this difference under laboratory conditions, indicating that the difference in flowering were due to genetic differences. Crosses between the populations showed no obvious incompatibility barriers, although slightly less seed was produced from A. odoratum crosses when the female came from the pasture population

16 compared to when it came from the mine population. This indicates that differences in flowering had not arisen due to fertilization incompatibilities. The difference in flowering time between the populations was also exaggerated close to the boundary between the populations, indicating that the difference in flowering was under selective pressure (McNeilly & Antonovics, 1968).

It is easy to imagine such selective pressure arising in the case of S. spicant var. fallax and S. spicant var. spicant in Iceland as both varieties seem well adapted to their habitats and their interbreeding would produce plants that were less likely to be successful due to ploidy or ecological requirements. Research on the reproductive ecology of S. spicant varieties in Iceland is essential in understanding the possibility of hybridization between S. spicant var. spicant and S. spicant var. fallax, and to what extent gene flow is possible between these varieties.

4.5 Models of differentiation between S. spicant var. fallax and S. spicant var. spicant

One of the great enigmas of S. spicant var. fallax has been how it became differentiated from the other S. spicant varieties. Previous experiments indicate that the difference between the two varieties is genetic and not ecological (Hallgrímsson, 1968; Löve & Löve 1968). Genetic differences can arise by many routes, some of which imply immediate reproductive barriers or phenotypic differences, such as changes in ploidy, but others act gradually over long time scales, such as genetic drift and constant selective pressure.

Generally, plant differentiation, and further on in time, speciation, are characterized by some form of reproductive isolation between previously interbreeding populations. Although reproductive isolation can arise through many routes, habitat differentiation and ecological oppurtunities often play a major role (Riesberg & Willis, 2007). When a founder population colonizes an ecological niche numerous factors can lead to subspeciation or speciation. For instance, when the founder population is small, it is likely to be subject to genetic drift that will differentiate it from the original population. This differentiation can also occur due to altered selective forces in different environments (Templeton, 2008), for example in Deildartunguhver plants that thrive in geothermal environments would be selected for but these same plants would be selected against in snow basins. Although the importance of adaptation in speciation is little contested, the context between adaptation and genetic differentiation is often quite unclear (Sobel et al., 2009).

The first hypothesis about the origin of S. spicant var. fallax, and the simplest explanation for the morphological differences between S. spicant var. fallax and S. spicant var. spicant, was that the fallax variety were an ecomorph of S. spicant. Although S. spicant var. fallax has not yet been grown from a spore in a different environment, this hypothesis has traditionally been rejected due to numerous transplantation experiments that have demonstrated that S. spicant var. fallax holds its characteristics after transplantation to different climates and soils (Hallgrímsson, 1968; Löve & Löve, 1968).

The second hypothesis, presented by Löve & Löve (1968), that S. spicant var. fallax were geographically isolated from S. spicant var. spicant can also be confidently rejected. Not only does S. spicant grow in at least 2-3 other locations in Borgarfjörður (Flora of Iceland, 2016;

17 Icelandic Institute of Natural History, 2017), but its spores were encountered in lake sediments from Borgarfjörður, which were dated from before the settlement of Iceland until the late Middle Ages (Erlendsson, 2007 p. 221). Since these spores were identified as S. spicant spores, their origin was probably not from Deildartunguhver, as the spores from S. spicant var. fallax appear to be morphologically different from the spores of S. spicant var. spicant and are unlikely to have matched the descriptions in identification keys. The spores encountered in lake sediments must therefore have been from S. spicant var. spicant plants. This suggests that a frequent historical encounter of S. spicant var. fallax and S. spicant var. spicant is evident and therefore rejects the claim that S. spicant var. fallax and S. spicant var. spicant are reproductively isolated due to geographical barriers. Furhtermore, an example of an isolated population of S. spicant from North America of a similar size as the population of S. spicant var. fallax has shown that intragametophytic selfing does only reach 40,3% in the most extreme case of geographic isolation (Soltis & Soltis, 1987, 1988) and geographic isolation is not a significant barrier to interpopulational reproduction.

The results from S. spicant var. fallax karyotype analysis suggest that there is a difference in ploidy between S. spicant var. spicant and S. spicant var. fallax. Differences in ploidy can act swiftly as a reproductive barrier (Riesberg & Willis, 2007) and enhance the colonization of new ecological niches (Soltis & Soltis, 2000). However, since S. spicant var. fallax is the diploid form of S. spicant it is tempting to consider it to be a more ancient form than other S. spicant varieties. If so, S. spicant var. fallax is likely an ancient relict that has survived for a long time in its habitat. It is probable that the established differences in morphology between S. spicant var. spicant and S. spicant var. fallax have both arisen quickly due to differences in ploidy, and gradually due to different ecological preferences and altered selective pressure. The differences in ploidy and straightforward differences in habit, frond morphology and ecology support this notion, as well as the differences in spore morphology observed here for the first time.

In conclusion I will therefore present a third hypothesis on the origin of S. spicant var fallax: Struthiopteris spicant var. fallax is an ancient relict that is starkly different from S. spicant var. spicant, both in ploidy and morphology. It is likely an established species, but not a variety of S. spicant, that is well adapted to its habitat and has remained there for a long time.

The results above are of great significance and must be confirmed in a seperate study. If confirmed, they give rise to many outstanding questions regarding the classification of Struthiopteris, the origin and evolution of S. spicant and how S. spicant var. fallax has survived in Iceland or recolonized it after the last glacial maximum. Hopefully, these elements will be addressed in upcoming research.

18 References

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19 Guðmundsson, Þ. (1940). Sjaldgæfar jurtir fundnar á Austurlandi. Náttúrufræðingurinn 10(3- 4): 163-165 (in Icelandic). Hallgrímsson, H. (1968). Nýjar athuganir á Deildartunguburknanum. Flóra 6: 75-76 (in Icelandic). Hernandez-Verdun, D. (2005). Nucleolus: from structure to dynamics. Histochemistry and cell biology 125(1): 127-137. Horjales, M., Redondo, N. & Pérez Prego, J. M. (1990). Nota Citotaxonómica sobre pteridoflora del noroeste de la peninsula Ibérica. Anales del Jardín botánico de Madrid 48(1): 82-84 (in Spanish). Icelandic Institute of Natural History (2017). Skollakambur (Blechnum spicant). Retrieved 22.07.2017 from http://www.ni.is/biota/plantae/monilophyta/skollakambur-blechnum- spicant (in Icelandic). Ingadóttir, Á. (supervised by). (1996). Válisti 1. Reykjavík, Náttúrufræðistofnun Íslands (in Icelandic). Kim, S., Lee, D. & Rayburn, A. L. (2015). Analysis of active nucleolus organizing regions in polyploid prairie cordgrass (Spartina pectinate Link) by silver staining. Cytologia 80(2): 249-258. Kristinsson, H., Þorvaldsdóttir, E. G., Steindórsson, B. (2007). Vöktun válistaplantna 2002- 2006 (fjölrit nr. 50). Náttúrufræðistofnun Íslands (in Icelandic). Löve, Á. (1948). Gróður nyrzt á Hornströndum. Náttúrufræðingurinn 18(3): 97-112 (in Icelandic). Löve, Á. & Löve, D. 1961. Some Chromosome Numbers of Icelandic Ferns and Fern-allies. American fern journal 51(3): 127-128. DOI: 10.2307/1546094 Löve, Á. & Löve, D. (1968). Cytotaxonomy of Blechnum Spicant. Collect. Bot. 7: 665-676 (Barcelona 1968). Manton, I. (1950). Problems of evolution and cytology in the pteridophyte. Cambridge, Cambridge University Press. Mazooji, A. & Salimpour, F. (2014). Spore Morphology of 34 Species of Monilophyta from Northern Parts of Iran. Annual research and review in biology and botany 4(6): 924- 935. McNeilly, T. & Antonovics, J. (1968). Evolution in closely adjacent plant populations – IV. Barriers to gene flow. Heredity 23: 205-218. DOI: 10.1038/hdy.1968.29 Passarelli, L. M. (2007). Estudios esporales en especies del grupo Blechnum penna-marina (Blechnaceae-Pteridophyta). Acta botanica Malacitana 32: 49-66 (in Spanish). Passarelli, L. M., Gabriel y Galán, J. M., Prada, C. & Rolleri, C. H. (2010). Spore morphology and ornamentation in the genus Blechnum (Blechnaceae), Grana 49(4): 243-262. DOI: 10.1080/00173134.2010.524245 Peck, J. H., Peck, C. J. & Farrar, D. R. (1990). Influences of Life History Attributes on Formation of Local and Distant Fern Populations. American fern journal 80(4): 126- 142. Penrod, K. A. & McCormick, L. H. (1996). Abundance of Viable Hay-Scented Fern Spores Germinated from Hardwood Forest Soils at Various Distances from a Source. American fern journal 86(3): 69-79.

20 Perrie, L. R., Wilson, R. K., Shepherd, L. D., Ohlsen, D. J., Batty, E. L., Brownsey, P. J. & Bayly, M. J. (2014). Molecular phylogenetics and generic taxonomy of Blechnaceae ferns. Taxon 63(4): 745-758. DOI: 10.12705/634.13 Riesberg, L. H. & Willis, J. H. (2007). Plant speciation. Science 317: 910-914. Sobel, J. M., Chen, G. F., Watt, L. R. & Schemske, D. W. (2009). The biology of speciation. Evolution 64(2): 295-315. DOI: 10.1111/j.1558-5646.2009.00877.x Soltis, D. E. & Soltis, P. S. (1987). Polyploidy and breeding systems in homosporous pteridophyta: a reevaluation. The American naturalist 130(2): 219-232. Soltis, P. S. & Soltis, D. E. (1988). Genetic variation and population structure in the fern Blechnum spicant (Blechnaceae) from Western North America. American Journal of Botany 75(1): 37-44. Soltis, P. S. & Soltis, D. E. (2000). The role of genetic and genomic attributes in the success of polyploids. Proceedings of the National academy of sciences 97(13): 7051-7057. Templeton, A. R. (2008). The reality and importance of founder speciation in evolution. Bioessays 30(5): 470-479. DOI: 10.1002/bies.20745 Tryon, R. M. & A. F. Tryon. (1982). Ferns and allied plants, with special reference to tropical America. Springer-Verlag. New York. Tucker, S., Vitins, A. & Pikaard, C. S. (2010). Nucleolar dominance and ribosomal RNA gene silencing. Current opinion in cell biolgy 22: 351-356. Wagner, W. H. Jr. (1974). Structure of spores in relation to fern phylogeny. Annals of the Missouri botanical garden 61(2): 332-353. Wagner, W. H. Jr. & Wagner, F. S. (1986). Detecting Abortive Spores in Herbarium Specimens of Sterile Hybrids. American fern journal 76(3): 129-140. Walker, T. G. (1973). Evidence from cytology in the classification of ferns. In: Jermy, A. C., Crabbe, J. A. & Thomas, B. A. The phylogeny and classification of ferns: 91-110. London, Academic press for the Linnaean society of London xiii 284 p. Puplished as supplement 1 (1973) 1-7. Cited through: Tryon, R. M. & Tryon, A. F. (1982). Wasowicz, P., Folcik, L. & Rostanski, A. (2017a). Typificatin of Blechnum spicant var. fallax Lange (Blechnaceae). Acta societatis botanicorum Poloniae 86(1): 3542. DOI: 10.5586/asbp.3542 Wasowicz, P., Gabriel y Galán, J. M. & Perez, R. P. (2017b). New combinations in Struthiopteris spicant for the European flora. Phytotaxa 302(2): 198-200. DOI: 10.11646/phytotaxa.302.2.11 Wolf, P. G., Sheffield, E. & Haufler, C. H. (1991). Estimates of gene flow, genetic substructure and population heterogeneity in bracken (Pteridium aquilinum). Biological journal of the Linnean society, 42: 407-423. Zenkteler, E. (2012). Morphology and peculiar features of spores of fern species occurring in Poland. Acta agrobotanica 65(2): 3-10.

21 Supplement

This is a list of the 16 samples collected for karyotyping.

Sample ID: RE_01 Location: Reykjanes under Trölladyngja. Time collected: 17:55, 05.07.2016 Information: The whole sample is believed to be from a single individual found in grassland, about 20 meters from a hot spring vent. The sample was taken from a cluster of leaves containing around 30 sterile leaves and two fertile leaves. No spores were visible but green sporangia were observed.

Sample ID: RE_02 Location: Reykjanes under Tölladyngja. Time collected: 18:03, 05.07.2016 Information: The sample was obtained from the same cluster as sample RE_01.

Sample ID: RE_03 Location: Reykjanes under Trölladyngja. Time collected: 18:20, 05.07.2016 Information: One individual was found in a 1-2 m2 cave with steam at 25°C. Frond length was 8 cm. No fertile leaves, spores or sporangia were observed.

Sample ID: LA_01 Location: Landmannalaugar under Brennisteinsalda. Time collected: 11:30, 06.07.2016 Information: One or two individuals were found together in geothermal soil among other vegetation at 36°C ground temperature. The cluster had 15-20 leaves, around 4 cm long. No spores, sporangia or fertile leaves were observed.

Sample ID: DE_01 Location: Deildartunguhver. Time collected: 11:45, 07.07.2016 Information: One individual plant bearing 13 fronds, 2-5 cm long. Spores were found on morphologically sterlie fronds but no spore sample was collected. The plant was growing among mosses on a warm rocky substrate.

Sample ID: DE_02 Location: Deildartunguhver. Time collected: 12:00, 07.07.2016 Information: One individual plant bearing 7 fronds, 2-3 cm long. No fertile leaves, spores or sporangia were observed. Found growing among mosses on a warm substrate.

Sample ID: DE_03 Location: Deildartunguhver. Time collected: 12:15, 07.07.2016 Information: One individual plant bearing 7 fronds, 2-4 cm long. Spores were found on morphologically sterile leaves. The spore sample used in this study was collected from this plant. The plant was growing among mosses on a warm substrate.

22 Sample ID: DE_04 Location: Deildartunguhver. Time collected: 12:30, 07.07.2016 Information: One individual plant bearing fronds 3-5 cm long. No fertile leaves, sporangia or spores were observed. Found growing on a warm substrate.

Sample ID: BJ_01 Location: Above Svanshóll in Bjarnarfjörður. Time collected: ~12:15, 08.07.2016 Information: Large or middle sized fern with fertile leaves but it is unsure weather they had sporangia or spores. Found in grassland on a mountain slope.

Sample ID: BJ_02 Location: Above Svanshóll in Bjarnarfjörður. Time collected: ~12:15, 08.07.2016 Information: A cluster of rosettes that had dead fertile leaves with spores or live fertile leaves with or without spores. Middle sized, 5-15 cm. Found in grassland on a mountain slope.

Sample ID: BJ_03 Location: Above Svanshóll in Bjarnarfjörður. Time collected: ~12:15, 08.07.2016 Information: A small plant, 5-8 cm. No fertile leaves, sporangia or spores were observed. Found in grassland on a mountain slope.

Sample ID: BJ_04 Location: Above Svanshóll in Bjarnarfjörður. Time collected: ~12:15, 08.07.2016 Information: Middle sized plant, 10-15 cm. No fertile leaves were observed on this plant nor other nearby plants. Found on a warm rocky substrate on a mountain slope.

Sample ID: BJ_05 Location: Above Svanshóll in Bjarnarfjörður. Time collected: ~12:15, 08.07.2016 Information: Middle sized, 10-20 cm. No fertile leves were found on this plant but nearby plants had fertile leaves. Found in grassland on a mountain slope.

Sample ID: BJ_06 Location: Above Svanshóll in Bjarnarfjörður. Time collected: ~12:15, 08.07.2016 Information: A large plant, 15-30 cm. Fertile leaves were found on this plant but no spores were visible. Found in grassland on a mountain slope.

Sample ID: HE_01 Location: Héðinsfjörður. Time collected: 16:00, 09.07.2016 Information: Found above a deserted farm near the main road. Fronds were 5-25 cm and many dead fronds were observed around the plant. The plant was found growing under Vaccinium bushes and mosses. Fertile leaves were developing with reddish hue.

23 Sample ID: HE_02 Location: Héðinsfjörður. Time collected: 16:05, 09.07.2016 Information: Plant found under Vaccinium bushes at the same location as HE_01.